Primitive Neural Stem Cells in the Mouse Brain

by

Rachel Leeder

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Institute of Medical Science University of Toronto

© Copyright by Rachel Leeder 2015

 

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Primitive Neural Stem Cells in the Mouse Brain

Rachel Leeder

Doctor of Philosophy

Institute of Medical Science University of Toronto

2015

Abstract

Neural stem cells (NSCs) reside in the tissue lining the lateral ventricles of the

adult mouse brain. At the top of the hierarchy are primitive (p)NSCs that arise in advance

of definitive (d)NSCs embryonically. After the discovery that pNSCs persist in the adult

mouse brain, I sought out to characterize pNSCs and determine whether they express the

pluripotency gene Oct4 in the adult brain as the do embryonically. Next, I addressed the

cell cycle time of pNSCs and whether they are activated to proliferate to repopulate

dNSCs after dNSC and downstream progenitor ablation. Finally, I identified cell type

specific markers of pNSCs and pharmacological methods to selectively target and

activate endogenous pNSCs. These selective markers can be used for future studies to

enrich for pNSCs and to develop future therapies to target pNSCs endogenously.

Together, this thesis presents evidence that pNSCs are an Oct4-expressing, reserve

population at the top of the NSC hierarchy capable of repopulating dNSCs.

 

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Acknowledgements

First, I would like to thank my supervisor Derek van der Kooy for welcoming me

into the lab and setting me out on my scientific career. I learned the important lessons of

critical thinking, coming to your own conclusions, your science is only as good as your

ability to communicate it, and when you know that you are right, take the bet. His

guidance but freedom to find my own way is what made my graduate career more than

about the science.

I thank my committee members Cindi Morshead and Janet Rossant who

continually challenged me to think harder and consider every scenario before coming to a

conclusion. I appreciate your input to shape my project and improve communication

skills over the years.

Thank you to our collaborators and those who contributed transgenic mice and

reagents (K. Hochedlinger, A. Tomlin S. Nishikawa, M. Sofroniew, A. Nagy). To the

Department of Comparative Medicine at the University of Toronto and in particular AJ

Wang for his endless help keeping my mice healthy to make these experiments possible.

The members of van der kooy lab and the 11th floor have been a huge help

throughout this project. Breakfast club, stemmies, and even lunchtime have been

opportunities to discuss, think and critically analyse. In particular, thank you to our

amazing lab manager and technician Brenda Coles for keeping the lab running and

making many last minute orders to save my experiments. To Sue Runciman who early on

gave me the great advice of “if your experiments aren’t working today, just go home,

they will work tomorrow,” which has become my motto. To the lab members who

graduated before me Margot Arntfield, Laura Donaldson, Lilian Riad-Allen, Brian

 

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DeVeale and Brian Ballios who offered help where they could and more importantly

made my time in the lab enjoyable. To the LIF team, Nadia Sachewsky and Wenjun Xu,

no one quite understands the trials of this project like they do. Finally, to Samantha

Yammine for swooping in at the end and helping any way she could to get my last

experiments done and being instrumental in my making it to the end of this process with

my sanity intact.

Finally, I would like to thank my family and friends for their support through this

process. My parents have always supported me and lent a sympathetic ear, and Michelle

for being an amazing friend. To my husband, Jamie, thank you for supporting me and

always being a wonderful distraction to keep me going.

 

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Table of Contents Abstract ii Acknowledgements iii

List of Figures and Tables vii List of Abbreviations ix Chapter 1: General Introduction 1 Discovery of Stem Cells 2 Stem Cell Criteria 4 Progenitors 5 Modes of Stem Cell Division 6 Pluripotency Genes 7 In Vitro ESC-Derived Neural Stem Cells 10 Induced Pluripotent Cells 11 iPSCs for Clinical Use 13 Early Embryonic Development 15 Neural Induction in the Embryo 17 Radial Glia in the Embryo 19 Emergence of Primitive and Definitive NSC Populations 20 Perinatal Neurogenesis 22 Adult Neural Stem Cell Discovery 23 Neural Stem Cell Isolation 27 Adult Neurogenesis in the Periventricular Region 29 Architecture of the NSC Niche 31 Neurogenesis in the Hippocampus 35 Increasing Cortex Size with the Outersubventricular Zone 37 Human Neurogenesis 40 Stem Cell Quiescence 42 NSC Activation 45 Cell Type Specific Markers 47 Cell Surface Profiling 49

Aims and Hypothesis 53 Chapter 2: Primitive neural stem cells in the adult mammalian brain 57 give rise to the GFAP expressing neural stem cells Abstract 58 Introduction 59 Methods 60 Results 63 Discussion 89

 

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Chapter 3: Quiescent primitive neural stem cells repopulate the ablated 109 definitive neural stem cell population in the adult mouse brain Abstract 110 Introduction 111 Methods 113 Results 116 Discussion 130 Chapter 4: Targeted activation of primitive neural stem cells in the mouse brain 135 Abstract 136 Introduction 137 Methods 139 Results 143 Discussion 162 Chapter 5: General Discussion 167 Implications for the NSC lineage 168 Repopulation of dNSCs after ablation 172 Other quiescent NSC hypotheses 173 Are all neurospheres NSC-derived? 179 How many pNSCs are really in the brain? 181 Cell cycle times 183 Comparing pNSCs to other stem cell hierarchies 184 Why target pNSCs for regeneration? 186 Are pNSCs present in the human? 187 Conclusions 188 Future directions 189 References 194

 

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List of Figures

Chapter 1

Figure 1. Adult pNSCs give rise to dNSCs in the adult mouse brain, 24 similar to the lineage in embryonic development

Figure 2. The NSC niche 32 Figure 3. Identification of cell surface proteins enriched specifically

on ESCs, pNSCs and dNSCs 50 Chapter 2

Figure 1. LIF responsive colonies are derived from the adult periventricular region 64

Figure 2. LIF colonies express Oct4 and integrate into the ICM of blastocysts 70

Figure 3. Oct4 expressing cells are present in vivo 74 Figure 4. GFAP-TK model specifically ablates dividing GFAP+

cells in vitro and in vivo 76 Figure 5. Infusion of AraC+GCV leads to complete but temporary

loss of neurospheres. 82 Figure 6. Numbers of adult derived pNSCs can be increased by injury

or LIF infusion 86 Figure 7. In vivo lineage analysis 90 Supplemental Figure 1. The periventricular region contains proliferating

LIF-R+ cells in vivo. 96 Supplemental Figure 2. Expression profile of AdpNSCs 98 Supplemental Figure 3. The effects of GCV in vitro and in vivo 100 Supplemental Figure 4. Proliferating GFAP+ cells returned with longer

survival times following AraC+GCV treatment 102 Supplemental Figure 5. A. Repopulation of proliferating cells after ablation

in GFAP-tk mice 104 Supplemental Figure 6. YFP-GFAPtk derived colonies for transplantation 106

Chapter 3 Figure 1. Label retention in pNSCs and dNSCs in H2B-GFP mice 118

Figure 2. Oct4fl/fl;Sox1Cre/Cre (Oct4CKO) mice are a pNSC loss of function model and had significantly reduced ability to repopulate dNSCs 122

Figure 3. Reduced proliferation and dNSC recovery in the periventricular region of Oct4fl/fl;Sox1Cre;GFAP-tk mice after ablation 126

Figure 4. pNSCs are activated to proliferate following AraC infusion 128

 

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Chapter 4

Figure 1. Characterization of pup-derived pNSCs 144 Figure 2. pNSCs markers in the walls of the lateral ventricle 148 Figure 3. FACS analysis of primary pup brain cells 152 Figure 4. Inhibition of C-Kit signaling increased pup-derived pNSC

neurosphere formation 154 Figure 5. ErbB2 inhibition increased pup-derived pNSC neurospheres 158

Figure 6. C-kit and ErbB2 inhibitors delivered into the lateral ventricle of adult mice increased pNSC neurosphere formation 160 Chapter 5

Figure 1. The NSC lineage and niche proposed in this thesis 170 Figure 2. A unified theory of the NSC lineage 176

 

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List of Abbreviations

a Activated BMP Bone morphogenic protein CSF Cerebrospinal fluid d Definitive E Embryonic day EFH EGF, FGF, and heparin EGH Epidermal growth factor ESC Embryonic stem cell FBS Fetal bovine serum FGF Fibroblast growth factor GCNF Germ cell nuclear factor GFAP Glial fibrillary acidic protein GFP Green fluorescent protein H2B Histone2B HSC Hematopoetic stem cell INM Interkinetic nuclear migration LIF Leukemia inhibitory factor MST Mitotic somal translocation NSC Neural stem cell NPC Neural precursor cell P Postnatal day p Primitive q Quiescent qPCR Quantitative polymerase chain reaction RG Radial glia RT Reverse transcription SEM Standard error of the mean SEZ Subependymal zone SFM Serum free media SVZ Subventricular zone TA Transit amplifying VCAM Vascular cell adhesion molecule

 

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Chapter 1

General Introduction

 

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Discovery of Stem Cells

The discovery of stem cells has changed the way we understand embryonic

development and regenerative potential of adult tissues. Stem cells enable the

investigation of embryonic development, cellular differentiation, and organ maintenance

into the adult. The dogma that cells proceed through a unidirectional lineage restriction

and become cell type restricted with age has come into question. Consequently, fixed

cells states and the lack of regenerative potential of adult human organs have been

challenged. The intrinsic potential of adult stem cells is coming to light and changing our

understanding of the somatic cell hierarchies and the field of regenerative medicine.

Much remains to be discovered, but the stem cell field is rapidly expanding and

redefining our understanding of adult somatic cells.

Although the modern stem cell field is relatively new, Ernst Haeckel first used the

term stem cell in 1868. Haeckel used the term stem cell to describe a unicellular organism

at the top of the phylogenetic tree, from which multicellular organs arose (Haeckel,

1868). In addition, he later used the term to describe the fertilized egg that gives rise to all

the cells in the organism (Haeckel, 1877). The first use of the term stem cell to describe a

unique cell in the embryo capable of giving rise to specialized cells was by August

Weissman in 1885. Weissman proposed that a cell called the germ-plasm segregates early

in embryonic development and remains distinct from somatic cells and is transmitted

from one generation to the next (Weismann, 1885). Theodor Boveri traced cell lineages

in the developing nematode and identified germ cells as the only cells to maintain their

chromatin into development. Although it was incorrectly concluded that this was a trait of

all germ cells, rather than a unique trait of the Ascaris nematode, it led to the

 

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identification of germ cells that transmit genetic material between generations. Boveri

correctly proposed that germ cells are stem cells (reviewed in (Ramalho-Santos and

Willenbring, 2007). Valentin Häcker next described a single cell inside the embryo that

would divide to give rise to one germline cell and one mesodermal cell in 1892. Finally,

Edmund Wilson is credited with popularizing the term stem cell, described as an

“unspecialized mother cell” when he wrote a book summarizing these early findings

(Wilson, 1896).

The adult somatic stem cell field began in 1961 with James Till and Ernest

McCullogh, when they discovered hematopoetic stem cells, unknowingly at the time.

This report described colony forming units in the spleen in a landmark paper originally

aiming to identify the sensitivity of bone marrow cells to irradiation (Till and McCulloch,

1961). Till and McCulloch observed a linear relationship between the number of bone

marrow cells injected into irradiated recipient mice and the number of nodules that

formed in the spleens of those recipient mice. Till and McCulloch correctly predicted that

single cells from donors gave rise to colonies, and they further noted that some of the

cells in the rapidly proliferating colonies were undifferentiated (Till and McCulloch,

1961). The nodules were observed to contain all the different blood lineages and

suggested that a single cell could make all the cells in the hematopoietic lineage. Later, it

was confirmed that each nodule arose from a single cell via examination of radiation-

induced chromosomal abnormalities (Becker et al., 1963). In addition, cells from the

nodules could be removed and delivered into a new mouse to generate new nodules

(Siminovitch et al., 1963). This was the first report of a single cell that could self-renew

and generate a colony of cells that included all the cells in a lineage. Neither they nor the

 

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scientific community initially recognized the implications of their findings, which was

the first report of an adult somatic stem cell.

Stem Cell Criteria

Two cardinal properties are essential to categorize a cell as a stem cell: self-

renewal and multipotency (Siminovitch et al., 1963; Till and McCulloch, 1980; Morrison

et al., 1997; Weissman, 2000). Self-renewal of a stem cell during every cell division

ensures a daughter identical to the parent cell will persist for the duration of an

organism’s, or more specifically, an organ’s, lifetime. Without self-renewal, the stem cell

population would become depleted through baseline proliferation/differentiation and in

response to injury, thus would not be available to maintain a tissue through the life of the

animal (Till and McCulloch, 1980). The exception to this rule is the embryonic stem cell

(ESC), which gives rise to the entire embryo but does not persist in development

(Martin, 1981; Gardner, 1985). However, ESCs can be maintained indefinitely in culture

and hence are considered stem cells. Stem cells can last the life of the animal and regulate

the frequency of their divisions (Cheung and Rando, 2013). Stem cells can enter a

quiescent cell state where the cell has left the mitotic cycle, referred to as a G0 state.

Quiescence is reversible, and the cell can be described as resting but can reenter the cell

cycle at any time (reviewed in (Li et al., 2010), and likely plays a role in preserving the

stem cell population into old age. Although stem cells may become quiescent, they retain

their ability to become activated and generate downstream progenitors.

The second property of a stem cell is the ability to give rise to multiple cell types.

A stem cell functions to generate the cells in an organ during development and maintains

 

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this ability through the life of the animal (Kleinsmith and Pierce, 1964). ESCs are

pluripotent and able to generate all the cells in the body (Martin, 1981). Adult stem cells

are multipotent, meaning they are able to generate multiple cell types within their tissue.

For example, a neural stem cell (NSC) can give rise to all cell types in the brain including

neurons, astrocytes and oligodendrocytes (Reynolds and Weiss, 1996). Although stem

cells retain the ability to generate all the cells in the adult tissue, adult stem cells do not

always exhibit this function endogenously due to other cells or circulating inhibitory

signals (reviewed in (Cheung and Rando, 2013)). However, they maintain this intrinsic

ability and multipotency can be tested when cells are grown in culture. Clonal assays are

crucial to the stem cell field, and neurosphere assay is essential to the NSC field, since it

permits testing of self-renewal and multipotency from a single-cell derived colony,

termed a neurosphere (Reynolds and Weiss, 1992). The neurosphere assay (as will be

described further later) tests the intrinsic stem cell potential and all experiments in this

thesis were performed at clonal culture density (Coles-Takabe et al., 2008). Exhibiting

the cardinal properties of self-renewal and multipotency is required for a cell to be

classified as a stem cell.

Progenitors

Studying stem cells is made more challenging due to their close relationship to

their downstream progenitors and thus we must be careful to distinguish the two

populations (reviewed in (Seaberg and van der Kooy, 2003)). Undifferentiated cells that

proliferate to generate downstream progeny, but do not self-renew long-term or generate

all the cells in the tissue are termed progenitors (Potten and Loeffler, 1990; Weiss et al.,

 

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1996). Progenitors are an essential component of regeneration as they can participate in

the expansion of a cell population without involvement of the stem cell itself. Most often,

progenitors are more abundant and have shorter cell cycle times than their stem cell

counterparts and hence have the ability to quickly expand a cell population (Lajtha,

1979). Stem cells and progenitors are sources to regenerate the downstream lineage alone

or in combination. In some organs, for example in the brain, the stem cells reside in a

specialized niche while their progenitors leave the niche to migrate away and generate

specialized cells in the regions they are needed (Morshead et al., 1994; Reynolds and

Weiss 1996; Doetsch et al., 1999). This ensures that the stem cells remain protected and

often quiescent. Essentially, progenitors do the “busy work” of the stem cells enabling the

stem cells themselves to remain in their niche and divide slowly over the lifetime of the

organism.

Modes of Stem Cell Division

There are two methods of division observed in stem and progenitor cells. These

two types of division enable a stem cell to respond to its environment to produce the

downstream progenitors needed while maintaining its own population (Mione et al.,

1997). The first type of division a stem cell can undergo is asymmetric division, whereby

it produces one daughter that is an exact duplicate of itself and another daughter that is a

more restricted progenitor or a differentiated cell (Fuerstenberg et al., 1998). This leaves

the stem cell unperturbed, and the progenitor can either proliferate further to expand the

population or differentiate to form a specialized, functioning cell. Asymmetric division is

more common under baseline conditions and adult organ homeostasis where stem cells

 

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must be protected to survive the life of the animal and thus need to replace themselves

and generate downstream progenitors (Zhang et al., 2004). For example, when a dNSC

divides it replaces itself and generates a downstream TA cell (Weiss et al., 1996;

Morshead et al., 1998).

The second division method of a stem or progenitor cell is to divide

symmetrically. In symmetric division, both daughters can be identical to the parent cell

thereby functioning to expand the stem cell population (Martens et al., 2000).

Alternatively a symmetric division can occur whereby both daughter cells are more

restricted progenitors or differentiated cells, which leads to exhaustion of the stem or

progenitor cell pool. During embryonic development, symmetric divisions expand the

NSC pool between E11-14 and then the division mode switches to asymmetric between

E14-17 (Martens et al., 2000). In the adult, injury or cell loss can induce stem and

progenitor cells to divide symmetrically to expand their population to prepare to

repopulate the damaged cell lineage, as observed following a stroke in the brain

(Arvidsson et al., 2002; Zhang et al., 2004; Kolb et al., 2007; Sachewsky et al., 2014).

Stem cells and progenitors can alternate between symmetric and asymmetric division to

respond to environmental cues and maintain their tissue.

Pluripotency Genes

Pluripotency genes are factors responsible for the potential of a stem cell to give

rise to all cells in the body. The pluripotency genes are expressed in embryonic stem cells

(ESCs) and turn off as a cell differentiates into a downstream stem or progenitor cell

(reviewed in (Niwa, 2007)). It is important that these pluripotency genes turn off as cells

 

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differentiate because they are responsible for the ability to generate multiple cell types,

which if left unchecked, can lead to tumor formation due to misproduction of cells in the

tissue (reviewed in (Kang et al., 2009)). Therefore pluripotency gene expression is tightly

regulated.

Oct4 is a key pluripotency gene that is first expressed at the eight-cell stage and

continues to be expressed in the inner cell mass (ICM) of the developing blastocyst. After

implantation, Oct4 is expressed in the epiblast cells and becomes restricted to the

neuroectoderm after gastrulation and then to the primordial germ cells by E9.5 (Rosner et

al., 1990; Okamoto et al., 1990; Schöler et al., 1990). Loss of Oct4 expression in the

developing blastocyst leads to ICM cells that are no longer pluripotent (Nichols et al.,

1998). ESCs with Oct4 expression reduced by at least half will differentiate into

extraembryonic trophoblast cells (Nichols et al., 1998; Niwa et al., 2000). On the other

hand, ESCs with at least twofold increase in Oct4 expression above the normal level will

differentiate into primitive endoderm or mesoderm (Niwa et al., 2000). Germ cell nuclear

factor (GCNF) is expressed in the anterior neuroectoderm and restricts Oct4 expression in

the embryo (Akamatsu et al., 2009), which leads to neural differentiation (discussed

further later). Therefore, the exact Oct4 expression levels are crucial to the fate of ESCs

and are tightly regulated in the embryo. Within this thesis, I will explore whether Oct4 is

expressed and required by other non-embryonic cells.

There are 11 Oct genes and they are members of the fifth class of the POU (Pit-

Onc-Unc) family. POU is a family of transcription factors conserved amongst all animals

but not identified in plants or fungi. Although the mouse has only one Oct4 isoform, the

human has three splice variants (Oct4A, Oct4B, Oct4B1) but it appears that only Oct4A

 

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has self-renewal function (Wang and Dai, 2010). Oct4 has been implicated to have a role

in chromatin modification, transcription regulation, DNA replication, post-transcriptional

modifications, and other functions. Despite its popularity as a pluripotency gene it

remains unknown to what extent Oct4 performs other roles in stem cells. Another

function of Oct4 is that it maintains viability and regulates proliferation of cells in the

primitive streak (Deveale et al., 2013). Therefore, aside from its well-known importance

in stem cell populations, many of the other roles of Oct4 remain to be elucidated.

Interestingly, the Oct4 promoter activity is directly controlled by an autoregulatory

system including Oct4 itself and Sox2 (Okumura-Nakanishi et al., 2005).

Sox2 is another gene implicated in pluripotency and cell proliferation. Sox2 is first

expressed in ESCs of the ICM and its expression is maintained in the neuroectoderm and

the primitive streak. Sox2 is essential in early embryonic development and Sox2

knockouts are lethal at implantation (Avilion et al., 2003; Niwa, 2007). Unlike Oct4,

Sox2 is highly expressed in the adult brain and is commonly expressed in neural stem and

progenitor cells (collectively referred to as neural precursor cells (NPCs)). Sox2 regulates

multiple transcription factors that influence Oct4 expression (Masui et al., 2007).

Inhibition of Sox2 with interfering RNA induces ESCs to differentiate, predominantly

into trophectoderm (Ivanova et al., 2006). Sox2 and Oct4 activate many of the same

transcription factors and act synergistically to promote their own expression.

Sox2 is widely expressed in NSCs and NPCs in the adult mouse brain. In this

thesis, I will challenge the wideheld belief that Oct4 is only expressed in somatic cells

embryonically (reviewed in (Niwa, 2007)). I will describe an Oct4-expressing NSC that

 

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suggests that the adult brain may have more potential for regeneration than previously

thought.

In Vitro ESC-Derived Neural Stem Cells

While it remains unclear how primitive (p)NSCs arise during embryonic

development, in vitro, ESCs differentiate directly into pNSCs in culture via a default

mechanism (Tropepe et al., 2001). This lineage relationship was elucidated with the

differentiation of ESCs in serum-free, low density, clonal cell culture conditions and it

was observed that the cells began to upregulate neural lineage genes. ESCs in minimal

culture conditions first pass through a pNSC fate via a default mechanism, and then

differentiate into dNSCs. The differentiation of ESCs to pNSCs can be enhanced with

TGF-ß inhibition, indicating that it is the removal of a repressive signal that enables

differentiation of ESCs into pNSCs rather than addition of an exogenous factor (Tropepe

et al., 2001). ESC-derived pNSCs are cultured in serum-free media (SFM) supplemented

with LIF. The generation of pNSCs from ESCs is low in frequency, with just 0.2% of

ESCs giving rise to a pNSC-derived neurosphere when plated in SFM plus LIF. The low

frequency of pNSCs is predominantly due to cell death, and addition of pro-survival

factors NAC and cAMP increased frequency 100-fold so that 20% of ESCs gave rise to

pNSC in culture (Smukler et al., 2006).

pNSCs derived from an ESC line are an intermediate cell type between ESCs and

dNSCs. In vitro pNSCs express Oct4, whereas dNSCs derived from those ESC-derived

pNSCs are Oct4– (Akamatsu et al., 2009). In addition, only pNSCs can integrate into the

ICM of a blastocyst and contribute to a chimeric embryo (Karpowicz et al., 2007). It was

 

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previously thought that pNSCs do not exist in the adult mouse brain, but as I will

discover in this thesis, adult-derived pNSCs exist and maintain these characteristics

previously reported in ESC-derived pNSCs.

Induced Pluripotent Cells

The phenomenon whereby an adult somatic cell is induced to dedifferentiate and

return to an undifferentiated state after overexpression of pluripotency genes, called an

induced pluripotent cell (iPSC), was first described by Takahashi and Yamanaka in 2006.

This discovery illuminated the fact that cell fates might be more dynamic than we

previously thought, and suggests that endogenous cells might have even more potential

for regeneration than previously understood. In this landmark study, which earned Shinya

Yamanaka along with John Gurdon, the Nobel Prize in Physiology or Medicine in 2013,

genes known to be essential to ESCs were overexpressed in adult fibroblast cells to

determine whether they could re-induce pluripotency. After a mixture of viruses

containing many genes successfully induced the dedifferentiation of cells in culture, the

gene combination was whittled down to leave 4 essential factors. These genes were Oct4,

Sox2, Klf4 and c-Myc, generally known as the Yamanaka factors (Takahashi and

Yamanaka, 2006). It was later determined that c-Myc could be removed from the

combination to reduce the likelihood of aberrant growth, and decrease cancer risk

(Wernig et al., 2008; Nakagawa et al., 2008). In addition, many other genes were

identified that promote dedifferentiation and could substitute for some of the Yamanaka

factors (Yu et al., 2007; Ichida et al., 2009; Heng et al., 2010; Ieda et al., 2010; Chen et

al., 2011; Gao et al., 2013). Next, reprogramming was successfully performed in human

 

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fibroblasts (Takahashi et al., 2007). Further studies have supported that ability to

dedifferentiate all adult cell types attempted, including mature B lymphocytes (Hanna et

al., 2008; Staerk et al., 2010), and validates the impact of this finding.

Reports have demonstrated that all cell types tested can dedifferentiate with some

combination of factors. Interestingly, NSCs are the only known adult cell type identified

that dedifferentiate with the introduction of only one gene, Oct4 (Kim et al., 2009a; Kim

et al., 2009b). This is likely a result of NSCs expressing endogenous Sox2, c-myc and

Klf4 and thus Oct4 is the only missing Yamanaka factor in this population. The ability to

differentiate NSCs with only Oct4 suggests that NSCs may have more potential than

other adult somatic cell types to contribute to endogenous regeneration if targeted and

activated in the adult mouse brain (discussed further later).

Due to the tumorigenic concern of pluripotent gene expression in iPSCs, alternate

methods to avoid the use of integrating vectors have been developed. Many of these

approaches include non-integrating vectors, RNA and small molecule approaches (Okita

et al., 2008; Shi et al., 2008; Warren et al., 2010; Yuan et al., 2011; Davis et al., 2013;

Warren and Wang, 2013; Ohnuki et al., 2014; Steichen et al., 2014; Varga et al., 2014).

These alternate approaches increase the applicability of iPSCs for clinical use since they

reduce tumorigenic concerns. The next step towards clinical use is increasing the

efficiency and frequency of reprogramming (Hanna et al., 2009) and scaling up cultures

using bioreactors (Dang and Zanstra 2005; Kirouac and Zandstra; 2008; Csaszar et al.,

2012). The danger of culture-induced modifications can be reduced with low passage

number to reduce copy number variants (Liu et al., 2014). Therefore, despite the large

 

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hurdles that remain to bring iPSCs to clinical use, progress is being made to make iPSC

therapies safe.

iPSCs for Clinical Use

The discovery of iPSCs has launched a new stem cell field that is moving quickly

towards clinical implications. iPSCs have the unique characteristic that they can be

derived from any cell in the body. A patient with a rare disease can provide skin cells

through a minimally-invasive skin biopsy that can be used to generate stem cell cultures

of that disease. For uncharacterized diseases, iPSC cultures can help identify genes or

signaling pathways responsible for the disease of interest (reviewed in (Gunaseeli et al.,

2010; Bellin et al., 2012)). iPSCs are a great advantage for rare diseases where cell

culture models and mouse models don’t exist. Once the causal gene is identified, mouse

models can be generated to potentiate future research.

Another example of clinical implications of iPSCs is the generation of patient-

specific iPSC cultures. Patient-specific iPSCs can be derived from a patient’s skin and

then differentiated into any cell type in the body. By deriving iPSCs from a patient, the

differentiated cell types in culture will be syngenic to those in the patient’s body and

harbor the same mutations (reviewed in (Inoue et al., 2014)). Therefore, pharmacological

agents can be tested on the cells of interest to determine efficacy in advance of being

administered to the patient (reviewed in (Ko and Gelb, 2014)). Alternatively, if a drug is

known to be efficacious but can cause dangerous side effects, it could be tested in cell

cultures (for example on patient-specific cardiac cells) to reduce the risk to the patient

 

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(Burridge and Zambidis, 2013; Scott et al., 2013). Therefore, patient-specific iPSCs have

many implications for future clinical use.

Drug screening and disease model development comprise immediate to short-term

applications of iPSCs, however many long-term possible applications also exist. One

long-term goal in the regenerative medicine field is to develop the technology to grow

organs from iPSCs for transplant into patients. These organs would have multiple benefits

over the current standard of donor organ transplant. iPSC-derived organs would be

patient matched, therefore reducing or eliminating concerns over HLA matching or tissue

rejection (reviewed in (Inoue et al., 2014)). Organs could be grown on demand on a

patient-by-patient basis. Currently organ donation programs have a severe shortage of

organs available, resulting in long patient wait times and deaths (Rosenblum et al., 2012).

Culturing organs from iPSCs could eliminate the supply shortage and provide organs to

all the patients that require them. Presently, iPSC-derived organs are in their preliminary

stages of development with researchers culturing organs in the lab for proof-of-concept

(Badylak et al., 2012). These cells are cultured in the lab for extended periods of time

leading to risks of contamination or proliferation-induced mutations due to the large

number of cells needed to populate a human organ. The next barriers will be cost and

safety to bring iPSC-derived organs to clinic.

While organ regeneration with iPSCs hold promise in the future for regenerative

therapies, targeting endogenous stem cells within a tissue has the potential to be realized

in the clinic sooner and with fewer mutagenic risks. The ability of endogenous stem cells

to proliferate and differentiate provides great potential for the development of novel

NSC-based therapies, without the risk of delivering foreign cells (Daley and Scadden,

 

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2008). Drugs that are previously used in the clinic can be repurposed for other treatment,

and hold the most promise for quick translation to the clinic. For example cyclosprin A is

an immune suppression drug already used in the clinic, and found to increase NSC

survival and increase colony size in vitro and number of proliferating cells in vivo (Hunt

et al., 2010; Erlandsson et al., 2011). Metformin is another drug used extensively in the

clinic and was also found to promotes neurogenesis to increase neuron production (Wang

et al., 2012). These two drugs are early examples of pharmacological methods to

manipulate endogenous NSCs. In the fourth chapter of this thesis, I will investigate novel

methods of targeting and pharmacologically activating endogenous NSCs to increase

neurogenesis in the mouse brain.

Early Embryonic Development

Embryonic development initiates with a spermatocyte fertilizing an oocyte

resulting in a zygote (Mulnard, 1967). The zygote divides symmetrically to generate an

embryo composed of 8 cells with equal potential, which are all totipotent and have the

ability to generate an entire embryo and placenta (Rossant, 1975; Rossant, 1976; Kelly,

1977). The cells at the 8-cell stage compact before dividing to produce a 16-cell morula.

The 16-cell and 32-cell morulae contain internal cells surrounded by a larger population

of external cells, which continue to separate and will form distinct cell lineages. The

pluripotent ESCs are derived from the ICM and each of these cells if removed, could

generate an entire embryo itself but do not contribute to the placenta or yolk sac (Rossant,

1975; Rossant and Papaioannou, 1984; Nagy et al., 1990; Nagy et al., 1993). ESCs can be

expanded indefinitely in culture in the presence of leukemia inhibitory factor (LIF). Even

 

16

 

in culture, ESCs continue to express the pluripotency gene Oct4 and have a high

proliferation rate.

The embryo at the 64-cell stage (E3.5) is comprised of ESCs surrounded by an

epithelial cell layer of trophectoderm cells, which will populate the placenta (reviewed in

(Rossant, 2001). Oct4 and Sox2 are expressed in the ICM but repressed in trophectoderm

cells that express Cdx2 and Eomes (Beck et al., 1995; Hancock et al., 1999). The

developing embryo undergoes cavitation, whereby the trophoblast cells secrete fluid into

the morula to create a large internal cavity called a blastocoel, and the embryo is then

referred to as a blastocyst (Mulnard and Huygens, 1978; Fernández and Izquierdo, 1980).

During the late blastocyst stage at approximately E4.5 the ICM segregates into two cell

types, which are the epiblast and a layer of cells surrounding it called the hypoblast or

primitive endoderm. The primitive endoderm layer delaminates from the ICM and lines

the blastocoel cavity, generating the extraembryonic endoderm, which will give rise to

the yolk sac (Beck et al., 1995). The extrembryonc endoderm cells can be isolated and

grown in culture as extra-embryonic endoderm (XEN) (Kunath et al., 2005). The epiblast

will separate into an embryonic epiblast and a layer of cells that will form the amnionic

cavity, which becomes fluid-filled to protect the embryo. The epiblast, trophoblast and

XEN cells comprise the three cell types that comprise the late blastocyst of embryonic

development.

During implantation (E4.5-5.5) the trophoblast cells that surround the blastocyst

invade into the uterine epithelium. The ICM secretes factors, including FGF, that support

the trophoblast cells so that they proliferate and adhere to the uterine wall (Tanaka et al.,

1998). The chorion forms the fetal portion of the placenta and the maternal portion arises

 

17

 

from uterine epithelium and is called the decidua. The primitive streak is a transient

structure that forms from the epiblast to give the embryo polarization with the distinction

of the anterior-posterior axis (reviewed in (Rossant and Tam, 2009)). Gastrulation begins

around E6.5 and the epiblast differentiates into the three germ layers including endoderm,

mesoderm and ectoderm (Snow, 1981; Beddington, 1982). Gastrulation initiates with the

epiblast cells undergoing a transition to mesenchymal cells, which then ingress between

the epiblast and the endoderm to become incorporated into the mesoderm or definitive

endoderm (Luckett, 1978). The post-implantation mouse embryo has an egg cylinder

motif compared to other mammalian embryos. At the end of gastrulation, the three germ

layers are present but the ectoderm is within the mesoderm and endoderm. Therefore, the

embryo must turn and bring the ectoderm to the outside of the embryo and the endoderm

inside to anatomically orient the embryo. This leaves the embryo oriented properly for

development with all three germ layer present.

Neural Induction in the Embryo

Neural induction occurs at approximately E7.5 when the ectoderm becomes

specified into neural and epidermal cells. The neural plate is the first primordial brain

structure to arise and it goes on to form a polarized, pseudostraified epithelium of

ectoderm. Initial theories suggested that neural cells were induced by a positive induction

signal, however it became clear in subsequent studies that neural specification of

ectoderm occurs through a default mechanism in the absence of extrinsic signals.

Ectodermal cells cultured at low density led to many cells expressing neural genes and

forming neural cell structure (Godsave and Slack, 1989; Sato and Sargent, 1989).

 

18

 

However, in vivo this neural default is regulated by inhibitor molecules including bone

morphogenic protein (BMP) (Smith and Harland, 1992; Lamb et al., 1993; Sasai et al.,

1994). Inhibition of BMP enables ectodermal cells to become neural (Hemmati-Brivanlou

and Melton, 1994; Hemmati-Brivanlou and Melton, 1997). Other signaling factors

including FGF and Wnt signaling are involved, but appear to function via downstream

BMP inhibition (Linker and Stern, 2004). Transforming growth factor (TGFß) signaling

is high in non-neural primordial regions of the developing embryo (de Sousa Lopes et al.,

2003; Yang et al., 2006). Loss of TGFß signaling causes neural tissue development,

suggesting that TGFß is involved in inhibiting the neural default (Camus et al., 2006).

Together, this suggests a common mechanism with the differentiation of pNSCs from

ESCs in vitro (Tropepe et al., 2001; Smukler et al., 2006).

Neurulation generates the neural tube from the neural plate starting at E8 in

mouse development. Before beginning to fold, the neural plate changes shape to become

more elongated with a broader rostral region and narrower caudal region. Neural tube

closure begins at the six to seven somite stage in mice (~E8.5) with the neural folds

closing at the hindbrain/cervical boundary then extending rostrally and caudally, finishing

by E10 (reviewed in (Copp et al., 2003)). Rostrally the neural tube folds from the median

hinge point whereas caudally it folds from the dorsolateral hinge point, mediated by

Sonic hedgehog signaling (Ybot-Gonzalez et al., 2002). After closing, the neural folds

fuse together via cell protrusions that interact to form adhesions to create the neural tube.

Apoptosis disrupts the connection between the surface ectoderm and the neural ectoderm

leaving the neural tube independent, at which point it becomes covered by a layer of

neuroectoderm. The neural tube is the embryonic precursor of the central nervous system.

 

19

 

Radial Glia in the Embryo

Radial glia (RG) arise from neuroepithelial cells before neurogenesis begins and

are responsible for building the layers in the cerebral cortex. The relationship between

NSCs and RG remains unclear, and interesting to investigate, although not addressed in

this thesis (discussed in future directions). RG are bipolar with the apical foot of its long

cellular process contacting the ventricle and a basal foot reaching out to the pial surface.

RG express many astrocyte markers including RC1, RC2, vimentin, nestin, and GLAST

(reviewed in (Bonfanti and Peretto, 2007). These immature glial cells guide neuronal

migration and act as scaffolding to escort newborn neural progeny from the ventricular

zone out to the cortical layers. Cortical layers II-VI are generated in an inside-out fashion,

with the inner cerebral layers populated first and then the cells populating the outer layers

migrate out through the previous layers to the tissue exterior to build the brain (reviewed

in (Kriegstein et al., 2006)).

RG undergo interkinetic nuclear migration (INM), whereby the nucleus travels a

long distance along the cellular process back to the ventricle to undergo division and then

migrates cortically between cell divisions (Misson et al., 1988). Newborn neurons

migrate along columns of clonally related RG out towards the cerebral cortex (Noctor et

al., 2001). RG divide both symmetrically and asymmetrically depending on the stage of

division. Early in embryonic development, they divide symmetrically to expand their

population and both daughters maintain contact with the ventricle to act as embryonic

NSCs and give rise to neural progeny. Later, RG divide asymmetrically with only one

cell maintaining the RG phenotype and remaining in contact with the ventricle while the

 

20

 

other is an intermediate progenitor that migrates away along the process (Kriegstein et al.,

2006). These progenitors populate the cortex and generate all the cortical layers in a

mainly inside-out fashion.

As development progresses and RG are no longer required, they become detached

from the ventricular surface and move towards the cortex via somal translocation. Once

removed from the ventricle, RG differentiate into astrocytes (Chanas-Sacre et al., 2000;

Noctor et al., 2008). It has been suggested that adult dNSCs arise from a subpopulation of

RG that persist into the adult brain (Merkle et al., 2004). Therefore, the exact fate of

embryonic RG and their relation to the NSC lineage remains unresolved.

Emergence of Primitive and Definitive NSC Populations

Embryonic pNSCs can be isolated as early as E5.5 during the egg cylinder stage

of embryonic development (Hitoshi et al., 2004). Embryonic-derived pNSCs express the

pluripotency gene Oct4 (Tropepe et al., 2001). In addition, they can integrate into the

ICM of a developing embryo when cultured with a morula in an aggregation experiment

and pNSCs can contribute to non-neural tissue development (Tropepe et al., 2001;

Hitoshi et al., 2004; Karpowicz et al., 2007). These observations that pNSCs seem to be

less restricted to the neural fate as compared to dNSCs led to their naming as primitive

NSCs due to their incomplete neural commitment. The term primitive has previously

been used to describe a stem cell that is predominantly tissue-restricted, but has limited

amounts of pluripotency (Morrison et al., 1997). Together, pNSCs exhibit indicators of

pluripotency not exhibited by definitive NSCs.

 

21

 

pNSCs are a small cell population that were previously thought to peak early in

development and decline in abundance between E7.5-8.5 (Hitoshi et al., 2004). It was

incorrectly concluded that the decline in abundance meant that pNSCs are only present

embryonically (Hitoshi et al., 2004). However, as I will go on to report in this thesis,

pNSCs persist in the adult brain as rare, Oct4-expressing cells that are upstream and able

to repopulate dNSCs.

dNSCs appear between E7.5-8.5 in the neuroectoderm and unlike pNSCs, do not

express the pluripotency gene Oct4 (Hitoshi et al., 2004). Notch signaling is required for

the differentiation of pNSCs to dNSCs and to maintain dNSCs (Hitoshi et al., 2004).

Oct4 suppression is essential to generate dNSCs, likely to enable pNSCs to differentiate

into or divide to give rise to dNSCs (Akamatsu et al., 2009). Germ cell nuclear factor

(GCNF) is an Oct4 transcriptional repressor that is expressed in the embryo by E6.5,

continues to be expressed in the neuroepithelium at E8.5, and is restricted to the germ cell

lineage by E9.5 (Chung and Cooney, 2003; Lan et al., 2003). GCNF expression

suppressed Oct4 and mice that are GCNF-/- continue to express Oct4 in the

neuroepithelium at E8.5, when it is suppressed in wildtype mice (Akamatsu et al., 2009).

GCNF-/- mice do not experience a depletion of pNSC-derived neurospheres between

E5.5-8.5, and instead give rise to an increased number of pNSC-derived neurospheres at

E8.5. In addition, GCNF-/- brains give rise to fewer dNSC-derived neurospheres than

wildtype controls at E8.5 (Akamatsu et al., 2009). pNSC-derived neurospheres from

GCNF-/- mice showed increased self-renewal with maintained Oct4 expression and gave

rise to more secondary neurospheres when passaged back into leukemia inhibitory factor

(LIF) conditions, but a reduced number of secondary neurospheres when passaged into

 

22

 

FGF to generate dNSCs (Akamatsu et al., 2009). Together, these GCNF experiments

elucidate that Oct4 repression is a key component for pNSCs to generate dNSCs and

supports that Oct4 expression is unique to pNSCs. I will investigate Oct4 expression in

adult pNSCs in chapter 2, and after identifying that adult pNSCs continue to express

Oct4, I will investigate whether they depend on its expression in chapter 3.

The dNSCs that arise first at E7.5-8.5 are basic fibroblast growth factor (FGF)-

dependent (Fig. 1A) (Tropepe et al., 1999; Karpowicz et al., 2007). Later in development,

another population of dNSCs arises at E8.5 that are epidermal growth factor (EGF)-

dependent (Karpowicz et al., 2007). The FGF-dependent dNSCs give rise to the EGF-

dependent NSCs at E12.5, and these are distinct cell populations in the developing

embryo (Fig. 1A). The addition of both EGF and FGF (and heparin) to culture media

(termed EFH) gives rise to an additive number of dNSCs as compared to EGF or FGF

alone from the E14.5 embryo (Tropepe et al., 1999). However, it appears that dNSCs

become sensitive to both mitogens as development progresses and adult dNSCs are

cultured in EFH conditions. The adult neural stem cell lineage and the discovery of

pNSCs in the adult neural lineage will be the focus of this thesis and introduced later (Fig

1B,C).

Perinatal Neurogenesis

Neurogenesis in the embryo proceeds through waves of generating predominantly

a single cell type. Neurons are the first differentiated cell type born in the brain and arise

early in embryonic development at around E10 (reviewed in (Finlay and Darlington,

1995). Astrocytes begin to appear immediately after birth, between postnatal day (P)0-7.

 

23

 

Oligodendrogenesis occurs last between P7-14. The birth of one cell type at a time might

suggest that independent progenitors are responsible for production of each cell type or

that the niche acts on and influences the fate of the neural stem or progenitor cells (will

be referred to as neural precursor cells, NPCs). Interestingly, dNSCs isolated in the

neurosphere assay peak perinatally (Hitoshi et al., 2004), which might reflect their heavy

participation in perinatal neurogenesis. In chapter 4, I investigate the perinatal abundance

of pNSCs and whether pup-derived pNSCs differ from adult-derived pNSCs. In the

discussion I will address possible implications for their involvement in populating the

brain.

Adult Neural Stem Cell Discovery

The adult mammalian brain was thought to be post-mitotic without any ability to

generate new neurons after development. This belief was held long after hematopoietic

stem cells (HSCs) were identified and accepted by the scientific community (Till and

McCulloch, 1961; Spangrude et al., 1988; Capel et al., 1990; Smith et al., 1991). The

dogma was that since the adult brain was thought to be incapable of incorporating

newborn neurons into existing circuits, it would not require stem cells. In addition, since

brain development proceeds chronologically via waves of neurogenesis, astrogenesis,

then oligodendrogenesis, it was postulated that a distinct progenitor was responsible for

the generation of each cell type and that those progenitors did not persist after their cell

type was populated (Gage et al., 1995).

This dogma that the brain is fixed and all of its cells are post-mitotic persisted for

many years. Proliferating cells in the adult brain were first reported in 1961, coincident

 

24

 

Figure. 1

 

25

 

Figure. 1. Adult pNSCs give rise to dNSCs in the adult mouse brain, similar to the

lineage in embryonic development. A. Emergence of the NSC lineage in the developing

embryo. LIF-dependent pNSCs fist appear at E5.5, FGF-dependent dNSCs arise at E8.5

and definitive EGF-dependent dNSCs appear last in development at E12.5 (Tropepe et

al., 1999; Karpowicz et al., 2007). B. The currently accepted adult NSC lineage. Adult

dNSCs proliferate slowly and give rise to TA cells, which divide to generate neuroblasts.

Neuroblasts migrate away from the NSC niche along the RMS and differentiate into

functional neurons in the olfactory bulb. C. Adult NSC lineage proposed in this thesis.

pNSCs persist into the adult mouse brain and give rise to dNSCs through the life of the

animal.

 

26

 

with the identification of colony forming units in the spleen. This first report of dividing

cells in the adult brain was by Smart who observed that thymidine-H3 was incorporated

into the subependyma of the adult mouse brain (Smart, 1961). Further, Smart

demonstrated that neuroglia in the subependyma incorporated thymidine-H3 and

underwent division (Smart, 1961). Soon after, Joseph Altman injected thymidine-H3 into

the rats with a needle-induced lesion and reported newborn glia and neurons in the brain

after injury (1962). In addition, thymidine-H3 labeled cells were observed in regions

distant from the injury, suggesting non-injury related proliferation in the adult brain

(Altman, 1962). Subsequently, electron microscopy visualized postnatally generated

neurons in the olfactory bulb and hippocampus (Kaplan and Hinds, 1977). Despite these

early reports, the concept of neurogenesis in the adult brain was at first dismissed and not

accepted by the scientific community.

While we now know early reports of cellular division in the adult brain are indeed

true, it was the behavioural data from songbirds that succeeded in shifting hypotheses on

adult neurogenesis. The ability of adult songbirds to learn new songs from year to year to

attract new mates suggested the possibility that new neural circuits were being

established. Furthermore, newborn neurons were observed in the higher vocal center, a

brain region associated with song learning (Goldman and Nottebohm, 1983; Alvarez-

Buylla et al., 1988). These studies used thymidine-H3 and retrograde fluorogold uptake to

determine the birthday of neurons in the vocal center, where it was surprisingly observed

that forebrain neurons were produced postnatally (Alvarez-Buylla et al., 1988).

Interestingly, neurons continued to be born after 8 months of age in songbirds, despite

that the vocal center overall volume stopped increasing (Alvarez-Buylla et al., 1988).

 

27

 

These new observations suggesting neural turnover and integration of newborn neurons

into the vocal center indicated that brain circuitry may be more plastic than previously

thought. While these pivotal studies led the field towards adult neurogenesis, more recent

studies have cast doubt on the link between newborn neurons in adulthood and new song

learning (Walton et al., 2012). Nonetheless, neurogenesis persists throughout the lifetime

of birds that do or do not learn new songs, and newborn neurons in the high vocal centre

into adulthood and continue to provide evidence adult neurogenesis.

Neural Stem Cell Isolation

The ability to grow and maintain NSCs in culture was a crucial step towards

characterizing NSCs in the brain. NSCs were first isolated and cultured by Reynolds and

Weiss (1992). They isolated tissue from the striatal region, including the walls of the

lateral ventricle, in adult mice and observed that rare cells generated free-floating

colonies when grown in culture with EGF and FGF. These colonies were comprised of

relatively undifferentiated cells that stained for the filamentous protein nestin (Reynolds

and Weiss, 1992). Another group also isolated neural precursors from the adult mouse

brain that proliferated in culture (Richards et al., 1992). These reports confirmed that

NSCs could be isolated from the adult mammalian brain and propagated in culture.

This marked the emergence of the neurosphere assay, whereby one NSC proliferates in

culture to generate a clonal neurosphere that is derived from a single cell.

Culturing NSCs enabled the discovery of NSC characteristics and testing of their

intrinsic potential outside of their niche. The proliferation of NSCs as described by

Reynolds and Weiss is dependent on mitogens, and yields colonies later termed

 

28

 

neurospheres that were determined to originate from a single cell (Reynolds and Weiss,

1992). Mixed cultures of GFP-labeled and RFP-labeled cells indicated that primary cells

must be plated at 10 cells/µl and left untouched during their culture period to generate

clonal, non-mixed, neurospheres (Coles-Takabe et al., 2008). The ability of a single stem

cell to give rise to a clonal colony demonstrates the proliferative capacity of stem cells.

Then, each individual colony can be passaged by dissociation to give rise to additional

neurospheres to demonstrate the critical stem cell criterion of self-renewal (Reynolds and

Weiss, 1996). A clonally-derived neurosphere can be plated in adherent culture

conditions in the presence of fetal bovine serum (FBS) and differentiated to determine

whether the cell of origin was multipotential, the final criterion to identify a stem cell.

The neurospheres isolated from the striatal region of the brain by Reynolds and Weiss

were able to generate neurons, astrocytes and oligodendrocytes (Reynolds and Weiss,

1996). These early studies to identify NSCs and develop a method of culturing them have

paved the way for the NSC field.

pNSCs and dNSCs are very different stem cell populations based on many

criteria, as will be discussed in this thesis, including their cell culture conditions and

neurosphere phenotype. All neurospheres are cultured in serum free media, but pNSCs

are grown in the presence of LIF whereas dNSCs are grown in EFH conditions. dNSCs

proliferate in the neurosphere assay to EFH neurospheres. In addition to cell culture

conditions, pNSC-derived and dNSC-derived neurospheres differ in appearance. pNSC-

derived neurospheres are very adherent and are between 50-100 µm in diameter whereas

dNSCs are less compact and larger at 100-200 µm in diameter. Both pNSCs and dNSCs

self-renew and are multipotential, but differ in phenotype and the proportion of neurons,

 

29

 

astrocytes and oligodendrocytes they generate when differentiated. In this thesis I will

investigate the differences between pNSC-derived neurospheres and dNSC-derived

neurospheres.

Adult Neurogenesis in the Periventricular Region

dNSCs are located in the subependyma and comprise just 0.2-0.4% of the

subependymal population (Morshead et al., 1994; Morshead et al., 1998) (Fig. 2A). These

dNSCs are responsible for and capable of repopulating the neural lineage after ablations

of downstream dividing neural cells (Morshead, 1994; Doetsch et al., 1999). dNSCs are

label-retaining and if labeled with BrdU or thymidine-H3 were identified a month later in

the periventricular region having retained their label (Doetsch et al., 1999). A defining

feature of dNSCs, although not unique to dNSCs, is GFAP expression (Doetsch et al.,

1999; Morshead et al., 2003). GFAP expression in dNSCs, while pNSCs are GFAP–, led

to the discovery of pNSCs in chapter 2 and is essential to the basis of this thesis.

Mice with Herpes Simplex Virus thymidine kinase expression driven by the

GFAP promoter (GFAP-tk) were used to selectively eliminate dividing GFAP-expressing

cells when ganciclovir (GCV) is administered (Bush et al., 1998). This mouse strain

utilizes a thymidine kinase of the herpes simplex virus transgene to phosphorylate GCV

and retain it in GFAP+ cells. When GCV is retained it is metabolized to toxic nucleotide

analogs, which interrupt nucleic acid synthesis and induce cell death in any infected cell

that undergoes mitosis. When GCV was infused into the brains of GFAP-tk mice or

added to neurosphere cultures derived from GFAP-tk mice, dNSC-derived neurospheres

 

30

 

were lost (Morshead et al., 2003). This kill paradigm is proliferation-specific and

eliminates all dividing GFAP-expressing cells, therefore all dNSCs.

Downstream of dNSCs are constitutively proliferating cells that comprise a larger

population and are approximately 10% of the subependymal cells (Morshead and van der

Kooy, 1992). These constitutively proliferating cells, also known as transit amplifying

(TA) cells, were shown to arise from dNSCs based on replication-incompetent

retroviruses (Doetsch et al., 1999). These cells are responsible for expanding the pool of

neural progenitors to ensure that newborn neurons are available when needed. TA cells

have a cell cycle time of approximately 12.7 hours (Morshead and van der Kooy, 1992).

However, while these cell divisions are symmetric expansions to increase the number of

progenitors, many of the newborn cells will undergo cell death. Approximately 60% of

newborn constitutively proliferating cells undergo apoptosis, 25% migrate along the

rostral migratory stream to the olfactory bulb, and just 15% remain in the supendyma

(measured after 6 days). The cells that remain in the periventricular region persist for 15

days (Morshead et al., 1998).

TA cells differentiate to generate neuroblasts, which are the cells that leave the

periventricular region to migrate away from the niche (Doetsch and Alvarez-Buylla,

1996). To identify the downstream progenitors that migrate, dNSCs were activated to

proliferate with AraC infusion and then infected with retrovirus. While after short

survival times only GFAP-expressing NSCs were labeled, after 5-6 days chains of labeled

migrating neuroblasts were present (Doetsch et al., 1999). Neuroblasts migrate along

chains of polysialylated NCAM (PSA-NCAM) to reach the olfactory bulb.

Transplantation and thymidine-H3 experiments indicated that cells in the periventricular

 

31

 

region migrate anteriorly along the RMS to the olfactory bulb where they differentiate

into GABAergic granule periglomerular cells (Lois and Alvarez-Buylla, 1994). Newborn

neurons arrive in the olfactory bulb within a week and then take up to a week to

differentiate into mature neurons. Integration of newborn neurons into the neural circuit

indicates the turnover that persists in the mature mammalian brain. The NSC niche is

summarized in Figure 2.

Architecture of the NSC Niche

As development progresses, NSCs move away from the ventricle into the

subependymal zone (SEZ). NSCs become separated from the ventricle by a layer of

ependymal cells that are ciliated and in contact with the cerebrospinal fluid (CSF).

Ependymal cells arise from NSCs between E14-16 but do not mature and extend their

cilia until after birth (Spassky et al., 2005). The coordinated beating of ependymal cilia

creates the current of CSF and directs its flow through the ventricles (Worthington and

Cathcart, 1963; Cathcart and Worthington, 1964).

Some groups have reported that ependymal cells divide and function as the NSCs

of the adult brain since they are located in the position of the embryonic RG. An

experiment whereby DiI was injected into the lateral ventricle labeled neurosphere-

initiating cells (Johansson et al., 1999). It was incorrectly concluded that neurospheres

must be ependymal-derived since DiI was injected into the CSF and only ependymal cells

would have access to the DiI to incorporate it. However, it became clear that

subependymal cells likely took up the DiI label through a basal process (Mirzadeh et al.,

2008), thus the labeling was non-specific. dNSCs have a cellular process that extends

 

32

 

Figure 2.

 

33

 

Figure 2. The NSC niche. A. Schematic of a coronal section of the mouse brain. NSCs

reside within the subependyma lining the lateral ventricles (shown in purple). Adapted

from Morshead et al., (1992). B. NSCs reside within the SEZ of the lateral ventricles.

dNSCs are separated from the ventricle by an ependymal layer (E), but maintain a basal

process that extends into the CSF to receive signals. dNSCs give rise to transit amplifying

(TA) cells that go on to generate neuroblasts (N), which migrate away from the niche.

Many astrocyte-like cells populate the niche (A).

 

34

 

from the cell body in the subependyma out to the lateral ventricle (Mirzadeh et al.,

2008), to respond to signaling molecules in the CSF and thus took up the thymidine-H3

label. Despite the initially conflicting report of NSCs being ependymal, ependymal cells

were confirmed to not have NSC potential as isolated ependymal tissue could not form

self-renewing colonies (Chiasson et al., 1999; Laywell et al., 2000; Spassky et al., 2005).

Ependymal spheres grew in the absence of mitogens and did not show stem cell

characteristics as they cannot be passaged and did not give rise to neurons (Chiasson et

al., 1999). However, the debate continued and other groups continued to report that

ependymal cells were NSCs despite the inability to meet stem cell criteria (Coskun et al.,

2008). This ependymal debate reinforces the importance of strict stem cell criteria as a

prerequisite when studying the NSC lineage. As I will discuss in the second chapter,

pNSCs meet the rigorous stem cell tests and thus are true stem cells.

dNSCs are most likely to reside in the anterior ventral and posterior dorsal regions

of the lateral wall of the ventricle (Mirzadeh et al., 2008). Cells isolated from these

regions gave rise to the largest neural clones, suggesting that these two regions include

dNSCs. The dorsolateral corner of the lateral ventricle is the site of the highest level of

proliferation and is home to TA cells (Morshead and van der Kooy, 1992). This corner

had the highest amount of BrdU incorporation with up to 33% of dorsolateral cells taking

up the proliferation label during 14 h of injections. The RMS pinches off from the

dorsolateral corner and generates chains of newborn neuroblasts that migrate away from

the niche to the olfactory bulb (Doetsch et al., 1997).

The surface of the lateral wall of the lateral ventricle is a series of ependymal cells

organized into pinwheels with the apical process of dNSCs extending through the center

 

35

 

of the pinwheel to the ventricular space (Mirzadeh et al., 2008). The process is in direct

contact with the CSF. This provides dNSCs with direct contact to signaling molecules

circulating in the CSF and enables them to respond to their environment. These apical

endfeet express vascular cell adhesion molecule (VCAM) (Kokovay et al., 2012). Loss of

VCAM expression leads to disruption of the niche, with loss of pinwheel structure, and

dNSCs proliferate but deplete their population (Kokovay et al., 2012). In addition, dNSCs

have a longer basal process that extends in the opposite direction of the ventricle and

contacts a blood vessel (Mirzadeh et al., 2008). This positions dNSCs to receive

environmental cues from both the CSF and the circulating blood, and is essential to their

ability to respond to their environment to maintain the neurogenic niche. This pinwheel

architecture was not observed, nor were the apical process of dNSCs reported, on the

medial wall (Mirzadeh et al., 2008), despite the fact that dNSC-derived neurospheres are

commonly isolated from this location. It remains unclear whether the medial wall derived

dNSCs have any phenotypic differences to lateral wall dNSCs.

Neurogenesis in the Hippocampus

Neurogenesis in the adult mouse brain is also reported in the dentate gyrus of the

hippocampus. During embryonic development, the cornu ammon and the dentate gyrus of

the hippocampus are populated by neurons that migrate from the neurogenic zone lining

the lateral ventricles. The dentate gyrus forms around E14 from the neuroepithelium as

cells migrate along RG toward the pial surface and form the ‘V’-shaped structure

(Altman and Bayer, 1990; Pleasure et al., 2000). As the hippocampus develops it seals off

the ventricle dorsally but remains in close contact with the lateral ventricle. Within the

 

36

 

dentate gyrus, cells proliferate to give rise to newborn granule neurons that integrate into

the granule layer of the hippocampus.

Hippocampal neurogenesis has been described in many mammals including the

mouse, rat, non-human primates, and humans. However, the presence of a true

hippocampal NSC in the adult brain remains under debate. Hippocampal NSCs do not

self-renew and hence cannot be classified a stem cell (Clarke and van der Kooy, 2011).

Hippocampal cultures are often contaminated with periventricular NSCs due to dissection

techniques that do not sufficiently exclude the nearby lateral ventricle tissue (Walker et

al., 2008; Bonaguidi et al., 2008). The posterior lateral ventricle touches the exterior of

the hippocampus and thus is difficult to remove from dissected dentate gyrus tissue.

Clarke et al., demonstrated that hippocampal NSCs only self-renew when cultured on an

embryonic cortical tissue explant (2011). This suggests that hippocampal neurogenesis is

supported by progenitors that maintain the ability to self-renew only perinatally, and lose

this ability in the adult (Seaberg and van der Kooy, 2002; Chechneva et al., 2005; Clarke

and van der Kooy, 2011). On the other hand, other groups have reported that

hippocampal NSCs do persist in the adult mouse brain (Parent et al., 1997; Eriksson et

al., 1998; Kukekov et al., 1999; Palmer et al., 1997; Mignone et al., 2004; Suh et al.,

2007; Gould, 2007; Lugert et al., 2010; Song et al., 2012). Due to the debate surrounding

NSCs in the hippocampus, this thesis will refer to hippocampal NPCs.

Sox2 is commonly used as a NPC marker and labeled a population of adult

hippocampal cells that give rise to both neurons and astrocytes in vivo (Suh et al., 2007).

In addition, these hippocampal precursors are activated in response to mitotic signals to

generate more downstream progenitors (Suh et al., 2007). Other groups have also

 

37

 

reported a hippocampal precursor that is Sox2+ and responds to exercise and epileptic

activity via Notch activity (Lugert et al., 2010). Quiescent hippocampal NPCs have been

reported that are nestin+ and RG-like (Song et al., 2012), and were reported to comprise

1% of the adult granule neuron population (Lagace et al., 2007). However, hippocampal

NPCs were observed to decrease with age (Encinas et al., 2011), which contrasts with the

stem cell criterion of self-renewal. Aside from the debate over self-renewal, hippocampal

precursors have many similarities to periventricular NSCs in the proteins they express

including Sox2, GFAP, nestin, and vimentin (Kempermann et al., 2004; Kriegstein and

Alvarez-Buylla, 2009) reviewed in (Suh et al., 2009). Subependymal dNSC and pNSC

markers will be discussed further in the fourth chapter of this thesis.

Increasing Cortex Size with the Outersubventricular Zone

A large question in the scientific field is how the large mammalian brain forms.

The relative brain size compared to body size is 15-fold higher in humans compared to

mice, and this increase is predominantly accounted for by an expanded cerebral cortex

(Finlay and Darlington, 1995). The size of the neocortex in humans is approximately

1000-fold larger than the mouse brain, with approximately 16 billion neurons (reviewed

in (Florio and Huttner, 2014)). The predominant architectural difference in animals with a

large neocortex is the presence of an outersubventricular zone, composed of outer RG

cells (Smart, 1961; Zecevic et al., 2005). The large numbers of cells needed to populate

the cerebral cortex are generated via outer RG cells in the outersubventricular zone, a

highly mitotic region apical of the subventricular zone that is only present in mammals,

predominantly primates, with large cortices. The outersubventricular zone is much

 

38

 

greater in size than the ventricular zone, and at gestational week 15 in the human

embryonic development accounts for 75% of neurogenic proliferation (Zecevic et al.,

2005). The cortex forms in an inside-out fashion with newborn cells migrating through

existing cell layers to populate the outer regions of the cortex (reviewed in (Kriegstein et

al., 2006). In this model, the inner layer (layer VI) is populated first and the outer cells

(layer II) of the cortex form last. In these higher order mammals, outer RG are

responsible for generating the progenitors that populate the neocortexes.

Interestingly, the outersubventricular zone is present in animals with and without

gyrification. It is hypothesized that gyrification emerged early in evolution and is actually

present in all major mammalian families and not solely in primates. Gyrification evolved

early in mammal evolution and lissencephaly emerged secondarily (Kelava et al., 2012;

Reillo and Borrell, 2012). The Radial Unit hypothesis proposed that cortical size is

dependent on the number of cortical radial units, thus the number of RG present.

However, while the gyrated brain has a higher abundance of RG, they are not the sole

mechanism responsible for gyrification. The abundance of outer RG does not predict the

amount of gyrification, thus sufficient outer RG may be a prerequisite but alone not

sufficient (Chenn and Walsh, 2002; Kelava et al., 2012). In addition to RG, other cells

that populate the cortex arise from ventral regions including the medial ganglionic

eminence and lateral ganglionic eminence. Another hypothesis proposed that bigger

brains in primates results from increased progenitor activity, but again progenitor

overproduction is not sufficient (Nonaka-Kinoshita et al., 2013). Possible mechanisms for

this increased proliferation include pseudostratification of the progenitor layer to increase

the population, and increase in symmetric expansion divisions (Fish et al., 2008).

 

39

 

Therefore, while the abundance of outer RG and their intermediate progenitors remain

essential to the development of large cortexes, the interplay of other components are also

involved. The expansion of the cerebral cortex is likely dependent on multiple features of

outer RG within the outersubventricular zone.

Similar periventricular architecture is observed in the primate brain as the rodent

brain. The primate brain is organized into a three-layer niche around the lateral ventricles

with an ependymal, hypocellular and astrocytic layer. A similar RMS structure exists,

composed of migrating neuroblasts. BrdU incorporation labeled neuroblasts that leave the

periventricular region and migrate to the olfactory bulb (Kornack and Rakic, 2001). Outer

RG cells are PAX6+, Sox2+ and highly proliferative, similar to RG in mouse development

(Hansen et al., 2010). The cells in the outersubventricular zone show high proliferation

and cell cycle reentry to mediate cortex growth (Lukaszewicz et al., 2005).

Different than subventricular RG, outer RG undergo mitotic somal translocation

(MST) rather than interkinetic nuclear migration. In MST the outer RG cell soma travels

along the basal fiber towards the cortex before undergoing cytokinesis (Hansen et al.,

2010). Despite maintaining a long cellular apical process, the outer RG apical process

lacks direct contact with the ventricle (Fietz et al., 2010; Hansen et al., 2010). Outer RG

cells divide with a horizontal cleavage plane with the basal daughter retaining the basal

fiber and the outer RG phenotype and the apical daughter becoming radially bipolar. The

outer RG moves towards the cortical plate with each division, continuously expanding the

outersubventricular zone (Hansen et al., 2010). The apical, non-RG daughter also

continues to proliferate, contributing to the large amount of cell production. Outer RG

give rise to both intermediate progenitors to expand the population as well as directly to

 

40

 

neuroblasts (Gertz et al., 2014). Together, the high amount of proliferation in both outer

RG and their progeny serves to rapidly expand the pool of newborn neuroblasts that

migrate away to populate the large cerebral cortex in mammals.

The evolution of the mammalian neocortex has been attributed to differences in

the size and cell types in the germinal zone, leading to increased cortical surface area.

Outer RG appear to be essential to build the large cerebral cortex, however, it remains

unclear if other cell types are present and involved in neocortical development. Although

not within the scope of this thesis, it remains to be determined if pNSCs are present in the

gyrificated brain and if so whether they exhibit any differences from pNSCs in the mouse

brain. Human ESCs in culture are not LIF-dependent and preliminary attempts to derive

pNSCs from human ESCs in culture suggested neural identity was closer to ESCs in

maintenance conditions (Chaddah et al., 2012). However, ‘naive’ ESCs are LIF-

dependent and may be able to generate human pNSCs (Hanna et al., 2010). This thesis

will focus on pNSCs in the perinatal and adult mouse brain, leaving aside the embryonic

brain and human brain for future studies.

Human Neurogenesis

Human neurogenesis is very difficult to study due to the obvious difficulties in

reaching a cell population in the middle of the brain of a living human. Experiments are

limited to using postmortem tissue or tissue isolated from brain regions during surgical

procedures, which usually implies that the region is tumorigenic (reviewed in

(Kempermann, 2013)). Other studies have investigated neurogenesis with proliferation

assays to take advantage of drugs delivered for other medical purposes, for example

 

41

 

during cancer treatment, or radioactive labeling induced by a radioactive exposure, for

example after an atomic bomb or nuclear plant meltdown (Kreisel, 1995; Souchkevitch,

1996; Bhardwaj et al., 2006; Kempermann, 2013). The radioactive labeling is

incorporated into proliferating cells and provides insight into neurogenesis in the human

brain.

The adult human brain is similar to the mouse in that it has an astrocytic ribbon in

the periventricular region separated by a layer of ependymal cells from the lateral

ventricle (Sanai et al., 2004). Again similar to the mouse, neurosphere-forming cells are

enriched in the rostral region of the ventricles. These human NSC-derived neurospheres

self-renewed and were multipotential, generating predominantly astrocytes again similar

to mouse dNSCs. Human HSCs were not isolated from any other brain regions, including

the other ventricles (Sanai et al., 2004).

Despite the many apparent similarities between mouse and human NSCs, the

scientific community continues to debate human migrating neuroblasts in vivo. Groups

have reported the absence of chains of migrating neuroblasts towards the olfactory bulb

in the human (Sinai et al., 2004). While other groups have observed a human RMS with

BrdU incorporating neuroblasts (Curtis et al., 2007). In addition, chains of migrating

neuroblasts have been reported in human infants under 18 months of age and becoming

reduced through childhood and absent in the adult (Sanai et al., 2011). It remains

unresolved whether a mouse-like RMS is present in the human brain. The implications of

this finding remains unclear as there is strong evidence that human NSCs persist in the

adult brain, but their contribution remains to be determined. The scientific debate over the

contribution of adult human NSCs continues, but is not addressed in this thesis.

 

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Stem Cell Quiescence

Self-renewal of a stem cell to maintain itself through the life of an organism is a

key criterion for classification of a stem cell. A stem cell may not proliferate throughout

the lifetime of the animal during normal tissue homeostasis, but it must maintain the

ability to proliferate as needed. Quiescence of stem cells and their ability to become

activated when need is essential for tissue maintenance, function, and regeneration

(reviewed in (Li et al., 2010)). Quiescence is actively maintained through a balance of

cell signals that include epigenetic, transcription and post-transcription modifications

(reviewed in (Cheung and Rando, 2013). To protect the stem cell population, cells may

proliferate less to prevent against stem cell exhaustion or to reduce their exposure to

replication-induced mutations. Stem cells depend on their intermediate progenitors to

proliferate and expand the pool of undifferentiated cells to meet the tissue’s needs. For

example, in the adult brain dNSCs proliferate slowly and their progeny, TA cells,

proliferate quickly (cell cycle times estimated at 15 days vs. 12.7 h) to expand the pool of

cells that can migrate away from the niche (Morshead et al., 1994; Morshead et al.,

1998).

From an evolutionary standpoint, reduced proliferation of stem cells with age may

have come about for multiple reasons. Possibly, cells go quiescent with age to protect

against proliferative exhaustion or they may limit their proliferation to prevent against

replication-dependent mutations (reviewed in (Wang and Dick, 2005; Orford and

Scadden, 2008)). Stem cells have the added risk that if they do undergo a mutation and

become cancerous they are more likely to form tumours, since they are already long-lived

 

43

 

cells that proliferate. Therefore, it would be beneficial to the organism to have reduced

stem cell proliferation with age to reduce the likelihood of a stem cell becoming

tumorigenic and harmful to the animal (Cheung and Rando 2013). While detrimental to a

tissue in need of regeneration in the short-term, controlled stem cell proliferation would

be advantageous to the organism’s lifespan and thus potential to reproduce in the long-

term.

NSC cycle times are difficult to determine on a population basis with the limited

cell surface markers and the techniques available to prospectively identify stem cells. The

best estimates predict that dNSCs have a cell cycle time of 15 days (Morshead et al.,

1998). This is based on the life span of the constitutively proliferating cells if dNSCs

divide at a rate of 1:1 to repopulate them. However, TA cells that work to expand the

pool of progenitors derived from NSCs may enable dNSCs to divide less frequently and

rely on the TA cells to repopulate the lineage (Morshead et al., 1998). TA cells have a

cell cycle time of 12.7 h based on BrdU incorporation (Morshead and van der Kooy,

1992). This large discrepancy between cell cycle times of cells separated by just one

hierarchal level suggests a mechanism exists whereby NSCs maintain low levels of

proliferation that is compensated for by their direct progeny. Further analysis of dNSC

cell cycle time, in addition to pNSC cell cycle time, will be discussed in the third

chapter of this thesis.

Other tissues maintain similar organizations whereby cells at the top of the

hierarchy proliferate slowly and their progeny expand the population to maintain a large

pool of progenitors. For example in the blood system, populations of long-term HSCs

proliferate very slowly and are estimated to divide only once every 5 months (Wilson et

 

44

 

al., 2008; Foudi et al., 2009). Long-term HSCs are quiescent under baseline conditions

and activated to proliferate in response to ablation paradigms that kill their downstream

proliferating cells (Wilson et al., 2008). These quiescent HSCs are the cells responsible

for repopulating the entire blood lineage when transplanted into an irradiated mouse

(Morrison and Weissman, 1994; Cheshier et al., 1999; Trevisan et al., 1996; Benveniste

et al., 2003). Immediately after irradiation, downstream proliferating cells are needed for

the mouse to survive the initial loss of all their blood cells, but only long-term HSCs can

engraft and repopulate the bone marrow long-term (Benveniste et al., 2010). Therefore,

the blood system is a stem cell model whereby an upstream quiescent stem cell divides

rarely and depends on the downstream cells to maintain the population, except in

response to injury or in a transplant model.

Despite the long-term survival advantage for a cell that proliferates less and

decreases its rate of mutation, it may be less advantageous when a tissue requires

newborn cells. For example, skeletal muscle stem cells (satellite cells) are quiescent cells

that are activated in response to muscle damage, but become less active with age and thus

exhibit decreased tissue regeneration (Conboy et al., 2005; Morrison and Spradling,

2008). Despite the age-induced quiescence in these stem cell populations, they maintain

the ability to proliferate given the right environment. Parabiotic pairings in mice, where

the circulatory system of a young mouse is surgically connected to the circulatory system

of an old mouse, exposed the old HSCs in the bone marrow to a young circulating

environment (Conboy et al., 2005). These experiments elucidated that old HSCs could be

activated to proliferate similar to young cells when surrounded by a young environment.

 

45

 

These experiments suggest that quiescence in aged stem cells is not intrinsic, but instead

is niche-dependent and can be overcome by the exposure to a factor from a young niche.

The same phenomenon occurs in old muscle stem cells in vivo and in NSCs in

vitro, whereby old aged-derived NSCs increase their proliferation when cultured in a

young environment (Piccin et al., 2014). If the factors inducing quiescence in the aged

niche or promoting proliferation in the young niche can be identified, they may be

implemented to promote endogenous regeneration in tissues that undergo little

regeneration, such as the brain. Identifying the signals that relieve quiescence would also

elucidate the regulatory network involved in maintaining quiescence. In addition, the

tendency for stem cells to reduce their proliferation and go quiescent in the adult might

suggest that abundances of NSC populations are underestimated, as some of the quiescent

cells will not be activated in culture to proliferate and generate a neurosphere. In this

thesis, I will investigate whether pNSCs are quiescent in the adult mouse brain and

methods of targeting and activating pNSCs.

NSC Activation

NSCs in the adult mouse brain respond to environmental cues and injury to

become activated and maintain the niche of the adult brain. Environmental cues can

activate NSCs to increase neurogenesis, either through the addition of a positive cue or

removal of an inhibitory cue. NSCs are known to be able to quickly respond to tissue

damage or ablation of their downstream progenitors. A non-specific kill paradigm with

thymidine-H3 ablated the rapidly proliferating TA cells, and it only took two days for

dNSCs to be activated to repopulate their downstream progeny (Morshead and van der

 

46

 

Kooy, 1992). In addition, this early study observed that stress (induced with anesthetic or

food deprivation) increased the number of proliferating, BrdU-incorporating, cells in the

dorsolateral corner of the lateral ventricles (Morshead and van der Kooy, 1992). Thereby

indicting that dNSCs and/or TA cells become activated in response to their environment.

NSCs are able to respond to damage in their environment and a balance of signalling

molecules in the niche moderates NSC and progenitor proliferation.

Exercise and an enriched environment are two external stimuli that can activate

neurogenic proliferation. Neurogenesis in response to exercise or environment has been

well documented in the dentate gyrus (van Praag et al., 2005; Lugert et al., 2010;

Kannangara et al., 2010). Exercise has also been reported to increase subependymal

neurogenesis, measured with BrdU incorporation in vivo and neurosphere formation in

vitro, in aged mice via exercise (Blackmore et al., 2009). This increased proliferation was

hypothesized to occur via growth hormone activation, as exercise increased growth

hormone and growth hormone receptor null mice did not experience an increase in

neurogenesis (Blackmore et al., 2009). Increased neurogenesis in the hippocampus has

been implicated to facilitate long-term potential, learning and memory (van Praag et al.,

1999; Imayoshi et al., 2008). Together, these studies indicate that NSC activation leads to

new neurons that integrate into neuronal circuits and functionally alter the adult

neurogenic niche.

NSCs also respond to brain injury including stroke (Arvidsson et al., 2002;

Thored et al., 2006). NSCs from the periventricular region migrate away from their niche

towards the infarct region of the cerebral cortex, where they differentiate into neurons

and glia to repair damaged tissue (Kolb et al., 2007). Despite this migration of NSCs to

 

47

 

damaged tissue, newborn neurons do not integrate into the network of neurons at the site

of injury or appear to meaningfully repair the tissue long-term (Arvidsson et al., 2002).

Apoptosis of newborn neurons is thought to result from the inflammation at the site of

injury, rather than an intrinsic problem of the newborn cells, leading to reduced

regeneration (Ekdahl et al., 2009). Another external stimulus that increases neurogenesis

is immunosuppression (Erlandsson et al., 2011). Cyclosporin A treatment increased NSC-

mediated recovery from stroke with increased tissue repair of the infarct region and

increased BrdU incorporation (Hunt et al., 2010; Erlandsson et al., 2011). NSCs are

activated in response to injury, particularly after a stroke, and give rise to newborn cells

in the adult brain.

Despite the fact that NSCs are activated via many mechanisms, their contribution

to endogenous repair is limited. It remains to be seen whether adjustments to activation,

integration or survival are needed to enable neurogenesis to contribute to endogenous

repair. The fourth chapter of this thesis will explore methods of targeting and

pharmacological methods to activate pNSCs, and potentially increase regeneration after

injury.

Cell Type Specific Markers

Identifying cell-type specific markers and methods to target individual cell

populations are key goals in NSC biology. Without selective cell surface markers it is

difficult to isolate cell populations, which makes it arduous to study those cell

populations. In addition, testing pharmacological interventions is less accurate if the cells

of interest cannot be isolated (Obermair et al., 2010; Miller and Kaplan, 2012). These

 

48

 

issues are magnified in rare stem cell populations, for example in pNSCs, where the

population of interest is underrepresented in the niche and its neighbours can mask any

pNSC-specific effects.

Much work has been devoted to identifying NSC-specific cell markers. NSCs

express PAX6 embryonically and its expression is essential as mutated cell lines and

PAX6-depleted mice showed reduced NSC self-renewal and decreased proliferation

(Gómez-López et al., 2011). GFAP was one of the first proteins identified to be expressed

in dNSCs (Morshead et al., 2003; Doetsch et al., 1999). NSCs also express Sox2, and

many studies incorrectly classify any Sox2+ cell a NSC. However, Sox2 is expressed in

many downstream proliferating progenitors and thus is a non-specific NSC marker. Sox2

is activated downstream of LIF-R signaling via the JAK-STAT pathway, and is also

expressed in pNSCs (as will be discussed in the second chapter of this thesis). Similarly,

nestin is often used as a NSC and progenitor cell marker but is even less specific than

Sox2 and has been reported to mark many proliferating cells in the brain and other

tissues. As well, nestin can become activated in primary culture in response to cell stress

further decreasing its specificity. Therefore, identifying more specific NSC markers

would be of great use to the NSC field.

Many cell markers have been indentified that are expressed in NSCs but are not

exclusive to NSCs. dNSCs are CD133+ and double sorting for GFAP+ CD133+ enriched

cell cultures to 70% dNSCs. After exposure to Hoechst, collection of the side population

for cells that effluxed the dye enriched dNSCs 7.5-fold over the unsorted population

(Kim and Morshead, 2003). NSCs are CD133+, Lewin X+ and have high aldehyde

dehydrogenase activity (Obermair et al., 2010). To add to the complication of identifying

 

49

 

selective NSC markers, the expression of some enriching genes were only effective at

certain points in development (Murayama et al., 2002). The issue with such cell markers

is that none are specific to NSCs and thus leave the NSC field without the ability to

isolate a pure population for prospective analysis.

This thesis focuses on characterizing pNSCs and identifying genes and cell

markers expressed in pNSCs. In the second chapter of this thesis, pNSCs are identified

and characterized to express Oct4. pNSCs are the sole adult somatic cell to express Oct4

and this provides an invaluable cell marker that enables selective pNSC to assess the role

of pNSCs in the adult mouse brain. In the fourth chapter I will investigate other cell

surface markers and methods to target and activate endogenous pNSCs.

Cell Surface Profiling

Specific cell surface markers are valuable tools to study any cell population, and a

previous study utilized ESCs to derive large quantities of pNSCs in culture to identify

and quantify cell membrane-bound proteins. DeVeale et al., isolated ESC-derived

primitive neurospheres to isolate cell surface proteins specific to ESC-derived primitive

neurospheres but not ESCs or ESC-derived definitive neurospheres (DeVeale et al.,

2014). Whole neurospheres were analyzed and therefore included both the stem cells and

progenitors in the cell surface profiling (Fig 3A). Despite the non-pure pNSC population

for analysis, this pool of cell surface proteins holds great potential to be tested for

selective pNSC markers and the ability to endogenously activate pNSCs.

To identify cell surface proteins in each population of ESCs, primitive

neurospheres and definitive neurospheres were collected for cell surface capturing

 

50

 

Figure 3

A.

B.

 

51

 

Figure 3. Identification of cell surface proteins enriched specifically on ESCs,

pNSCs and dNSCs. A. ESCs in culture were used to generate pNSC-derived

neurospheres and dNSC-derived neurospheres for cell surface protein analysis. B. A mass

spectrometry based screen of in vitro ESC-derived primitive neurospheres identified cell

surface markers upregulated in primitive neurospheres compared to ESCs and dNSC-

derived neurospheres. These cell surface markers provide potential targets to isolate and

manipulate pNSCs in the mouse brain. Adapted from DeVeale et al., (2014).

 

52

 

(DeVeale et al., 2014). To prepare the cells the colonies/neurospheres were dissociated,

the extracellular proteins were oxidized, then the cells were lysed and the nuclei were

pelleted. The supernatant was recovered to collect the cell membranes and cytoplasm and

centrifuged to isolate the microsomal pellet. From this pellet, isolated cell membrane

proteins enriched in one cell type over the other cell types were identified. For the

purpose of this thesis, I focused on the cell surface proteins enriched at least 5-fold in

pNSC neurospheres (Fig 3B). In the fourth chapter of this thesis I will test the cell surface

markers identified on ESC-derived primitive neurospheres, to validate their relevance to

mouse derived pNSCs in vitro and in vivo. These specific cell markers will provide novel

pharmacological methods of targeting and activating pNSCs in the brain.

 

53

 

General Aims

In this thesis I will characterize pNSCs, investigate their role in the adult mouse

brain and identify selective pNSC markers. Previously, it was thought that pNSCs were

only present during embryonic development, however, as I will go on to discuss in this

thesis we recently discovered that pNSCs persist as rare cells in the adult mouse brain. I

will characterize adult-derived pNSCs to investigate gene and protein expression,

particularly the pluripotency factor Oct4. I will take advantage of in vitro clonal analysis

to assess the self-renewal and multipotency of the pNSC population. Next, I will assess

the lineage relationship between pNSCs and dNSCs. I will estimate the cell cycle time of

pNSCs as compared to dNSCs using a label-retention model. A pNSC loss of function

mouse model will be employed to ablate dNSCs and their downstream progeny and test

the requirement for pNSCs in dNSC repopulation. Finally, I aim to identify selective cell

surface markers of pNSCs to improve our ability to target and pharmacologically

manipulate this cell population. This knowledge will expand our understanding of the

NSC lineage in development and the adult mouse brain. Exploring the neural hierarchy is

important to draw conclusions on the cell types present in the NSC lineage and develop

methods of targeting these cell types for therapeutic purposes.

Overall Hypothesis

Primitive NSCs comprise a reserve stem cell population in the mouse brain that

expresses Oct4, is predominantly quiescent, is upstream of GFAP+ dNSCs, and

proliferates in response to injury or downstream lineage ablation.

 

54

 

Specific Hypotheses and Aims

1) pNSCs persist as an Oct4-expressing population upstream of dNSCs in the

adult mouse brain

After identification that pNSCs persist in the adult mouse brain I will characterize

the multipotency and gene expression profile of this population in comparison to

dNSCs. I will assess the pluripotency of pNSCs based on Oct4 expression by qPCR,

immunohistochemistry, FACS analysis, and their ability to contribute to chimeric

blastocysts. In addition, I will attempt to quantify pNSCs with Oct4+ cell flow

analysis and visualize Oct4+ cells in the periventricular region of the adult mouse

brain. This chapter will comprise the initial discovery and characterization of pNSCs

in the adult mouse brain.

2) pNSCs are slow cycling under baseline conditions and activated to

repopulate dNSCs following dNSC ablation in the adult mouse brain

I will assess the cell cycle time of pNSCs using the Histone2B-GFP mouse model

that takes advantage of label-retention in non-dividing cells. I posit that pNSCs are

responsible for dNSC repopulation in injury/ablation paradigms and will perform

dNSC ablations in pNSC loss of function mice to test the requirement for pNSCs in

dNSC recovery. This chapter will speak to the quiescent nature of pNSCs as a reserve

stem cell population that is activated by the ablation of their downstream progeny.

 

55

 

3) Pup-derived pNSCs are more abundant than adult-derived pNSCs and

provide a valuable population for the identification of cell surface markers

specific to pNSCs

Identification of pNSC-specific cell markers is limited due to the rarity of pNSCs

in the adult brain. pNSCs are 30-fold more abundant in the postnatal day 7 brain and I

will compare their equivalence to adult pNSCs based on self-renewal, multipotency

and gene expression profiles. Cell surface markers identified in a previous mass

spectrometry based screen of ESC-derived pNSCs compose a valuable list of targets

to test on mouse brain-derived pNSCs and I will confirm their efficacy on pup-

derived pNSCs in vitro followed by adult-derived pNSCs in vivo. This chapter will

validate the equivalence of pup- and adult-derived pNSCs and the ability to employ

cell surface markers identified in culture to target pNSCs in the adult mouse brain.

 

56

 

 

57

 

Chapter 2

Primitive neural stem cells in the adult mammalian brain give rise to the GFAP expressing neural stem cells

This chapter has been published:

Sachewsky N, Leeder R, Xu W, Rose K, Yu F, van der Kooy D, Morshead C (2014)

Primitive Neural Stem Cells in the Adult Mammalian Brain Give Rise to GFAP-

Expressing Neural Stem Cells. Stem Cell Reports 2:6, 810-2

My contributions to this manuscript:

Figure 1C; Figure 2A, D, F; Figure 3B; Supplemental Figure 2A, C

 

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Abstract

Adult forebrain definitive neural stem cells (NSCs) comprise a subpopulation of

GFAP expressing subependymal cells that arise from embryonic FGF dependent NSCs

first isolated from the developing brain at E8.5. The embryonic FGF dependent NSC is

derived from a LIF responsive, Oct4 expressing primitive NSC (pNSC) first isolated at

E5.5. We report the presence of a rare population of pNCSs in the periventricular region

of the adult forebrain. Adult derived pNSCs are GFAP negative, LIF responsive stem

cells that display pNSC properties including Oct4 expression and the ability to integrate

into the inner cell mass of blastocysts. Adult derived pNSCs (AdpNSCs) generate self-

renewing, multipotent colonies that give rise to definitive GFAP+ NSCs in vitro and

repopulate the subependyma following the ablation of GFAP+ NSCs in vivo. These data

support the hypothesis that a rare population of pNSCs is present in the adult brain and

are upstream of the GFAP+ NSC.

 

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Introduction

Neural stem cells (NSCs) in the adult brain reside in the periventricular region

where they generate progeny that migrate along the rostral migratory stream (RMS) to

the olfactory (OB) and become interneurons (Chojnacki et al., 2009; Garcia et al., 2004;

Mirzadeh et al., 2008; Morshead et al., 1994). Numerous studies suggest that adult

forebrain NSCs comprise a subpopulation of the GFAP expressing (GFAP+) cells within

the subependyma (SE) lining the lateral ventricles (termed type B cells) (Capela and

Temple, 2002; Chiasson et al., 1999; Doetsch et al., 1999a; Morshead et al., 2003). The

GFAP+ NSCs generate clonally derived, multipotent, self-renewing colonies (termed

neurospheres) in the presence of growth factors (epidermal growth factor (EGF) and

fibroblast growth factor 2 (FGF2)) in vitro (Doetsch et al., 1999a; Garcia et al., 2004;

Morshead et al., 2003). The GFAP+ adult NSCs are derived from embryonic definitive

NSCs and both share similar properties including FGF2 and EGF responsiveness, self-

renewal, and multipotentiality (Hitoshi et al., 2004; Tropepe et al., 2001). GFAP

expression in definitive NSCs occurs during development after embryonic day 16.5

(Imura et al., 2003) and continues into adulthood. Leukemia inhibitory factor (LIF)

dependent primitive (p)NSCs are present at E5.5 and give rise to FGF2 responsive NSCs

beginning at E8.5, which then go on to generate the GFAP+, neurosphere forming

definitive NSCs in the adult brain.

We have found a rare population of pNSCs in the adult mammalian brain (termed

AdpNSCs). We isolated cells from the adult periventricular region that generate clonally

derived, self-renewing, and multipotent colonies in vitro in the presence of LIF. These

LIF colonies can be passaged to give rise to GFAP+, neurosphere-forming cells in the

 

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presence of EGF and FGF2. Most interestingly, the LIF colonies expressed Oct4 in vitro

and contributed to the inner cell mass of developing blastocysts following morula

aggregation, which are characteristics attributed to pNSCs derived from ESCs (Hitoshi et

al., 2004; Tropepe et al., 2001). We observed Oct4 expression in periventricular tissue

using qPCR of primary cells and in wholemount sections from adult brains. Further, we

asked whether these adult derived pNSCs could generate the GFAP+, neurosphere

forming NSCs in vivo. We took advantage of a transgenic mouse that expresses Herpes

Simplex Virus thymidine kinase under control of the GFAP promoter (GFAP-tk mice),

which enabled the selective ablation of proliferating GFAP+ cells in vitro and in vivo

(Bush et al., 1998; Bush et al., 1999; Imura et al., 2003; Morshead et al., 2003) following

exposure to the antiviral agent Ganciclovir (GCV). We used multiple ablation paradigms

and found that following an initial and complete loss, GFAP+ NSCs invariably recovered

over time, thereby confirming the presence of a GFAP negative cell upstream of the adult

NSC in the lineage. Additionally, adult mice that are effectively AdpNSC-null and do not

generate LIF colonies are unable to repopulate the GFAP+ NSC population following

ablation. Hence, these findings demonstrate the presence of a rare population of Oct4+

pNSCs in the adult forebrain whose progeny include the GFAP+ type B cells that are

indeed neurogenic in vivo (Doetsch et al., 1999a) and form neurospheres in vitro

(Morshead et al., 2003).

Methods

Animals. Animals were maintained in the Department of Comparative Medicine

at the University of Toronto in accordance with institutional guidelines.

 

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Cell culture. Mice were sacrificed by cervical dislocation. Brains were dissected

and the periventricular region was cultured as previously described (Chiasson et al.,

1999). For neurosphere assays, cells were plated in serum-free media in EGF, FGF, and

Heparin or in LIF alone. For adherent colonies, cells were plated on mouse embryonic

fibroblasts (MEFs) in standard ESC media supplemented with LIF and passaged once

weekly as previously described (Tropepe et al., 2001). EFH neurospheres or LIF colonies

were counted at 7-10 days in vitro.

Surgery. For all surgical procedures, animals were anesthetized with 1-5%

isoflurane and injected with ketoprophen (5 mg/kg). Stroke was induced by removing the

skull and dura in the region bound by -0.5mm, +2.5mm (AP) and +0.5mm, 3.0mm (M/L)

relative to bregma. Saline soaked cotton swab was used to remove pial vessels. 200 µM

GCV and 2% AraC was infused via mini osmotic pump (Alzet 1007D) with cannulas

placed at 0.2mm A/P, 0.7mm M/L, and 2.5mm D/V relative to bregma. Transplanted

animals received 1µl of 800 cells injected at 0.5mm M/L, 1.5mm A/P, and 2.5mm D/V

relative to bregma.

qPCR. ESCs, LIF colonies, EFH neurospheres were collected into Buffer RLT

with β-mercapthenol. Samples were processed as per manufacturer’s directions using the

RNeasy Micro Kit (Qiagen, Mississauga, ON), including treatment with the RNase-free

DNase Set (Qiagen). cDNA synthesis was carried out with Superscript III First Strand

Synthesis System (Invitrogen, Carlsbad, CA). qPCR was carried out on a 7900HT Fast

 

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Real-Time PCR System (Applied Biosystems). Cycling conditions consisted of initial

activation (2 min at 50oC, then 10 min at 95oC), followed by 40 cycles of 15s at 95oC, 1

min at 60oC, followed by 15s at 95oC, 15s at 60oC and 15s at 95oC.

Tissue preparation and immunohistochemistry. Mice were sacrificed with an

overdose of sodium pentobarbital and perfused transcardially with ice cold PBS followed

by 4% PFA, post-fixed, and cryoprotected in 20% sucrose. For immunohistochemistry,

sections re-hydrated with PBS and membranes were permeabilized with 0.3% Triton-X in

PBS for 20 minutes at RT. For BrdU imaging, DNA was denatured with 1N HCl at 65oC

for 30 minutes. Sections were blocked with 10% NGS (normal goat serum) or 10% NDS

(normal donkey serum (Sigma)) in PBS for 1 hour at RT before incubation with primary

antibodies at 4oC overnight followed by incubation of secondary antibodies for 1 hour at

37 oC. Wholemount sections were derived from Oct4-GFP adult mice as previously

reported (Mirzadeh et al., 2010). Staining was visualized on an AxioVision Zeiss UV

microscope and Nikon 200 microscope or Olympus Fluroview FV1000 confocal laser

scanning microscope.

Statistics. Data are represented as mean ± sem unless otherwise stated. Statistical

analyses were performed by GraphPad Prism 5 (GraphPad Software, Inc.) using ANOVA

with Bonferroni’s multiple comparison test or Student’s T-test unless otherwise stated.

 

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Results

Multipotent and self-renewing LIF responsive colony forming cells are present in

the periventricular region in vivo

Mouse neural development indicates the presence of a LIF-dependent pNSC that gives

rise to FGF2-dependent NSCs in the early embryo (Akamatsu et al., 2009; Bauer and

Patterson, 2006; Doetsch et al., 1999b), thus we investigated the potential continued

presence of a LIF responsive cell in the adult forebrain. We observed LIF receptor

positive (LIF-R+) cells in the ependyma and SE of the adult brain, similar to previous

observations (Bauer and Patterson, 2006) (Fig. S1A). We prepared neurosphere cultures

at clonal densities (Coles-Takabe et al., 2008) from adult mice in the presence of LIF

alone, identical to conditions used to isolate pNSCs from the early embryonic brain

(Hitoshi et al., 2004). We observed a rare population of free-floating spherical colonies of

cells with well-defined borders in LIF-only conditions (1.5 ± 0.2 per 40,000 cells) that

were >50 µm in size but typically less than 150 µm (Fig. 1A). The low incidence of adult

LIF-only colony formation is consistent with rare frequency of pNSCs during early

development (Hitoshi et al., 2004). Individual adult LIF colonies could be passaged into

LIF for > 4 passages and differentiated into neurons, astrocytes and oligodendrocytes,

thereby displaying the properties of self-renewal and multipotentiality (Fig. 1B). Adult

LIF colonies differentiated into all three neural phenotypes with equal frequencies (Fig.

1C), similar to in vitro ESC-derived pNSCs (Fig. 1C’). This is significantly different

from adult EFH neurospheres where astrocytes comprise the vast majority of

differentiated progeny (Hunt et al., 2010). Hence, the differentiation profiles support the

conclusion that pNSCs, regardless of the age of derivation, are more similar to each other

 

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Figure 1

 

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Figure 1. LIF responsive colonies are derived from the adult periventricular region

A. Spherical, free-floating colonies (50–150 µm in diameter) were observed in LIF and

compared to neurospheres grown in EFH (>100 µm diameter). B. LIF responsive

colonies gave rise to neurons (BIII tubulin+, red), astrocytes (GFAP+, green) and

oligodendrocytes (O4+, green). C. Differentiation profile of LIF colonies revealed that

oligodendrocytes, neurons, and astrocytes are produced at equal frequency upon

differentiation (similar to what is seen in ES-derived pNSC colonies (C’) (n ≥ 3 colonies

per group, >400 cells per colony were counted). D. Free-floating LIF colonies could be

isolated at all timepoints through development and into old age (n>4 per timepoint). Data

represent mean + SEM. Scale bars = (A) 40 µm, (B) 50 µm.

 

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than to the definitive neural stem cells. Notably, dissections of the striatum and cortex did

not result in LIF colony formation. Taken together, these data reveal that the adult brain

periventricular region contains a stem cell population that is distinct from the definitive

GFAP+, EFH responsive NSC in terms of its differentiation potential and LIF

dependence.

To determine whether this LIF-R+ stem cell population was distinct from the adult

GFAP+ NSCs, we employed a transgenic mouse that expresses thymidine kinase from the

GFAP promoter (GFAP-tk) thereby permitting the specific ablation of dividing GFAP+

cells in the presence of GCV in vivo and in vitro (Morshead et al., 2003). GCV is taken

up by all cells and phosphorylated by the transgene leading to the accumulation of toxic

metabolites and cell death when the GFAP expressing cells undergo mitosis. When

primary cultures from GFAP-tk mice were grown in standard neurosphere conditions

(EFH), no neurospheres formed in the presence of GCV (Fig. 3A) indicating that

neurospheres were derived from GFAP+ cells as previously observed (Morshead et al.,

2003). In sharp contrast, primary LIF colonies from GFAP-tk mice formed even in the

presence of GCV in vitro and at the same frequency as LIF colonies from non-transgenic

(NT) controls (1.1 ± 0.5 versus 1.3 ± 0.6 colonies per 40 000 cells, GFAP-tk and NT

respectively), indicating that the LIF responsive colonies are derived from a GFAP

negative cell. Most interestingly, we found that individual colonies grown in LIF alone,

from GFAP-tk and NT mice, could be passaged into EFH indicating that LIF colonies

(derived from GFAP– cells) were able to generate GFAP+ neurosphere forming cells

during colony formation. Notably, LIF colonies from GFAP-tk mice could not be

passaged into EFH+GCV indicating that the secondary neurospheres that formed in EFH

 

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were derived from GFAP+ cells. Together, these data reveal that a LIF-R+, GFAP–,

colony forming stem cell is present in the adult brain and is able to generate GFAP+ type

B cells that form EFH neurospheres in vitro.

We examined the lineage relationship between the LIF responsive free-floating

colonies, and EFH neurospheres, and EGF-only neurosphere. Neurospheres were derived

from wildtype animals under standard neurosphere conditions (free-floating in serum-free

media containing EGF, FGF2 and Heparin (EFH) or EGF-only). Individual clonally

derived neurospheres were collected, dissociated and replated to assess secondary

neurosphere formation. Invariably, individual primary EFH neurospheres (derived from

the GFAP+ adult NSCs) passaged into EFH, generated secondary neurospheres that were

subsequently propagated through > 5 passages (n=18/18 neurospheres). In contrast,

individual primary EFH neurospheres never passaged into LIF only conditions (n=0/18).

These findings indicate that the GFAP+ adult NSC does not give rise to AdpNSCs.

We asked if EGF-only neurospheres could be passaged into LIF conditions to

form secondary neurospheres and again, we never observed secondary neurospheres from

individual EGF only neurospheres (n=0/18 individual neurospheres). Most importantly,

and in contrast to previous reports that EGF neurospheres are derived from GFAP- transit

amplifying cells (type C cells) (Doetsch et al., 2002), our EGF-only primary

neurospheres from GFAP-tk animals never passaged into EGF+GCV (n=0/12), indicating

that EGF-only neurospheres are derived from a GFAP expressing adult NSC. Taken

together, these results indicate that our adult derived LIF colonies are derived from

GFAP– stem cells that can give rise to the GFAP– adult NSCs and further, that the reverse

relationship is not true; GFAP+ adult NSC do not give rise to AdpNSCs in vitro.

 

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We further asked whether this adult population of LIF responsive NSCs was seen

through embryonic development and into adulthood. Previous work demonstrated that

embryonic primitive NSCs declined by E8.5 to barely detectable levels (Hitoshi et al.,

2004); however, they did not extend their findings to later times in embryogenesis.

Herein, we isolated primary periventricular tissue from various developmental stages,

starting at E8.5, and cultured the cells in LIF conditions. We have shown that LIF

responsive free-floating colonies can be isolated from E8.5 at rare frequencies (Fig. 1D)

consistent with what was shown by others (Hitoshi et al., 2004) and further, that the

population expands in numbers in late embryogenesis and the early post-natal period,

followed by a decrease into adulthood that is maintained into old age (>22 month old

animals) (Fig. 1E). Hence, the LIF responsive pNSC can be isolated from the developing

and adult brain.

LIF colonies express Oct4 in vitro and in vivo and integrate into the inner cell mass

of blastocysts

We asked if the LIF-R+ colony forming cells in the adult brain had properties

similar to pNSCs derived from the embryonic brain. pNSCs derived from the E5.5-8.5

embryo or from ESCs display properties of pluripotency including the expression of Oct4

(Akamatsu et al., 2009) and pNSCs derived from ESCs have the capacity to integrate into

the inner cell mass (ICM) of blastocyst chimeras (Tropepe et al., 2001). We grew adult

derived LIF colonies in ESC conditions (on mouse embryonic fibroblast feeder cells

(MEFS)) and generated colonies morphologically similar to ES cell colonies. The adult

LIF colonies express Oct4 as seen by qPCR, immunohistochemistry, and GFP expression

 

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when cells are derived from Oct4-GFP transgenic mice (Fig. 2A-C). We made morula

aggregates using transgenic YFP expressing cells from adult mice grown as (1) LIF

colonies on MEFs, (2) neurospheres in standard EFH conditions (negative control) and

(3) YFP expressing ESCs (positive control). Consistent with previous studies, ESCs

integrated at a high frequency (71.7%) while adult EFH neurosphere derived cells did not

integrate into the developing blastocysts in vitro (Karpowicz et al., 2007). Most

strikingly, adult LIF colony derived cells also integrated into the ICM, albeit at a much

lower frequency (2.5%) (Fig. 2D). The rare frequency of integration may be the result of

the heterogenous population within a LIF colony consisting of both pluripotent AdpNSCs

and definitive GFAP+ NSCs. This likely leads to an underestimation of the true numbers

of AdpNSCs that can integrate into chimeric blastocysts. To date, we have not observed

any AdpNSC derived chimeras that survive until E9.5 after transfer back to

pseudopregnant mice, perhaps consistent with the lower level of Oct4 expression in

AdpNSC colonies compared to ESCs and embryonic pNSCs (Fig. 2A).

We grew adult LIF colonies in LIF alone (no feeders) from Oct4-neo transgenic

mice, which harbor a neomycin resistance cassette knocked into the Oct4 locus, thereby

conferring neomycin resistance in Oct4 expressing cells. After passaging individual

Oct4-neo primary LIF colonies, we observed the formation of secondary LIF colonies in

the presence of neomycin revealing that LIF colonies were derived from Oct4 expressing

cells (Fig. 2E). Primary neurospheres derived in EFH could never be passaged in the

presence of neomycin as predicted. This complete depletion of EFH neurospheres, but

unchanged numbers of LIF colonies, indicate appropriate antibiotic selection of Oct4+

 

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Figure 2

 

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Figure 2. LIF colonies express Oct4 and integrate into the ICM of blastocysts

A. qPCR Oct4 expression in ESCs, pNSC, adult LIF colonies grown in ES conditions,

and adult neurospheres grown in EFH (n=3 independent samples/group, in triplicate). B.

LIF colonies from Oct4-GFP mice grown in ESC conditions express Oct4 (green) (C-i)

Control ES colonies and (C-ii) adult LIF colonies from YFP expressing mice grown in

ES conditions express Oct4 (red). D. ESCs and adult LIF colony cells (green) integrate

into the ICM of blastocysts. E. LIF colonies derived from Oct4-neo transgenic mice, but

not from control animals, form secondary colonies in G418 (n=12 passaged

colonies/group). F. qPCR Oct4 expression from the periventricular region of adult brain

compared to control (n=4/group). Data represent mean + SEM. Scale bars = (B) 100 µm,

(Ci) 50 µm, (Cii) 25 µm, (D) 50 µm.

 

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cells in the presence of G418. If variable expression of the transgene was occurring, we

also would expect there to be some Oct4 cells that did not possess the antibiotic

resistance gene and therefore would die in the presence of the antibiotic. Notably, no

such death was seen. Taken together these findings reveal that adult derived LIF colonies

display similar properties to pNSCs from the developing embryo.

To ensure that the Oct4 expression observed in vitro was not an artifact of

culturing periventricular cells, we looked for Oct4 expression in vivo. Given that

AdpNSCs represent an exceedingly rare population of cells and their Oct4 expression is

extremely low, we performed qPCR on primary dissected periventricular tissue. We

observed significant Oct4 mRNA expression in the periventricular tissue compared to the

complete lack of expression in cortical tissue from the adult brain (Fig. 2F). We also

looked for the presence of GFP expressing cells in the periventricular region from Oct4-

GFP expressing mice and observed rare GFP cells in wholemount sections of the lateral

ventricle periventricular region (Fig. 3A-B, Video S1). We identified rare GFP

expressing cells in the periventricular region (Fig. S2B), which co-localized with LIF-R

but not GFAP or ß-catenin, consistent with our in vitro findings. Furthermore, we

performed FACS analysis of primary dissections of the periventricular region from adult

Oct4-GFP mice to estimate the number of Oct4+ AdpNSCs. FACS analyses revealed that

0.08% of the sorted cells were Oct4+; suggesting that approximately 80 cells in the

periventricular region are AdpNSCs (100 000 cells are obtained per brain dissection).

This estimate is higher than might be expected from the neurosphere assay and suggests

that we may have not optimized our AdpNSC culture conditions and/or the increased

sensitivity of FACS to levels of GFP expression leads to the isolation of GFP+ cells that

 

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were not visualized in wholemount sections. To further supplement our FACS findings,

we utilized an imager coupled with flow cytometry to visually identify single cells. Live

cell staining identified GFP+ and LIF-R+ populations from Oct4-GFP mice (Fig. 3C-i-ii).

We used fixed cells from Oct4-GFP mice and showed that all GFP+ and LIF-R+ cells are

always GFAP– (Fig. 3C-iii-iv), consistent with our wholemount data (Fig. S2B). Most

important, we never observed a GFP expressing cell from control tissue (the

periventricular region of CD1 control mice or the cortex of Oct4-GFP mice). Together,

these findings demonstrate that Oct4 expressing cells are present in the periventricular

region of the adult brain in vivo.

To further characterize the AdpNSCs, we examined the gene expression of

AdpNSCs using qPCR on LIF colonies, EFH neurospheres and ESCs. We looked at

additional markers of pluripotency including Nanog, Sox2, Klf4, TERT and c-myc (Fig.

S2A). We observed that LIF colonies express detectable levels of Nanog, which was

undetectable in EFH neurospheres. As well, LIF colonies express mRNA levels of Klf4

and c-myc equivalent to ESCs (0.88 and 1.7 fold relative to ESCs, respectively). Sox2 and

TERT were also identified in LIF responsive colonies (0.54 and 1.7 fold expression

relative to ESCs, respectively). Examination of proneural genes including Sox1, Notch,

Nestin, and CD133 (Fig. S2A) revealed that LIF colonies express significantly lower

mRNA levels of the neural markers Sox1, Notch and Nestin compared to EFH

neurospheres. CD133 was similar in LIF colonies and EFH neurospheres (Fig. S2A).

Thus, we report that adult LIF colonies have higher expression of pluripotency markers,

and lower levels of definitive NSC markers, making

 

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Figure 3

 

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Figure 3. Oct4 expressing cells are present in vivo. A. Wholemount sections derived

from Oct4-GFP mice reveal Oct4+ (green) periventricular cells (Dapi, blue punctate

nuclei) optimized to detect GFP expression in high magnification images in the adult

brain (blood vessels (red)). B. Wholemount sections of the periventricular region of Oct4-

GFP adult mice reveal Oct4+ (green) cells labeled with Hoechst (blue). Insets show

increased magnification of the Oct4+ cells. Orientation markers provided within images.

Scale bar = 20 µm. C. Image stream images showing (i-ii) Oct4+ LIF-R+ live cells and

(iii-iv) Oct4+ LIF-R+ GFAP– from fixed cells from Oct4-GFP mice.

 

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Figure 4

 

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Figure 4. GFAP-TK model specifically ablates dividing GFAP+ cells in vitro and in

vivo. A. GCV dose response curve in vitro (n=2 independent experiments). B. 2 or 7 day

GCV infusion in vivo followed by immediate sacrifice with cells plated in EFH in the

absence (-) or presence (+) of GCV in vitro (n ≥ 5 mice/group). 7 day GCV infusion (n ≥

3mice/group) followed by plating in (C) EFH to assay GFAP expressing adult NSCs or

(D) LIF-only to assay for AdpNSCs. E. BrdU+ cells in the SE following 2 day GCV

infusion (n=3 mice/group). F. EFH neurosphere assay following 21-day GCV infusion

(n=3 mice/group). *p ≤ 0.05. Data represent mean + SEM.

 

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them more ESC-like. Taken together, the immunohistochemistry, wholemounts, and

qPCR data reveal that AdpNSCs are LIF-R+/Oct4+/GFAP–/Sox2+/Nestin+/CD133–.

Adult derived primitive NSCs repopulate GFAP expressing NSCs following ablation

with GCV in vivo

Based on the NSC lineage from embryonic development into adulthood, we asked

whether AdpNSCs gave rise to GFAP+ neurosphere forming NSCs in the adult brain. To

address this question, we performed in vivo experiments to examine the potential for

AdpNSCs to contribute to SE repopulation using the GFAP-tk mouse. Previous studies

using GFAP-tk transgenic mice led us to predict that intraventricular infusion of GCV

would effectively create a GFAP+ NSC depleted mouse as cells are killed when

proliferating (Bush et al., 1998; Bush et al., 1999; Imura et al., 2003; Morshead et al.,

2003). In vitro, we observed a complete loss of clonal EFH neurosphere formation from

the SE of GFAP-tk mice in the presence of 20 µM GCV with no effect of GCV on the

number (Fig. 4A) or size (Fig. S3A) of NT littermate control EFH neurospheres. In vivo,

intraventricular GCV infusions into GFAP-tk and NT mice for 7 days, followed by

immediate sacrifice, resulted in GFAP+ NSCs depletion with a >99.5 ± 0.5% loss in

clonal EFH neurosphere formation from GFAP-tk versus NT mice (Fig. 4B-C), similar to

previous findings (Garcia et al., 2004; Morshead et al., 2003). However, when the

neurosphere assay was performed from GFAP-tk mice that survived for various times

post GCV infusion, the numbers of EFH neurospheres returned over time, reaching 30%

of control levels by day 14 (7 days after GCV treatment) (Fig. 4C) despite an initial

99.5% depletion in EFH neurosphere numbers at the time of sacrifice (1 of 6

 

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animals examined had 1 EFH neurosphere). A similar return of EFH neurospheres was

seen after 2 days of GCV infusion (Fig. S3C). Significantly, in vitro exposure to GCV

completely and invariably eliminated EFH neurosphere formation from GFAP-tk mice at

all times examined (at immediate sacrifice and during EFH neurosphere recovery post-

infusion), indicating that the returning EFH neurospheres were derived from GFAP+

cells. Furthermore, neurospheres never grew in EGF-alone immediately following the

GCV infusions or when cultured in EGF+GCV at later survival times, indicating that

GFAP–, EGF responsive progenitor cells (Doetsch et al., 2002) were not responsible for

the return of neurospheres over time (Fig. S3B). Interestingly, following GCV infusion

for 2 or 7 days in vivo, the numbers of AdpNSC derived LIF colonies that form in vitro

was unchanged upon immediate sacrifice and at later survival times post-GCV (Fig. 4D).

These findings indicate that the LIF colonies are derived from a GFAP– cell as they are

not depleted by the GCV ablation. Further, the LIF population divides asymmetrically to

repopulate the GFAP+ NSCs in vivo since the number of LIF colonies does not expand

following the ablation. Consistent with the return of EFH neurospheres in vitro, the

numbers of proliferating cells (BrdU+) in vivo in GCV infused GFAP-tk mice increased

with longer survival times (Fig. 4E).

We extended the length of GCV infusion in vivo to 21 days, 50% longer than the

estimated 15-day cell cycle time of slowly dividing adult NSCs (Morshead et al., 1998) to

eliminate the possibility that a rare GFAP+ NSC escaped the GCV treatment and

repopulated the SE. We observed a complete 100% loss of GFAP+ NSC derived, EFH

neurosphere formation following 21 days of GCV; however, EFH neurospheres still

returned with longer survival times (Fig. 4F). The invariable return of GFAP+ NSCs over

 

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time supported the hypothesis that an earlier cell in the lineage, that was GFAP–, was able

to repopulate the SE.

In vivo ablation of GFAP+ cells following AraC activation does not lead to a

permanent loss of neural stem cells

We performed more rigorous attempts to permanently deplete GFAP+ NSCs in

vivo using a well-established paradigm to induce GFAP+ NSC proliferation prior to the

administration of GCV in vivo. Previous work has shown that infusion of an anti-mitotic

agent kills the rapidly proliferating NSC progeny in the SE and leads to the recruitment

and division of the GFAP+ NSC with repopulation of the SE within 8-10 days (Doetsch et

al., 1999b; Morshead et al., 1994). Based on these findings, we infused cytosine β-D-

arabinofuranoside (AraC) intraventricularly for 7 days followed immediately by GCV

infusion during the time when GFAP+ NSCs are proliferating to repopulate the SE

(Doetsch et al., 1999b; Morshead et al., 1994). Similar to previous findings (Doetsch et

al., 2002), the numbers of EFH neurospheres observed following AraC infusion alone

was initially depleted but quickly returned to control values (Fig. 5A). Indeed, the

numbers of GFAP+ NSCs in AraC treated animals was 1.6-fold greater than saline

infused controls at 3 days post-infusion (day 10 sacrifice) indicating an initial

overcompensation as the surviving GFAP+ NSCs underwent expansionary divisions to

repopulate the SE. In contrast, GFAP-tk mice receiving AraC+GCV treatment for 3

days revealed a 100% loss of neurosphere formation upon immediate sacrifice (day 10)

and a slower return of GFAP+ NSCs. EFH neurosphere formation inevitably returned at

longer survival times but did not return to control levels at the longest time examined

 

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(51.4 + 18.6% of controls by day 42) (Fig. 5B). Extending GCV infusion to 7 days

following AraC revealed a similar initial 100% loss of EFH neurosphere formation,

followed by the return at longer survival times (21.5 + 11.5% of NT controls by day 43).

Most importantly, the addition of GCV in vitro resulted in a complete and invariable loss

of EFH neurosphere formation from GFAP-tk mice at all survival times indicating that

the in vitro EFH neurospheres returning over time are generated from GFAP+ cells. Thus,

despite using multiple well-established kill paradigms to specifically ablate dividing

GFAP+ cells in vivo, we were unable to permanently deplete clonal EFH neurosphere

formation, indicating that GFAP+ cells recovered in vivo over time.

The loss of EFH neurosphere forming cells, albeit temporary, should also result in

an initial loss of proliferating NSC progeny. We examined the numbers of proliferating

cells in vivo following the 7-day AraC + 7-day GCV infusion. As predicted, we observed

an initial loss, followed by a complete recovery, of the numbers of proliferating cells by

day 42 post-treatment in the SE, RMS and olfactory bulbs (Fig. S4, S5A). Further,

following the AraC+GCV infusion, we observed proliferating LIF-R+ cells in the SE

(Fig. S1B-D). Hence, despite the in vivo depletions with AraC+GCV treatment, the

numbers of GFAP+, neurosphere forming cells and their progeny in vivo returned over

time.

We asked whether the inability to achieve permanent depletion of EFH

neurospheres in vitro, or proliferating cells in vivo, was due to GCV degradation in

GFAP+ NSCs over time, thereby leading to neurosphere formation with longer survival

times. We determined the length of time that GCV remains effective in killing

 

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Figure 5

 

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Figure 5. Infusion of AraC+GCV leads to complete but temporary loss of

neurospheres. A. AraC only infusion for 7 days into GFAP-tk mice resulted in a 99 ±

0.5 % loss in neurospheres relative to saline infused controls when sacrificed

immediately following infusion (day 7). Neurospheres returned to control values by 3

days post-AraC (n=3-6 mice/group/time point). B. AraC+GCV infusion eliminated TK

neurosphere formation immediately following the infusion (day 10), but returned with

longer survival times (n= 3-5 mice/group). Controls are NT mice that received infusions.

*p<0.0001. Data represent mean + SEM.

 

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proliferating GFAP+ cells once it is taken up by cells. Astrocyte monolayers from early

postnatal cortices of GFAP-tk and NT mice demonstrated that GCV remains toxic to cells

for at least 10 days following a single 16 hour exposure to the drug (Fig. S6B-C); a time

exceeding the in vivo survival times post infusion when we observed the return of

neurospheres in vitro. Hence, a loss of GCV toxicity cannot account for the return of

neurospheres over time. Thus these data provide strong support for the presence of an

upstream stem cell that is GFAP– and capable of generating GFAP+ NSCs following

ablation in vivo.

Finally, the lineage relationship between AdpNSCs and GFAP+ adult NSCs was

studied using a transgenic Floxed Oct4-Sox1Cre mouse that allows for specific ablation

of the Oct4 population in cells that express the neural gene Sox1. Floxed Oct4-Sox1Cre

mice are devoid of AdpNSCs and never give rise to LIF colonies in vitro whereas the

littermate controls generate normal numbers of LIF colonies (2.8+0.4 per 40,000 cells),

and EFH neurospheres are not changed (25.3+3.7 vs 27.5+4.0 neurospheres per 5,000

cells from transgenic vs. littermate controls, respectively). The floxed Oct4-Sox1Cre mice

have normal numbers of EFH neurospheres likely because Sox1 expression turns on after

pNSCs and definitive NSCs are present in the developing brain (Wood and Episkopou,

1999) hence the Oct4 allele is excised after the neural lineage is established. Most

interesting, following ablation of the EFH neurospheres, preliminary data reveals a lack

of GFAP+ adult NSC repopulation (0+0 EFH neurospheres vs. 10.2+0.1 EFH

neurospheres per 5 000 cells from transgenic vs. littermate controls, respectively). These

findings indicate that AdpNSCs are necessary for repopulating the EFH responsive,

 

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GFAP+ adult NSC in vivo following ablation in the adult brain and support the hypothesis

that GFAP+ adult NSCs are the progeny of the AdpNSCs.

Transplanted LIF responsive colonies, devoid of GFAP expressing adult NSCs,

contribute to neurogenesis in vivo

Based on our hypothesis that AdpNSCs generate GFAP+ adult NSCs, we predict

that AdpNSC progeny would contribute to neurogenesis in vivo. We performed

transplantation experiments to test our hypothesis using populations of AdpNSCs that do

not contain GFAP+ progeny prior to transplantation. GFAP-tk mice were crossed to YFP

reporter mice to generate mice (YFP-GFAP-tk mice) that ubiquitously express YFP and

permit the selective ablation of dividing GFAP+ cells in the presence of GCV. When LIF

colonies from YFP-GFAP-tk mice were isolated in vitro, the total numbers of LIF

colonies was the same in the presence or absence of GCV and not different from NT YFP

controls grown in GCV; however, the YFP-GFAP-tk LIF colonies were smaller in size

than control LIF colonies likely due to the ablation of the GFAP expressing progeny

within the colony (Fig. S6A-B). As predicted, YFP-GFAP-tk LIF colonies grown in the

presence of GCV never generated EFH neurospheres whereas those grown in the absence

of GCV, or LIF colonies grown in the presence of GCV from NT littermate controls,

always gave rise to EFH (Fig. S6C). Thus, the YFP-GFAP-tk derived LIF colonies did

not contain GFAP+ neurosphere forming cells prior to transplantation. We generated

single cell suspensions from 2-week LIF+GCV colonies and transplanted 800 YFP+ cells

into the anterior SE of wild-type CD1 mice. We examined the mice at 48 hours post-

injection or 14 days post-injection. At early sacrifice, 4.6% of transplanted

 

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Figure 6

 

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Figure 6. Numbers of adult derived pNSCs can be increased by injury or LIF

infusion. A. The numbers of LIF colonies isolated following GCV infusion (3 days)

(n>22 mice/group) or AraC+GCV infusion (n=5 mice/group). B. Stroke injured mice

generate significantly more LIF colonies at 4 days post-stroke, returning to control values

by day 11 (n=6-12 mice/group/time). Significance determined by one-way ANOVA with

post-hoc Bonferroni test, * indicates p<0.05 from LIF naïve to LIF Day 4, # indicates

p<0.05 from EFH naïve to EFH Day 7. The numbers of EFH neurospheres significantly

increases at 7 days post-stroke (n>3 mice/group). C. Four day LIF infusion results in

significant increase in LIF colonies but not (D) EFH neurospheres (n=6 mice/group).

Significance determined by Student’s T-test (p<0.05), unless otherwise stated. Data

represent mean + SEM.

 

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YFP+ cells had survived and were observed at the injection site (Fig. 6A). At day 14 post-

transplantation we observed YFP+ cells migrating along the RMS (Figure 7C-i-ii, Figure

S6D-i), residing in the OB (Fig. 7C-iii) as well as differentiating into neuronal like

morphology (Fig. S6D-ii-iii). These data further support the hypothesis that AdpNSCs

are able to contribute to neurogenesis in vivo.

The adult derived primitive NSC population is activated and expanded following

stroke or LIF infusion

The AdpNSC population comprised a very rare population of cells with an

average of 5.6 + 2.5 colonies per brain from naïve controls, with similar numbers from

GFAP-tk mice when grown in the presence or absence of GCV, and from AraC+GCV

treated GFAP-tk and NT mice (Figure 6A). The lack of expansion in numbers suggests

that AraC+GCV treatment results in asymmetric divisions of the AdpNSC to repopulate

the GFAP+ NSC during regeneration of the SE thereby maintaining their absolute

numbers. We reasoned that an expansion of this rare AdpNSC population might occur in

an injury model such as stroke where there is no depletion in the GFAP+ adult NSC

fraction (Zhang et al., 2004). We previously have demonstrated that the pial vessel

disruption (PVD) model of stroke results in increased numbers of EFH neurospheres post

stroke (Erlandsson et al., 2011). We used the PVD model to determine if LIF colony

formation was also increased following injury. Mice received a stroke lesion on day 0

and the number of AdpNSC derived LIF colonies was examined at 4, 7 and 11 days post-

stroke. We observed a significant 5.4 fold increase in the number of AdpNSC LIF

colonies at day 4 with a return to control numbers by day 7 post-stroke. Stroke also

 

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resulted in a significant 3.8 fold increase in the number of EFH neurospheres at day 7

post-stroke (Fig. 6B). These data suggest that AdpNSCs are sensitive to environmental

cues that result following injury. Most interesting, the AdpNSCs expansion occurs more

rapidly and prior to the definitive GFAP+ NSC expansion, supporting the hypothesis that

the AdpNSC resides upstream of the GFAP+ adult NSC.

Many types of injuries to the nervous system are accompanied by a rapid and

transient increase in LIF expression hence we asked whether increased LIF signaling

played a role in the activation of AdpNSCs in our stroke model (Banner et al., 1997;

Suzuki et al., 2005 Bauer et al., 2003). Intraventricular infusion of LIF for 4 days resulted

in a significant 2.5 fold increase in the number of LIF colonies from adult mice (Fig. 5C)

with no increase in the numbers of EFH responsive neurospheres (Fig. 6D). These data

indicate that the AdpNSC pool expands in response to LIF in vivo.

Discussion

Our results demonstrate the existence of a rare, pNSC in the adult brain that

expresses Oct4 and has the ability to integrate into the inner cell mass of blastocyst

chimeras. This LIF responsive population acts as a reserve pool capable of repopulating

the neural lineage in the SE in vivo. Similar to its embryonic counterpart, the AdpNSC is

a GFAP–, LIF-R+ cell from the periventricular region of the brain. The inability to

permanently ablate the GFAP+ neurosphere forming cells following a complete initial

loss, suggests that the AdpNSC is upstream of the GFAP+ adult NSC (Fig. 7D). This

lineage relationship is supported by both in vitro and in vivo findings. In vitro, the

passaging of the LIF colonies into standard adult neurosphere conditions (EFH) reveals

 

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Figure 7

 

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Figure 7. In vivo lineage analysis. A. Distribution of YFP+ cells at 48 hours post-

transplant (n=4 mice). B. Distribution of YFP+ cells at 14 days post-transplant (n=7

mice). C. Representative images of cells (i-ii) along the RMS and (iii) in the OB after 14

days. Insets show higher magnification of YFP+ cells. D. Adult pNSCs are GFAP

negative, responsive to LIF and express the pluripotency marker Oct4. The self-renewing,

multipotent AdpNSCs give rise to EFH responsive, definitive adult NSCs. The definitive

adult NSCs are neurogenic in the adult brain. Data represent mean + SEM. Scalebars=

(C-i) 200 µm (C-ii) 70 µm and (C-iii) 200 µm.

 

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that LIF colonies give rise to GFAP+, neurosphere-forming cells. In vivo, LIF colony

derived cells devoid of GFAP+ cells at the time of transplantation are able to migrate

along the RMS and contribute to neurogenesis. Further, we showed that Floxed Oct4-

Sox1Cre mice that completely lack AdpNSCs are unable to repopulate the GFAP+,

neurosphere forming NSCs following ablation in vivo. Hence, based on the studies

herein, we propose that the AdpNSC proliferates in response to injury and gives rise to

GFAP+ adult NSCs that repopulate the SE following their ablation in vivo.

There are several pieces of evidence that indicate that GFAP+ NSCs can be

ablated completely in vivo following GCV treatment in GFAP-tk mice and that they are

subsequently reconstituted from a GFAP– cell in the neural stem cell lineage. First, zero

neurosphere forming cells were observed immediately following GCV treatment for 21

days, as well as following AraC+GCV treatment in GFAP-tk mice. Second, GCV

remains toxic to GFAP+ cells for times exceeding those infused, yet inevitably GFAP+

NSC derived clonal neurospheres returned in vitro and their proliferating progeny

returned in vivo. Third, the neurospheres that returned at longer survival times post-GCV

infusion were lost if GCV was added in vitro in all instances, and, therefore, must have

been derived from GFAP+ NSCs. Fourth, the observation that EGF alone did not support

neurosphere formation immediately after GCV infusion indicates that the neurospheres

were not derived from transit amplifying cells. Finally, the finding that the kinetics of the

return of GFAP+ NSCs was dramatically different in paradigms that did not completely

eliminate neurosphere formation (i.e. AraC treatment alone) versus when there was a

complete loss of GFAP+ NSCs (AraC+GCV treatment) suggests that different cell

sources may be responsible for repopulation of the SE. Together, these data support the

 

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hypothesis that the GFAP– (resistant to GCV treatment), AdpNSC is able to repopulate

the GFAP+ NSC population.

The numbers of GFAP+ NSCs did not return to control levels at even the longest

survival times examined (approximately 1 month after AraC+GCV treatment). One

possibility is that we simply did not wait long enough to observe the complete return to

control values. Alternatively, the AdpNSCs may only be able to proliferate a limited

number of times as has been suggested by studies showing that serial transplantation of

hematopoietic stem cells for repopulation has been limited to 5-7 rounds (Harrison et al.,

1978; Harrison and Astle, 1982). The fact that the AdpNSC was not killed by the AraC

treatment demands that the cell has a relatively long cell cycle time, in line with studies

identifying slowly cycling cells in other systems (Fuchs, 2009). Indeed, reports suggest

that the blood system has a population of long-term label retaining cells that proliferate

only once every 4-5 months, translating into approximately 5 divisions in the life of the

animal (Foudi et al., 2009; Wilson et al., 2008). This dormancy may be important for

maintaining “stemness” or may be a property of the “master” stem cells that are activated

in times of stress or injury (Fuchs, 2009). The existence of an AdpNSC that can be

activated in response to injury suggests that these slowing cycling cells, rare AdpNSCs

are called upon to divide only rarely to generate the GFAP+ adult NSCs that are

responsible for maintaining adult neurogenesis under baseline conditions.

We propose that the AdpNSC is CD133– based on the lack of colocalization of

Oct4 with β-catenin; however, others have proposed the presence of a stem cell-like

CD133+ cell in the adult brain. It has been reported that quiescent CD133 expressing,

ciliated ependymal cells respond to injury by entering into cell cycle and contributing

 

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cells to tissue regeneration (Carlen et al., 2009). The activated ependymal cells were

unable to self-renew to maintain their population, suggesting that they may represent a

non-stem cell response to injury (Carlen et al., 2009). The lack of self-renewal capacity

of the CD133 positive ependymal cells is distinctly different from the AdpNSCs which

are able to self renew in vivo as illustrated by their expansion in number following LIF

infusion and stroke. CD133 has been shown to be present on the apical surface of GFAP+

adult NSCs (Mirzadeh et al., 2008) and we also detect CD133 in LIF colonies; however,

we suggest this CD133 expression is due to the presence of GFAP+ progeny within the

LIF colony. Further, we do not see colocalization of Oct4 with β-catenin, which has been

shown to identify ependymal cells in wholemount sections of the lateral ventricle

periventricular region (Mirzadeh et al., 2008). Taken together, these finding suggest that

AdpNSCs do not correspond with the CD133+ ependymal cells previously studied.

In conclusion, we have identified a rare pNSC that is present in the adult brain.

The adult derived pNSC has characteristics of pluripotency, including Oct4 expression

and the ability to integrate into the inner cell mass of blastocyst chimeras. The progeny of

the AdpNSC include the GFAP+, neurogenic, neurosphere forming, type B NSCs present

in the adult SE, similar to pNSCs derived from the embryonic brain. We propose this

LIF-R+ pNSC may be an additional target for the development of regenerative medicine

strategies in the adult CNS.

 

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Supplemental Figure 1

 

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Supplemental Figure 1. The periventricular region contains proliferating LIF-R+

cells in vivo. Confocal images showing the presence of LIF-R+ cells within the SE in

naïve control mice. A. A subpopulation of SE LIF-R+ cells (green) are proliferative

(Ki67+, red). B. After AraC+GCV infusions (7 days each), Ki67+ cells (red) are seen in

the SE of TK mice. A subpopulation of Ki67+ cells are LIF-R+ (arrows). C. Sox2 (red) is

widely expressed in both the ependyma and SE and colocalizes with LIF-R (arrows). D.

Some LIF-R+ cells also express GFAP (red) in the subependyma of AraC+GCV treated

TK mice (arrows). Scale bar =15 µm Blue = Hoechst labeled nuclei.

 

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Supplemental Figure 2

 

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Supplemental Figure 2. Expression profile of AdpNSCs. A. Gene expression profile

of AdpNSC derived LIF colonies (orange bars) and EFH neurospheres (green bars)

compared to ESCs assayed by quantitative qPCR (n=3 independent replicates per group).

Significance between LIF colonies and EFH neurospheres is denoted by (*) and

significant difference to ESCs is denoted by (#), calculated based on the log value by 2-

way ANOVA with Bonferroni post-hoc test (p<0.05). B. Immunohistochemical analysis

of wholemount sections from Oct4-GFP mice reveal (i) Oct4-GFP (arrows) does not co-

localize with GFAP but does co-localize with (ii) LIF receptor. (iii) Oct4-GFP does not

co-localize with β-catenin. Scalebars = 10 µm. C. Representative images of FACS

analysis on (i) periventricular cells derived from naïve adult Oct4-GFP animals as well as

a (ii) positive control using actin-GFP mice, (iii) negative control using CD1 mice, and

(iv) transgenic control using Oct4-GFP cortex to set appropriate gates (n=2 independent

experiments). Data represent mean + SEM.

 

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Supplemental Figure 3

 

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Supplemental Figure 3. The effects of GCV in vitro and in vivo. A. Exposure to 20

µM GCV in vitro does not affect the size of neurospheres from NT littermate controls

(shown) or C57Bl/6 controls (data not shown) (n=3 independent experiments). B. 2 day

GCV infusion (n ≥ 4 mice/group) followed by sacrifice at various times post-infusion

results in a return of EFH neurosphere formation over time. C. GFAP– type C progenitor

cells are not responsible for the return of neurospheres in vitro as EGF responsive

neurospheres could be isolated from untreated control mice (NT, n=6), saline-infused

mice (S, n=3), and NT mice that received 7d GCV (n=3), AraC + GCV (n=2), or 21d

GCV (n=2). EGF-responsive neurospheres could not be isolated from TK mice following

7 day GCV (n=4), AraC + GCV (n=3), or 21 day GCV (n=3) exposure in the absence of

GCV in vitro. Data represent mean + SEM.

 

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Supplemental Figure 4

 

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Supplemental Figure 4. Proliferating GFAP+ cells returned with longer survival

times following AraC+GCV treatment. (i, ii) Representative confocal images showing

GFAP+ and Ki67+ cells in the SE of TK (i) and NT (ii) mice immediately following

AraC+GCV infusions (7 days each) (day 14). (iii, iv) By day 42, post-infusion

GFAP+/Ki67+ SE cells are observed in both TK and NT mice. Scalebars = 10 µm.

 

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Supplemental Figure 5

 

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Supplemental Figure 5. Repopulation of proliferating cells after ablation in GFAP-

tk mice. A. An initial loss of BrdU+ cells in the subependyma (SE) and rostral migratory

stream (RMS) of TK mice is followed by a return to control levels by day 42. Data

represents mean ± s.e.m. (n=3-4 mice per group, 5 sections/mouse per region). *p<0.05.

B. GCV remains toxic to dividing GFAP+ cells at least 10 days after exposure. Astrocyte

cultures generated from TK and NT mice were exposed to GCV for 12 hours, re-plated in

fresh media and maintained as monolayer cultures for up to 21 days. On day 5, 8, 10 and

21 after GCV exposure, cells were induced to divide by passaging and plating in standard

neurosphere conditions (EFH). TK cell cultures failed to generate neurospheres up to 10

days post GCV pulse. Even at 21 days post GCV exposure, only rare neurospheres

formed from TK cultures (5 versus 746 neurospheres per 360 000 cells plated from TK

versus NT cultures respectively). (n ≥ 2 independent experiments/timepoint). C.

Representative pictures of cultures from TK and NT cultures. At all times examined, the

GCV exposed NT and TK cultures had viable, healthy cells in the standard EFH

conditions. Anti-GFAP immunostaining shows astrocytes in TK and NT derived cultures

at day 10 prior to dissociation and plating in the EFH. Data represent mean + SEM.

Scale bars: all TK cultures 200 µm; NT cultures at days 5 and 10, 100 µm, day 8, 200

µm. GFAP staining, 200 µm.

 

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Supplemental Figure 6

 

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Supplemental Figure 6. YFP-GFAP-tk derived colonies for transplantation. A.

YFP-GFAP-tk (YFP-TK) mice generate YFP positive LIF colonies in the presence of

GCV similar to non-transgenic littermate controls (YFP-NT) grown in GCV. B. The

frequency of YFP-TK derived LIF colonies in the presence of GCV is not different from

YFP-NT littermate controls; however, YFP-TK LIF colonies were smaller in size (n=3

mice/genotype). C. Only YFP-NT derived LIF colonies grown in GCV passage into EFH

conditions indicating that the LIF colonies YFP-TK derived LIF colonies do not contain

GFAP+ neurosphere forming cells (n=3 animals per genotype). D. Representative images

of cells (i) migrating, (ii-iii) differentiating, and (iv) expressing the neuroblast marker

DCX at 14 days post-transplant. Data represent mean + SEM. Scalebars = 20 µm.

 

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Chapter 3

Quiescent primitive neural stem cells repopulate the ablated definitive neural stem cell population in the

adult mouse brain

This chapter has been submitted for publication:

Leeder R, Yammine S, Xu W, Sachewsky N, Morshead C, van der Kooy D. Quiescent

primitive neural stem cells repopulate the ablated definitive neural stem cell population in

the adult mouse brain.

 

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Abstract

Adult primitive neural stem cells (pNSCs) are rare, LIF-dependent, GFAP–, Oct4+

cells that reside in the mouse forebrain subependyma. Label retention experiments in

Histone2B (H2B)-GFP mice indicated that adult pNSCs are predominantly quiescent,

dividing approximately once every 3-5 months. Adult pNSCs express the pluripotency

gene Oct4 and Oct4fl/fl;Sox1Cre/Cre (Oct4CKO) mice did not generate pNSC-derived

neurospheres, providing a pNSC loss of function mouse model. Four weeks after GFAP+

definitive (d)NSC ablation, Oct4CKO mice had significantly fewer dNSC-derived

neurospheres, proliferating cells and Sox2+GFAP+ cells in the periventricular region,

suggesting that pNSCs are necessary for dNSC repopulation. In addition, pNSCs exit

quiescence upon ablation of their downstream progeny, as an antimitotic agent infused

into H2B-GFP mice to ablate downstream progeny caused quiescent pNSCs to proliferate

and dilute their label. In conclusion, pNSCs are primarily quiescent and upstream of

dNSCs, but proliferate following downstream lineage ablation to repopulate the adult

neural lineage.

 

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Introduction

NSCs reside within the periventricular region of the adult mouse brain. dNSCs are

a population of GFAP+, multipotent cells that self-renew and also referred to as type B

cells (Doetsch et al., 1999; Morshead et al., 1994; Morshead et al., 2003). In the adult

mouse brain, GFAP+ dNSCs divide to produce transit amplifying cells that give rise to

neuroblasts, which migrate along the rostral migratory stream to the olfactory bulb where

they differentiate into interneurons (Doetsch et al., 1999). Adult dNSCs are epidermal

growth factor (EGF)- and fibroblast growth factor (FGF)-dependent and proliferate to

form clonal neurospheres in vitro (Reynolds and Weiss, 1992). This population of dNSCs

can be specifically ablated by administration of ganciclovir (GCV) to mice that express

the herpes simplex virus thymidine kinase under the GFAP promoter (GFAP-tk) (Bush et

al., 1998). Infusion of GCV into the lateral ventricle killed proliferating dNSCs and

depleted dNSC-derived neurospheres (Morshead et al., 2003), however the dNSC

population returned over time upon removal of GCV (Sachewsky et al., 2014).

We postulated that dNSC repopulation occurs due to the presence of an additional

NSC type upstream of dNSCs. In the developing mouse embryo, primitive (p)NSCs can

be isolated from the mouse embryo as early as embryonic day (E)5.5, in advance of

dNSCs that are first present at E7.5 (Hitoshi et al., 2004). Interestingly, pNSC-derived

neurospheres can be passaged into dNSC-derived neurospheres, but the reverse is not true

(Sachewsky et al., 2014), suggesting that pNSCs lie upstream of dNSCs in the NSC

lineage. We recently reported that pNSCs persist into the adult brain as a rare population

of GFAP– adult pNSCs (Sachewsky et al., 2014), and postulated that this distinct cell

 

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population is upstream dNSCs in vivo and thus responsible for the repopulation of ablated

dNSCs in the adult brain.

pNSCs generate clonal pNSC-derived neurospheres in the presence of leukemia

inhibitory factor (LIF) in vitro. Unlike dNSCs, they express low levels of Oct4 based on

qPCR, transgene expression, flow cytometry, immunostaining, drug selection, and

morula aggregation experiments (Sachewsky et al., 2014). In addition, cell surface

markers have been identified to distinguish pNSCs from dNSCs (DeVeale et al., 2014).

pNSCs can be passaged to self-renew or into dNSC culture conditions to produce dNSC-

derived neurospheres, which then express GFAP and are killed if derived from GFAP-tk

mice and exposed to GCV (Sachewsky et al., 2014). As pNSCs are rare in the adult

mouse brain and generate small pNSC-derived neurospheres, we hypothesized that they

may also be a predominantly quiescent population. In many stem cell lineages,

quiescence is hypothesized to be a mechanism of preventing stem cell exhaustion and/or

protecting against replication-related mutations (reviewed (Wang and Dick, 2005; Orford

and Scadden, 2008)). This is well characterized in the rare, long-term repopulating

hematopoietic stem cells (HSCs), which reside at the top of the HSC hierarchy (Foudi et

al., 2009; Wilson et al., 2008; Anjos-Afonso et al., 2013).

To investigate differences between the pNSC and dNSC populations, we used

doxycycline (DOX)-inducible H2B-GFP mice to assess the cell cycle times in these two

cell populations. We identified label-retaining cells in the subependyma as pNSCs, and

label-retaining pNSC-derived neurospheres could still be obtained after a one-year chase.

Therefore, pNSCs persist as a quiescent cell population in the adult mouse brain. pNSCs

express Oct4 and we assayed mice with a conditional knockout of Oct4 (Oct4fl/-

 

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fl;Sox1Cre/Cre mice, hereafter referred to as Oct4CKO mice) and observed that pNSCs

seemed dysfunctional as they did not give rise to pNSC-derived neurospheres. We

therefore used Oct4CKO mice as a loss of pNSC function mouse model to ask whether

pNSCs are responsible for the repopulation of ablated dNSCs in vivo. Oct4CKO mice

exhibited significantly reduced repopulation of dNSC-derived neurospheres following

antimitotic treatment as compared to control mice. Furthermore, after 4 weeks of

recovery from antimitotic treatment, Oct4CKO mice had significantly fewer proliferating

(EdU+) and Sox2+GFAP+ cells in the periventricular region. This suggests that pNSCs

are upstream in the neural lineage, and that Oct4 is required for pNSCs to repopulate

dNSCs. Finally, after administration of an antimitotic drug to H2B-GFP mice, pNSCs

diluted their labeled histones, thus confirming they are activated to proliferate in response

to ablation of their progeny. We conclude that pNSCs are a quiescent population

upstream of dNSCs that become activated following dNSC ablation to repopulate dNSCs

and the downstream neural lineage.

Methods

Mouse strains. H2B-GFP mice were a kind gift from Dr. K. Hochedlinger (Foudi et

al., 2009). Oct4fl mice were a kind gift from Dr. A. Tomlin (Kehler et al., 2004). Sox1Cre

mice were a kind gift from Dr. S. Nishikawa (Takashima et al., 2007). GFAP-tk mice

were a kind gift from Dr. M. Sofroniew (Bush et al., 1998). C57BL/6 mice were

purchased from Charles River. All mice were maintained in the Department of

Comparative Medicine at the University of Toronto and in accordance with the Guide to

the Care and Use of Experimental Animals and approved by the Animal Care Committee.

 

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Label retention analysis. H2B-GFP received doxycycline (DOX) in the drinking

water (2mg/ml in 1% sucrose) for 6 weeks starting at 6-10 weeks of age, followed by

various chase periods without DOX exposure (Foudi et al., 2009).

Ablation paradigms. Oct4CKO and Oct4CKO-tk mice were anaesthetized with 3-5%

isoflurane and injected with Ketoprofen (3 mg/kg). A cannula was implanted into the

lateral ventricle (+0.2 mm anterior, +0.7 mm lateral, depth of 2.5 mm below the skull,

relative to bregma) and connected to a mini osmotic pump placed subcutaneously on the

back (Alzet 1007D, Direct Corp.). Oct4CKO received a 14-day infusion of 4% AraC

(Sigma) at 0.25 µl/hr. Oct4CKO-tk mice received a 7-day infusion of 2% AraC at 0.5 µl/hr

followed immediately by a 3-day infusion of 200µM GCV at 1 µl/hr (Sigma). H2B-GFP

mice that received AraC were allowed 3 days to recover after DOX exposure and then

infused for 7 days with 2% AraC at 0.5 µl/hr.

Proliferation analysis. EdU (Life Technologies A10044) was added to the drinking

water (0.2 mg/ml in 1% sucrose) for 7 days during the last week of recovery after AraC

and GCV infusion in Oct4CKO-tk and Oct4Ctl-tk mice.

Primary dissections and neurosphere cultures. The neurosphere assay was

performed as previously described (Chiasson et al., 1999). Cells were plated at clonal

density of 10 cells/µl (Coles-Takabe et al., 2008) in 24 well culture plates (Nunclon).

pNSC-derived neurospheres were plated in serum free media (SFM) supplemented with

 

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leukemia inhibitory factor (LIF, 10 ng/ml). dNSC-derived neurospheres were grown in

SFM supplemented with epidermal growth factor (EGF, 20 ng/mL; Sigma), basic

fibroblast growth factor (FGF, 10 ng/mL; Sigma) and heparin (2 µg/mL; Sigma).

Neurospheres were counted after 7-10 days in vitro. Primitive neurospheres are 50 µm or

larger in diameter and definitive neurospheres are 100 µm or larger in diameter.

Immunohistochemistry. Mice were sacrificed with sodium pentobarbital and

perfused transcardially with cold PBS followed by 4% PFA. Brains were post-fixed

overnight at 4oC then cryoprotected in 30% sucrose. Coronal sections (14 µm) were

prepared on a cryostat (-16oC). Tissue was permeabilized with 0.5% TritonX and blocked

in 10% NGS. Primary antibodies, chicken anti-GFP (Aves Lab #GFP-1020, 1:500),

rabbit anti-Sox2 (Abcam ab97959, 1:1000), and mouse anti-GFAP (Millipore MAB3402,

1:500), were incubated overnight at 4oC. Secondary antibodies, 488 goat anti-chicken

(Alexa, A11039, 1:400), 568 goat anti-rabbit (Alexa A11036, 1:400), and 647 goat anti-

mouse (Alexa A21236, 1:400), were incubated for 30 min at room temperature. EdU was

detected with Click-iT EdU Alexa Fluor 488 Imaging Kit (Life Technologies C10337).

Nuclei were counterstained with Hoescht. Staining was imaged on an Olympus Fluoview

FV1000 confocal laser scanning microscope.

Statistics. Data are represented as means ± SEM unless otherwise stated. Statistical

analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla,

CA) and Microsoft Excel. ANOVA with Bonferroni’s multiple comparison tests and

Student’s t-test were performed with a significance level of 0.05.

 

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Results

pNSCs are a quiescent stem cell population in the adult mouse brain

To compare the label retention and thus cell cycles of pNSCs and dNSCs, H2B-

GFP mice received DOX for 6 weeks to label periventricular cells. After DOX removal

cells diluted the GFP label as they proliferate (Fig. 1A), thus leaving quiescent cells

labeled over long chase periods (Foudi et al., 2009). Immediately following DOX

exposure (time 0), cells lining the lateral ventricle were labeled (Fig. 1B), as were pNSC-

derived and dNSC-derived neurosphere. Clonal neurospheres with any GFP expression

(including mottled expression) were considered labeled since we were interested in the

initial neurosphere-forming cell and the H2B-GFP label dilutes as the cells proliferate in

culture to form neurospheres. Mottled GFP staining in clonal neurospheres suggests

different cell cycle times of different precursor cells within the neurospheres. For these

experiments, we assumed that: 1) pNSCs and dNSCs were labeled equally in vivo. 2) All

cells dilute the label by half with every cell division. 3) There is not asymmetric

segregation of histones perhaps contrary to the immortal strand hypothesis. 4) Labeled

pNSCs and dNSCs dilute the label equally in vitro and form neurospheres where even

single GFP+ cells can be visualized.

Immediately after 6 weeks of DOX exposure 44 + 4% of dNSCs and 41 + 9% of

pNSCs were labeled (Fig 1C). The equal proportion of initially labeled pNSC- and

dNSC-derived neurospheres supports the unbiased, replication-independent labeling in

the H2B-GFP mouse model. In addition, we suggest that the equal percentages of pNSC-

and dNSC-derived neurospheres initially labeled after DOX exposure indicates that the in

vitro cell divisions and differences in neurosphere size do not affect our ability to detect

 

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GFP+ cells within the sphere. The H2B-GFP model indicated that dNSCs quickly diluted

out their label while pNSCs retained their label over longer chase periods (Fig. 1C).

dNSCs significantly diluted their label from 44% labeled at baseline to 17 + 2%, 2 + 1%

and 0.2 + 0.1% after a 1-, 4- and 12-month chase, respectively. pNSCs did not reduce

their label during the first month of chase, then decreased to 33 + 3% and 9 + 3% labeling

after a 4- and 12-month chase, respectively (Fig. 1C).

To estimate pNSC and dNSC cell cycle times, we expressed the percentages of

labeled neurospheres as a percentage of spheres initially labeled. For example after 1

year, 9% of the total population of pNSC-derived neurospheres was GFP+, which is

approximately 22% of the population of pNSC-derived neurospheres that was initially

labeled immediately after DOX exposure (Fig. 1D). Since at 12 months post-DOX

exposure, the percentage of labeled dNSC-derived neurospheres was 0.2 + 0.1% (1 GFP+

neurosphere from 1 of 14 mice analyzed) and not significantly different from 0%, we

presumed that the dilution of label happened well before 12 months in dNSCs. If we

exclude this dNSC 12-month time point from, this increased the R2 value of the line of

best fit from 0.95 to 0.99. The GFP dilution was plotted on a logarithmic scale to confirm

that the label diluted exponentially, further demonstrating the label retention in pNSCs

(Fig. 1D). From the lines of best fit on the dilution graph (R2 value of 0.94 for pNSCs),

we set the y intercept to 50% to calculate the amount of time (x) required to dilute the

H2B-GFP label by half. Assuming that we can calculate the time required to lose half the

GFP label in vivo from our neurosphere assays, we calculated the cell cycle of dNSCs to

be 24 days (0.8 months) and pNSCs to be 5.1 months. More generally, this suggests that

 

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Figure 1

 

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Figure 1. Label retention in pNSCs and dNSCs in H2B-GFP mice. A. DOX was

delivered in the drinking water for 6 weeks followed by 1-, 4-, and 12-month chase

periods. B. DOX-induced GFP labeling in cells surrounding the lateral ventricle of H2B-

GFP mice (scale bar = 50 µm). C. During the chase periods, dNSCs divided more

frequently and diluted their GFP label while pNSCs retained labeling longer, expressed

as a percentage of total neurospheres isolated (2-way ANOVA F(3,54)=3.8, p=0.001,

n=4-8 mice per timepoint). D. GFP-labeled neurospheres expressed as a percentage of the

initial neurospheres labeled immediately after DOX exposure.

 

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pNSCs dilute their label 6 times slower than dNSCs, which can be used to make a

separate, more conservative cell cycle time estimate that is less dependent on the

neurosphere assay as an accurate readout of in vivo cell divisions. Given the 6 fold

difference in cell cycle times and previous calculations that the cell cycle time of dNSCs

in vivo is about 15 days (Morshead et al., 1998), we can perform a more conservative

estimate that pNSCs divide once every 3 months. Based on these two methods of

analyses, we conclude that the cell cycle time of pNSCs is 3-5 months.

pNSCs are depleted in mice lacking Oct4 expression in Sox1+ cells

As Oct4 expression is unique to pNSCs, we sought to determine whether loss of

Oct4 expression affects pNSCs using Sox1-driven Cre expression to induce a conditional

loss of Oct4 in all neural cells (Fig. 2A). Oct4CKO mice appeared phenotypically normal.

pNSC-derived neurospheres were absent from Oct4CKO, while Oct4wt/fl;Sox1cre (Oct4Ctl)

mice generated a normal abundance of pNSC-derived neurospheres not different from

wild type controls (Fig. 2B). This suggests that Oct4 is required by pNSCs, at least for

neurosphere formation. In contrast Oct4CKO mice gave rise to a normal abundance of

dNSC-derived neurospheres (Fig. 2C). dNSCs appeared unaffected in Oct4CKO mice,

possibly because Oct4 is not excised until after Sox1 expression begins at E7.5 (Wood

and Episkopou, 1999), therefore, pNSCs and dNSCs are already established in the brain

before Oct4 protein is removed. The absence of pNSC-derived neurospheres from

Oct4CKO suggests a requirement for Oct4 expression in pNSCs to form pNSC-derived

neurospheres, though it is unclear whether it is required for pNSC survival, proliferation,

or cell identity.

 

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pNSCs are required for dNSC-derived neurosphere repopulation after dNSC

ablation

To determine whether the loss of pNSC-derived neurospheres in Oct4CKO mice

resulted from a loss of pNSC function in vivo and whether pNSCs are required for

repopulation of dNSCs, we ablated dNSC and neural progenitor populations to determine

whether dNSCs recover in Oct4CKO mice. AraC, an antimitotic drug, was infused directly

into the lateral ventricle of Oct4CKO and Oct4Ctl mice continuously for two weeks to

ablate all proliferating cells, including neurosphere-initiating dNSCs (Fig. 2D). After

AraC treatment, the ability of pNSC loss of function (Oct4CKO) mice to repopulate dNSCs

after a 2- and 4-week recovery period was assessed using a neurosphere assay. The

dNSC-derived neurosphere repopulation was reduced significantly after 4 weeks of

recovery in Oct4CKO mice vs. Oct4Ctl control mice, which recovered to 24 + 5% and 91 +

8% of naïve baseline, respectively (Fig. 2D). Therefore, Oct4 expression is required for

pNSC ability to repopulate ablated dNSCs.

AraC infusion did not induce a complete loss of dNSCs, and since even a few

remaining dNSCs could be capable of proliferating to recover the dNSC population

(Morshead et al., 1994), we sought a better ablation protocol. We crossed the Oct4CKO

strain to GFAP-tk mice to take advantage of a more extensive ablation protocol using

GCV (Sachewsky et al., 2014). We infused AraC for 7 days to ablate all dividing

progenitors cells and push dNSCs into cycle to increase the efficiency of the subsequent

3-day GCV infusion (Fig. 2E). After initial ablation, dNSC-derived neurospheres were

completely absent after a 2-week recovery in Oct4CKO;tk mice, and had returned to just

 

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Figure 2

 

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Figure 2. Oct4fl/fl;Sox1Cre/Cre (Oct4CKO) mice are a pNSC loss of function model and

had significantly reduced ability to repopulate dNSCs. A. Mice are homozygous for

Cre recombinase insertion to the Sox1 locus, and the Oct4fl allele that has exon 1 flanked

by loxP sites. i. Constitutive Cre expressed, drive by the Sox1 promoter, drives excision

of the critical exon of Oct4, causing the condition knockout (ii). B. dNSC-derived

neurospheres were not affected by loss of Oct4 expression. C. pNSC-derived

neurospheres were absent from Oct4CKO, while Oct4Ctl are similar to wildtype control

mice. N=8. D. Oct4CKOand Oct4Ctl mice received a 2-week AraC infusion to ablate

dNSCs and progenitors. Oct4CKO had significantly reduced repopulation of dNSC-derived

neurospheres compared to Oct4Ctl (2-way ANOVA F(2,18)=12.45, p=0.001, N=4). E.

Ablation in Oct4CKO-tk mice with 7 days of AraC infusion followed by 3 days of GCV

infusion induced a further reduction in the dNSC and progenitor population. Oct4CKO-tk

did not exhibit any dNSC repopulation after 2-week recovery and significantly reduced

repopulation after 4-week recovery compared to Oct4Ctl (2-way ANOVA F(2,13)=11.42,

p=0.001, N=3).

 

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2.7 + 1.8% of untreated control neurosphere levels after a 4-week recovery, a significant

reduction compared to Oct4Ctl;tk mice (Fig. 2E). Strikingly, half of the Oct4CKO mice who

received this treatment had 0 dNSC-derived neurospheres 4 weeks post-ablation, and the

other half only gave rise to rare dNSC neurospheres (average of 5 neurospheres per 40

000 cells, a 2.5% recovery). This same effect was seen when we allowed mice to recover

from ablation for 8 weeks, suggesting this lack of repopulation without functioning

pNSCs is permanent and that Oct4+ pNSCs are required for dNSC repopulation

Fewer EdU+ and Sox2+GFAP+ cells are present in Oct4CKO;tk mice after ablation

After AraC/GCV ablation in Oct4CKO;tk mice, EdU was administered in the

drinking water during the last week (days 21-28) of recovery to label proliferating cells

(Fig. 3A). Non-ablated wildtype mice, Oct4Ctl;tk mice and Oct4CKO;tk mice received EdU

to compare the amounts of proliferation in the subependyma. EdU, Sox2, and GFAP were

quantified in the periventricular region after a 4-week recovery from AraC/GCV ablation.

After 4-week recovery, Oct4CKO;tk mice had significantly fewer EdU+ cells in the

subependyma of the forebrain as compared to Oct4Ctl;tk and untreated controls (Fig. 3B,

C). Quantification of Sox2+GFAP+ cells revealed significantly fewer double positive cells

in proximity to the lateral ventricle in Oct4CKO;tk mice compared to Oct4Ctl;tk mice and

untreated controls (Fig. 3D, E). The number of Sox2+GFAP+ cells includes the dNSC

population, and is consistent with the reduced repopulation of dNSC-derived

neurospheres in Oct4CKO;tk following AraC/GCV treatment (see Fig. 2E, 3E).

Sox2+GFAP+ cells that were also EdU+ were quantified to identify proliferating

dNSCs. A greater proportion of Sox2+GFAP+ cells that were present were EdU+ in

 

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Oct4CKO;tk mice compared to Oct4Ctl;tk (Fig. 3F). There were fewer EdU+ proliferating

cells and fewer Sox2+GFAP+ cells, but of the Sox2+GFAP+ cells that were present a

higher percentage were proliferating compared to Oct4Ctl;tk. This increased proliferation

within the reduced Sox2+GFAP+ population suggests that in the absence of functioning

pNSCs, non-dNSC Sox2+GFAP+ precursors may undergo more proliferation to attempt to

repopulate the neural lineage. Despite attempts through increased proliferation of the few

remaining Sox2+GFAP+ cells, the periventricular region of Oct4CKO;tk pNSC loss of

function mice are unable to repopulate the ablated dNSCs.

pNSCs become activated and proliferate after antimitotic ablation

To confirm that pNSCs leave the quiescent state and begin to proliferate

following downstream neural lineage ablation, we returned to the H2B-GFP mouse

model. Cells were labeled with DOX exposure; 3 days later AraC was infused into the

lateral ventricles for 7 days, then mice recovered for 21 days (Fig. 4A). In this paradigm,

cells activated to proliferate following AraC infusion will dilute their label compared to

control mice that did not receive AraC. While pNSCs usually retain the same amount of

labeling after 1 month, mice that received AraC gave rise to significantly fewer labeled

pNSC-derived neurospheres than control mice that did not receive AraC (Fig. 4B). This

indicates that pNSCs were induced to proliferate following AraC infusion. In contrast,

dNSC-derived neurospheres were equally labeled from 1-month control mice and 1-

month with AraC mice (Fig. 4B). This does not preclude the proliferation of dNSCs, but

rather may suggest that most of the dNSC repopulation after AraC treatment arose from

labeled pNSC precursors that passed on their labeling in vivo. The dilution of the H2B-

 

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Figure 3

 

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Figure 3. Reduced proliferation and dNSC recovery in the periventricular region of

Oct4fl/fl;Sox1Cre;GFAP-tk mice after ablation. A. Mice received AraC/GCV ablation

followed by a 4-week recovery with EdU delivered in the drinking water during the last

week of recovery (days 21-28). B. C. Oct4fl/fl;Sox1Cre;GFAP-tk mice had fewer EdU+

cells in the periventricular region as compared to Oct4wt/fl;Sox1Cre;GFAP-tk after

recovery (2-way ANOVA F(2,13)=21.31, p=0.0001, N=3). D. Visualization of

GFAP+Sox2+ cells and EdU+ coexpression. Regular arrow indicates EdU+Sox2+GFAP+,

short arrow indicates EdU+Sox2+GFAP–, arrowhead indicates EdU+Sox2–GFAP+. E.

Fewer GFAP+Sox2+ cells were observed in Oct4fl/fl;Sox1Cre;GFAP-tk mice as compared

to Oct4wt/fl;Sox1Cre;GFAP-tk and wildtype control (2-way ANOVA F(2,11)=7.40,

p=0.01, N=3). F. An increased proportion of Sox2+ GFAP+ cells were EdU+ in Oct4fl/-

fl;Sox1Cre;GFAP-tk compared to Oct4wt/fl;Sox1Cre;GFAP-tk suggesting that dNSCs

proliferate to contribute to dNSC repopulation (2-way ANOVA F(2,12)=5.17, p=0.02,

N=3).

 

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Figue 4

 

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Figure 4. pNSCs are activated to proliferate following AraC infusion. A. H2B-GFP

mice received DOX in the drinking water for 6 weeks then allowed to recovery for 3

days. AraC was infused into the brains for 7 days to ablate proliferating cells, followed

by a 21-day recovery. B. pNSCs do not dilute their label within 1 month without ablation,

but after exposure to AraC significantly diluted their label, indicating proliferation (2-

way ANOVA F(2,12)=36.22, p=0.001, N=3). dNSCs did not show reduced labeling after

AraC treatment compared to control 1 month chase.

 

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GFP label in pNSCs but not dNSCs after AraC treatment supports a lineage model where

pNSCs are upstream of dNSCs and proliferate to repopulate dNSCs after dNSC ablation.

Discussion

pNSCs are a rare stem cell population in the adult mouse brain, which is quiescent

but proliferates to repopulate the ablated dNSC population. Oct4CKO mice served as a

pNSC loss of function mouse model and were utilized to test the repopulation of dNSCs

after ablation of dNSCs and downstream progenitors. Oct4CKO;tk mice exhibited a

significantly reduced dNSC-derived neurosphere repopulation, and significantly fewer

EdU+ cells and Sox2+GFAP+ cells (comprising dNSCs). We suggest that the small

amount of repopulation of dNSCs and their progeny observed at 4 weeks post-ablation in

the pNSC loss of function mice likely resulted from dNSCs that were not ablated in our

AraC/GCV paradigm. This idea is supported by the observation that some Oct4CKO;tk

mice had zero and some had a few dNSC-derived neurospheres after 4 and 8 weeks of

recovery, with zero dNSCs presumably being the mice with complete ablations. In

addition, the increased Sox2+GFAP+EdU+ cells in Oct4CKO;tk compared to Oct4Ctl;tk

suggests that other Sox2+GFAP+ precursors are proliferating and may be compensating to

repopulate the progenitor pool in the absence of functioning pNSCs. In further support of

the involvement of wild type pNSCs to repopulate dNSCs, pNSCs were observed to

proliferate after AraC infusion in a H2B-GFP mouse model. Together, these data suggest

that pNSCs become activated after antimitotic treatment, and exit quiescence to

proliferate to repopulate the downstream neural lineage. Therefore, these data indicate

that pNSCs fit into a stem cell hierarchy that is similar to those reported in other lineages

 

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where rare, quiescent cells reside at the top of the lineage and act as a reserve stem cell

pool (Wang and Dick, 2005; Orford and Scadden, 2008).

Recovery of ablated dNSCs and downstream progenitors after AraC or AraC/GCV

in Oct4CKO;tk mice supports a NSC hierarchy whereby pNSCs give rise to dNSCs

(Sachewsky et al., 2014). It appears that dNSCs are capable of maintaining their own

population in the absence of ablation, since otherwise untreated Oct4CKO did not give rise

to any pNSC-derived neurospheres, but generated a normal abundance of dNSC-derived

neurospheres. Despite the ability to maintain their own population at baseline, dNSCs

require pNSCs for dNSC repopulation following dNSC ablation. After AraC/GCV

ablation in control mice, the best recovery of dNSC-derived neurospheres was

approximately 70% of the naïve baseline number of dNSC clonal neurospheres. This is in

agreement with past reports that dNSCs only recover to about 75% of baseline levels

after two consecutive ablations (Morshead et al., 1994; Sachewsky et al., 2014), and may

reflect an insufficient recovery time or a limited ability of adult dNSCs to recover, as

reported in the blood stem cell lineage (Harrison et al., 1978). Alternatively, a negative

feedback signal may exist whereby downstream postmitotic progeny, which are more

abundant in the adult niche, signal back and limit adult dNSC repopulation.

Long-term label retention indicated that pNSCs are quiescent in the adult mouse

brain and we estimated that they divide approximately once every 3-5 months, which

corresponds to 5-8 divisions in the lifetime of a mouse. This is similar to label-retaining

primitive HSCs (also referred to as long-term or dormant HSCs) that divide once every

4.8 months (Wilson et al., 2008; Foudi et al., 2009). These primitive HSCs are normally

quiescent, but become activated following exposure to 5-fluorouracil (5-FU), an

 

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antimitotic drug, to repopulate the downstream hematopoietic lineage (Wilson et al.,

2008). Primitive HSCs also are very rare but have the greatest repopulation ability

amongst blood stem cell types after transplantation (Wilson et al., 2007). In addition to

the commonalities of long cell cycle time and rarity between these two primitive cell

populations, primitive HSCs are negative for the common HSC marker CD34 (Anjos-

Afonso et al., 2013; Wilson et al., 2007). This may be considered similar to pNSCs being

negative for the traditional NSC marker, GFAP, and could reflect a mechanism whereby

lineage-related brain stem cells have different characteristics to limit their sensitivity to

similar stresses and maintain a reserve population. Therefore pNSCs fit into a stem cell

hierarchy where rare, quiescent cells reside at the top of the adult stem cell hierarchy

serving as a reserve pool of stem cells to repopulate the downstream lineage.

pNSCs express Oct4 in the adult mouse brain (Sachewsky et al., 2014), and the

present report demonstrates that Oct4 is necessary for pNSC function. Another study

reported the absence of Oct4 expression in the mouse brain and that conditional

inactivation of the Oct4 alleles did not result in any phenotypic abnormalities or changes

in subependymal Ki67+ labeling (Lengner et al., 2007). In contrast, we previously

reported Oct4 expression specifically within the adult subependyma (Sachewsky et al.,

2014) and herein demonstrate that Oct4 expression is crucial to pNSCs and the

repopulation of dNSCs after dNSC ablation. In agreement with Lengner et al., we did not

observe a difference in the abundance of EdU+ cells in the periventricular region of

Oct4CKO;tk untreated mice versus C57BL/6 mice, and only observed a difference in EdU

expression after antimitotic treatment. It remains unclear how the loss of Oct4 results in a

pNSC loss of function mouse model. Oct4 may maintain pNSCs through survival, self-

 

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renewal, or conservation of multipotentiality. Greater understanding of the molecular

biology surrounding Oct4 expression in pNSCs would be valuable to further our

understanding of the role of Oct4 in the adult mouse brain.

We report a population of quiescent pNSCs that are upstream and repopulate

dNSCs in the adult mouse brain. Despite reports of ependymal cells activated after injury

to act as stem cells (Carlén et al., 2009; Magnusson et al., 2014), pNSCs are not

ependymal-derived, based on the subependymal position of Oct4-GFP cells in

periventricular wholemounts and on the findings that pNSC-derived neurospheres self-

renew and are not ciliated (Sachewsky et al., 2014). We performed cell cycle analysis on

pNSCs and dNSCs with the H2B-GFP mouse model, which holds great benefit over

BrdU label retention models, since the H2B model is not biased towards proliferating

cells to take up the initial label. Further, we propose that pNSCs are upstream of the

recently described quiescent (q)NSCs (Codega et al., 2014) and pre-GEPCOT cells

(Codega et al., 2014; Mich et al., 2014), which we suggest may be subtypes of GFAP+

dNSCs or B1 cells. Pre-GEPCOT cells were reported to be upstream of GEPCOT cells,

which are GlastmidEGFRhighPlexinB2highCD24−/lowO4/PSA-NCAM−/lowTer119/ CD45−

(Codega et al., 2014; Mich et al., 2014). pNSCs are the only NSC type that are Oct4+ and

GFAP–; they are the most rare, slowest proliferating and have the lowest rate of

neurosphere formation. Pre-GEPCOT cells are slowly proliferating with low neurosphere

forming abilities and have mixed GFAP expression, and are the most abundant in the

niche (Mich et al., 2014). Quiescent (q)NSCs as defined by Codega et al. are the first cell

population in the lineage to be exclusively GFAP+, maintain a low rate of neurosphere

formation (Codega et al., 2014)). Therefore, pNSCs, Pre-GEPCOTS and qNSCs are all

 

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characterized by low neurosphere forming ability, although it remains possible that only a

subpopulation of cells positive for the classifying cell markers function as stem cells.

Downstream, activated NSCs are more mitotically active, have EGF-R expression, and

mixed mid/high GFAP expression (Codega et al., 2014). It remains unclear which of

these populations are independent or overlapping cell populations, and whether some cell

types may be different states of the same cell population. We suggest that pNSCs reside

at the top of the NSC lineage and future studies, including lineage tracing under baseline

conditions, would be beneficial to consolidate our report with these other quiescent NSCs

to directly illuminate their lineage relationship.

 

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Chapter 4

Targeted activation of primitive neural stem cells in the mouse brain

This chapter has been submitted for publication:

Leeder R, DeVeale B, van der Kooy D. Targeted activation of primitive neural stem cells

in the mouse brain.

 

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Abstract

Primitive neural stem cells (pNSCs) are the earliest appearing members of the

NSC hierarchy. NSCs generate all cells in the neural lineage and hold great potential if

activated to contribute to brain regeneration. Thus, pNSCs are an ideal population to

target to promote endogenous NSC activation. pNSCs arise early in embryonic

development and persist into the adult mouse brain as rare Leukemia Inhibitory Factor

(LIF)-responsive cells. We hypothesized that pup-derived pNSCs are more abundant but

otherwise comparable to adult-derived pNSCs, and can be used to identify selective

markers and activators of endogenous pNSCs. We tested the self-renewal ability,

differentiation capacity and gene expression profile of pup-derived pNSCs and found it to

be comparable to adult-derived pNSCs. Next, we used pup pNSCs to test

pharmacological methods that could be used to develop novel strategies of promoting

endogenous brain repair. We modulated cell surface proteins that were enriched on in

vitro ESC-derived primitive neurospheres, C-Kit and ErbB2, to activate pNSCs. C-Kit

and ErbB2 inhibition selectively affected pNSCs and not dNSCs in vitro, and when

infused directly into the adult brain in vivo. Targeting pNSCs with C-Kit and ErbB2

modulation provides a valuable strategy to activate the earliest cell in the neural lineage

to contribute to endogenous brain regeneration.

 

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Introduction

Identifying cell-type specific markers is a key goal in NSC biology. The lack of

selective cell markers currently limits efforts to isolate pure cell populations or test

selective pharmacological interventions (Obermair et al., 2010; Miller and Kaplan, 2012),

a problem that is magnified in rare cell populations. pNSCs are LIF-dependent, express

Oct4 and arise at day 5.5 of embryonic development in advance of FGF-dependent

dNSCs that arise at embryonic day 7.5 (Hitoshi et al, 2004; Akamatsu et al., 2009). Adult

pNSCs are rare Oct4+ cells in the adult forebrain periventricular region that arise first in

the NSC lineage and give rise to dNSCs and downstream neural and glial progenitors

(Sachewsky et al., 2014). pNSCs produce clonal LIF-dependent neurospheres in culture

that can be passaged to self-renew or give rise to dNSCs (also termed type B cells) that

are GFAP+ and EGF/FGF-dependent (Doetsch et al., 1999; Morshead et al., 2003).

Despite the presence of NSCs, the brain exhibits limited endogenous repair to

counteract neurodegenerative disease or heal after injury. Pharmacological compounds to

activate endogenous NSCs and guide their differentiation could improve brain

recovery/repair (Miller and Kaplan, 2012). Therapeutic benefit might be achieved by

activating pNSCs to lead to large expansion of dNSCs and their progeny from the top of

the neural lineage, which could then be guided to differentiate into needed cell types. We

identified pNSC-specific cell surface markers and tested candidate pNSC-mediators on

the relatively abundant pup population to identify activators of endogenous adult pNSCs.

A previous mass spectrometry-based screen identified cell surface proteins

expressed on embryonic stem cell (ESC)-derived pNSCs (DeVeale et al., 2014). In vitro,

pNSCs arise from ESCs passaged into serum free culture conditions containing LIF and

 

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proliferate to generate clonal primitive neurospheres (Tropepe et al., 2001). C-Kit and

ErbB2 were upregulated on pNSCs relative to upstream ESCs and downstream dNSCs

(DeVeale et al., 2014). C-kit is expressed in the olfactory bulbs, hippocampus and cortex

in the adult mouse brain and on neural progenitors in culture (Motro et al., 1991;

Erlandsson et al., 2004). C-kit is a tyrosine kinase receptor that is activated by Steel, mast

cell growth factor and stem cell factor (SCF), and activates PI3-kinase, STAT, and

RAS/MAPK pathways (Motro et al., 1991; Keshet et al., 1991; Blom et al., 2008). ErbB2

is an EGF-R family member that is expressed in the periventricular region and cortex of

the adult mouse brain (Fox et al., 2005). ErbB2 receptors are activated when they cross-

phosphorylate each other and activate downstream tyrosine kinases (Fox et al., 2005).

The ErbB2 receptor preferentially targets Ras/MAPK and to a lesser extent PI3-kinase

(Yarden and Sliwkowski, 2001). Once validated in vivo, these cell surface proteins will

provide novel candidates for pNSC-specific therapies.

To validate C-Kit and ErbB2 in vivo, we first characterized postnatal day 7 (P7)

pup-derived pNSCs to test their equivalence to adult-derived pNSCs. Pup-derived pNSCs

were comparable to adult pNSCs in terms of self-renewal, differentiation potential, and

gene expression profile. Cell sorting demonstrated that pup pNSCs arose from an Oct4+,

GFAP–, nestinmid population. Next, we tested candidate molecules (DeVeale et al., 2014)

to identify those that affected the ability of P7 pup-derived pNSCs, but not dNSCs, to

form clonal LIF neurospheres in culture. Both C-Kit and ErbB2 inhibitors selectively

increased pNSC-derived neurosphere formation. We confirmed the specificity of their

activation using siRNAs. Finally, we delivered the pharmacological inhibitors directly

into the lateral ventricle of adult mice to confirm their effectiveness at expanding the

 

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pNSC population in vivo. These in vitro to in vivo experiments suggest that markers and

inhibitors validated in the pup population have consistent effects on endogenous pNSCs

at the top of the adult NSC lineage. These cell surface molecules enable selective

targeting and activation of pNSCs in the adult brain.

Methods

Mouse strains. Oct4-GFP mice were a kind gift from Dr. A. Nagy (Viswanathan

et al., 2003). Nestin-GFP mice were a kind gift from Dr. G. Enikolopov (Mignone et al.,

2004). GFAP-GFP mice were purchased from Jackson (#010835). CD1 mice were

purchased from Charles River. All mice were maintained in the Department of

Comparative Medicine University of Toronto in accordance with the Guide to the Care

and Use of Experimental Animals and approved by the Animal Care Committee.

Primary dissections and neurosphere cultures. The neurosphere assay was

performed as previously described (Chiasson et al., 1999). Mice were sacrificed by

cervical dislocation, brains were removed and lateral ventricles walls were dissected.

Cells were plated at clonal density of 10 cells/µl (Coles-Takabe et al., 2008) in 24 well

culture plates (Nunclon). Primitive neurospheres were plated in serum free media (SFM)

(Tropepe et al., 1999), supplemented with leukemia inhibitory factor (LIF, 10 ng/ml).

Definitive neurospheres were grown in SFM supplemented with epidermal growth factor

(EGF, 20 ng/mL; Sigma), basic fibroblast growth factor (FGF, 10 ng/mL; Sigma) and

heparin (2 µg/mL; Sigma). Neurospheres were counted after 7-10 days in vitro. Primitive

neurospheres are > 50 µm and definitive neurospheres are > 100 µm in diameter.

 

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Pharmacological inhibitors dissolved in DMSO and siRNAs were added to the

cell culture media at the time of plating. Gleevec (a form of Imatinib) is a C-Kit inhibitor

(Toronto Research Chemicals, G407000), stem cell factor (SCF) a C-Kit ligand (R&D

Systems, 455-MC) and an ErbB2 inhibitor (VWR, CA95061-882) were used at varying

doses. Smart pool siRNAs included C-Kit siRNA (Dharmacon, ON-TARGETplus Kit

siRNA, LU-042174-00), ErbB2 siRNA (Dharmacon, ON-TARGETplus ErbB2, LU-

064147-00) and scramble siRNA (Dharmacon, ON-TARGETplus Non-targeting siRNA

#1, D-001810-01) were transfected using DharmaFECT 1 Transfection Reagent

(Dharmacon).

Neurosphere differentiation. After 7 days in culture, neurospheres were

individually picked and plated onto Matrigel coated plates (4% Matrigel in SFM, Sigma).

Spheres were differentiated for 7 days in the presence of 1% fetal bovine serum (Life

Technologies), and fixed in 4% paraformaldehyde (PFA) (Sigma) at room temperature.

Stem cell self-renewal assay. To test self-renewal, individual neurospheres were

picked, transferred to an Eppendorf tube containing 200 µl SFM with growth factors,

triturated 30-50 times with a pipette tip, and transferred into an additional 300 µL of

media in a 24 well plate. The number of secondary neurospheres per primary

neurosphere was counted after 7-10 days in vitro.

Immunostaining and imaging. Wholemounts were performed as previously

described (Mirzadeh et al., 2008). Briefly, brains were dissected to expose the lateral

 

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ventricle and fixed overnight in 4% PFA at 4oC. Wholemounts were washed,

immunohistochemistry performed, then dissected and the lateral ventricle wall was

placed onto a microscope slide with aquamount and a coverslip.

Fixed cells were stored in St-PBS at 4oC. Cell cultures were permeabilized with

0.3% Triton-X in PBS for 5 minutes at room temperature for internal proteins. Samples

were blocked with 10% NGS (normal goat serum (Jackson Labs)) in St-PBS (with 2%

TritonX for wholemounts) for 1 hour at room temperature. Primary antibodies were

diluted in blocking solution at 4oC overnight for plated cells and 48h for wholemounts.

Secondary antibodies were incubated again in blocking solution for 40 minutes at room

temperature for differentiated spheres and 48 hours at 4oC for wholemounts. Nuclei were

counterstained with Hoescht (1:1000, Sigma).

The primary antibodies were rabbit anti-GFAP (Sigma, G9269, 1:400), mouse

anti-O4 (Millipore, MAB345, 1:200), rabbit anti-GFAP (Sigma, G9269, 1:400), mouse

anti-βIII tubulin (Sigma, T8660, 1:400), chicken anti-GFP (Aves Lab, GFP-1020, 1:500),

rabbit anti-Sox2 (Abcam, ab97959, 1:500), rabbit anti-ß-catenin (Sigma, C2206, 1:500),

rabbit anti-LIF-R (Abcam, ab101228, 1:500). Secondary antibodies included: 488 goat

anti-chicken (Alexa, A11039, 1:400), 488 goat anti-mouse (Alexa, A11029, 1:400), 488

goat anti-rabbit (Alexa, A11034, 1:400), 568 goat anti-mouse (Alexa, A11031, 1:400),

568 goat anti-rabbit (Alexa, A11036, 1:400). Nuclei were stained with Hoescht (Sigma,

33258, 1:1000). Staining was visualized on an AxioVision Zeiss UV microscope and

Nikon 200 microscope or Olympus Fluoview FV1000 confocal laser scanning

microscope.

 

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Quantitative PCR. ESCs, ESC-derived pNSCs, pNSC pup- and adult-derived

colonies, pNSC pup-derived neurospheres, and dNSC pup- and adult-derived

neurospheres were collected in Buffer RLT with β-mercaptoethanol. RNA was extracted

using the RNeasy Micro Kit (Qiagen), including treatment with the RNase-free DNase

Set (Qiagen). cDNA was synthesized with Superscript III First Strand Synthesis System

(Invitrogen). qPCR was performed on a 7900HT Fast Real-Time PCR System (Applied

Biosystems). Cycling conditions were: 2 minutes at 50oC, 10 minutes at 95oC, followed

by 40 cycles of 15 seconds at 95oC, 1 minute at 60oC, 15 seconds at 95oC. Ct values were

normalized to GAPDH and expressed relative to ESCs. The Taqman Probes used were:

GAPDH (Mm03302249_g1), Oct4/Pou5fl (Mm03053917_g1), Sox2 (Mm03053810_s1),

Klf4 (Mm00516104_m1), Nanog (Mm02019550_s1), Mash1/Ascl1 (Mm03058063_m1),

Nestin (Mm00450205_m1), Sox1 (Mm00486299_s1) (Applied Biosystems).

FACS. Cells were isolated from primary dissections of Oct4-GFP, GFAP-GFP,

and nestin-GFP mice for FACS analysis using a FacsAria (BD Biosciences). Cells were

counterstained with propidium iodide (2.5 µg/µl, BD Biosciences) to assess viability and

exclude dead cells. CD1 mice were used as negative control and actin-GFP mice were

used as positive control to set analysis gates.

Intracerebral infusion. CD1 mice were anaesthetized with 3-5% isoflurane and

injected with Ketoprofen (3 mg/kg). A cannula was implanted into the lateral ventricle

(+0.2 mm anterior, +0.8 mm lateral, and depth of 2.5 mm below the skull, relative to

bregma) and connected to a micro-osmotic pump placed subcutaneously on the back

 

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(Alzet 1003D, Direct Corp.). Mice received a 3-day infusion of 400 µM C-Kit Inhibitor

(Toronto Research Chemicals) or 130 µM ErbB2 Inhibitor (VWR) delivered at 1 µl/hr.

Statistics. Data are represented as means ± SEM unless otherwise stated and as

biological replicates. Statistical analyses were performed using GraphPad Prism 5

(GraphPad Software, Inc., La Jolla, CA) and Microsoft Excel. ANOVA with

Bonferroni’s multiple comparison tests and Student’s t-test were performed with a

significance level of 0.05.

Results

Pup-derived pNSCs are comparable to adult-derived pNSCs

Primary dissections of P7 pup brains generated 30-fold more pNSC neurospheres

than adult brain dissections, 1.3 + 0.2 (mean + SEM) adult-derived neurospheres vs. 43 +

3 (mean + SEM) pup-derived neurospheres per 40 000 cells plated. Thus P7 pups were

used to generate pNSCs in this study. To confirm that pup-derived pNSCs are equivalent

to adult-derived pNSCs, we compared morphology, self-renewal, differentiation, and

gene expression between the two populations. Similar to adult-derived pNSCs

(Sachewsky et al., 2014), P7 pup-derived pNSCs formed clonal, free-floating

neurospheres > 50 µm in diameter with clearly defined borders, while P7 pup-derived

dNSCs are > 100µm in diameter (Fig. 1A). Single sphere passaging demonstrated that

pup- and adult-derived clonal pNSC colonies self-renewed at the same frequency, 0.44 +

0.03 (mean + SEM) per pup neurosphere passaged vs. 0.38 + 0.06 (mean + SEM) per

 

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Figure 1

 

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Figure 1. Characterization of pup-derived pNSCs. A. Morphology of a clonal pNSC-

derived neurosphere and dNSC-derived neurosphere from P7 mouse brain, scale bars =

50µm. B. Pup-derived pNSCs differentiate to give rise to ßIII+ neurons, GFAP+

astrocytes and O4+ oligodendrocytes. C. Differentiation profile of pNSC neurospheres

derived both postnatal day 7 pups (n=7-9 neurospheres from 3 independent experiments).

D. Differentiation profile of pup-derived dNSCs neurospheres (n=7-10 neurospheres

from 3 independent experiments). E. Pup- and adult-derived pNSC colonies express

similar levels of stem cell and neural genes. qPCR of pNSC colonies/spheres and dNSC

spheres are expressed relative to ESCs and normalized to GAPDH. Pup-derived pNSCs

express similar levels of the pluripotency factors Oct4, Klf4, and Nanog, equivalent

nestin expression, and increased Mash1 and Sox1 mRNA compared to adult-derived

pNSCs . (2-way ANOVA F(30,84)=13.6 p=0.001 n=3, only select statistical significances

are shown). UD = undetectable

 

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adult neurosphere passaged (Chi-square p=0.52). The self-renewal ability indicates the

same frequency of stem cells in pup- and adult-derived pNSC colonies.

We confirmed that pup-derived pNSCs are multipotent and that clonal

neurospheres gave rise to neurons, astrocytes and oligodendrocytes in culture (Fig.1B).

Similar to adult-derived pNSCs (Sachewsky et al., 2014), pup-derived pNSCs produced

fewer GFAP+ astrocytes than adult- and pup-derived dNSCs and their differentiation

profile was markedly different from pup-derived dNSCs (Fig. 1C, D). Pup-derived

pNSCs and dNSCs gave rise to an increased proportion of oligodendrocytes compared to

adult-derived pNSCs and dNSCs (Sachewsky et al., 2014). This may reflect a

contribution to the wave of oligogenesis that occurs predominantly between postnatal

days 7-21 during mouse brain development (Sauvageot and Stiles, 2002). Pup pNSC-

derived neurospheres gave rise to neurons, astrocytes and oligodendrocytes confirming

that they are a multipotent stem cell population, similar to adult pNSCs.

Next, qPCR for pluripotency and neural genes confirmed that pup-derived pNSCs

had gene expression similar to adult-derived pNSCs. We compared ESC-derived

primitive neurospheres, adult-derived pNSC colonies, and adult-derived and pup-derived

pNSCs colonies and neurospheres. Colonies are adherent and grown on a feeder layer in

the presence of serum whereas neurospheres are free floating in serum free media, both

cultures are only supplemented with LIF. Regardless of age of origin, pNSC-derived

colonies and neurospheres had higher expression of pluripotency genes Oct4, Klf4, and

Nanog, with lower expression of the neural genes Mash1 and nestin compared to dNSC

neurospheres (Fig. 1E). pNSCs had lower Sox2 expression than dNSCs, a gene

implicated in stem cell proliferation (Julian et al., 2013; Surzenko et al., 2013),

 

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suggesting that pNSCs might have reduced proliferative capacity compared to dNSCs.

The characterization of pup-derived pNSCs confirms that they are comparable to adult-

derived pNSCs and their increased abundance make them a valuable population to study

methods of targeting endogenous adult pNSCs.

Pup pNSCs are Oct4+, Sox2+, LIF-R+, ß-catenin+

Adult pNSCs are Oct4+, Sox2+, LIF-R+, ß-catenin- (Sachewsky et al., 2014). We

prepared wholemounts of the lateral ventricle from P7 Oct4-GFP pups to determine

whether these proteins are expressed in pup pNSCs. In the pup periventricular region,

Oct4+ cells were identified and colocalized with Sox2 and LIF-R (Fig. 2A-B). In contrast

to adult pNSCs, Oct4+ cells coexpressed ß-catenin in the pup brain (Fig. 2C). ß-catenin

stained the ventricular surface extensively and labeled fewer cells deeper in the SEZ

where it colocalized with Oct4. This may indicate a difference between the pup and adult

pNSCs, or may have resulted from the fact that ß-catenin is membrane bound while Oct4-

GFP is cytoplasmic and thus they did not appear to overlap in the previous study

(Sachewsky et al., 2014). Interestingly, Oct4+ cells often resided in close proximity to

blood vessels (Fig, 2B, 2C). Thus, the pup brain contains a population of Oct4+, Sox2+,

LIF-R+, ß-catenin+ cells in the periventricular region.

pNSCs can be isolated from dNSCs based on Oct4, GFAP and nestin expression

In addition to visualizing pNSCs in the periventricular region based on Oct4

expression to identify protein expression based on immunohistochemistry, we performed

FACS in conjunction with the neurosphere assay to determine genes expression in

 

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Figure 2

 

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Figure 2. pNSCs markers in the walls of the lateral ventricle. A. Wholemounts were

prepared from Oct4-GFP pups, stained with anti-GFP and counterstained for (B) Sox2

(C) LIF-R, and (D) ß-catenin. Inset shows boxed region of Oct4+ cell at higher

magnification. Scale bars = 10µm. Arrows indicate cells of interest. Arrowheads indicate

blood vessels.

 

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sphere-initiating pNSCs. This technique was not successful in adult-derived pNSCs due

to their rarity (Sachewsky et al., 2014). First, we confirmed that pup-derived sphere

initiating pNSCs are Oct4+. We performed FACS analysis on primary cells from the

periventricular region of Oct4-GFP P7 pups to isolate Oct4+ cells (Fig. 3A). Oct4– and

Oct4+ cells, along with an ungated control, were plated in the neurosphere assay to assess

for pNSCs and dNSCs. pNSC-derived neurospheres arose exclusively from the Oct4+

population at significantly increased frequency over the ungated control, confirming that

pNSCs are Oct4+ (Fig. 3B). dNSC-derived neurospheres arose exclusively from the Oct4–

population, confirming that dNSCs are Oct4– (Fig. 3C). We conclude that Oct4

expression is exclusive to pNSCs and a defining difference from dNSCs in the pup brain.

To confirm that pNSCs are GFAP– in contrast to dNSCs, we performed FACS on

primary cells from GFAP-GFP mice to isolate GFAP– and GFAP+ cells (Fig. 3D). As

previously reported (Sachewsky et al., 2014) we observed that pNSCs are GFAP– and are

significantly increased over the ungated control (Fig. 3E). Pup dNSCs were GFAP+ (Fig.

3F), as reported previously in adult dNSCs (Doetsch et al., 1999; Chiasson et al., 1999;

Morshead et al., 2003). The low amount of cross contamination between the positive and

negative bins likely resulted from gating to maximize viability at the expense of purity.

This did not cause cross contamination in our Oct4-GFP or nestin-GFP sorts but may

have due to low GFAP expression in dNSCs as compared to GFAP expression in

astrocytes. Therefore, we confirmed that pNSCs are GFAP–, in contrast to dNSCs that are

GFAP+.

Subsequently, we used the nestin-GFP mouse strain to determine nestin

expression in pNSCs. Nestin is commonly used as a neural stem/progenitor marker but is

 

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also expressed in many immature and proliferating cells (Gilyarov, 2008). We sought to

determine whether nestin is expressed in pNSCs, as it is in dNSCs. We performed

primary dissections and isolated cells based on low, middle, or high nestin expression

(Fig. 3G). pNSC-derived neurospheres arose exclusively from the nestinmid population, a

significant increase over the ungated control (Fig. 3H). dNSC-derived neurospheres arose

overwhelmingly from the nestin-high population, also a significant increase over ungated

control (Fig. 3I). Therefore, pNSCs and dNSCs have different levels of nestin expression

and reduced nestin is a defining feature of pNSCs relative to dNSCs.

Cell surface markers identified on ESC-derived pNSCs target pup-derived pNSCs

in vitro

A previous mass spectrometry-based analysis identified novel cell surface

markers upregulated on ESC-derived pNSCs, as compared to ESCs and dNSCs (DeVeale

et al., 2014). In this study, C-Kit and ErbB2 receptors were upregulated on ESC-derived

pNSCs and their inhibition affected pNSC-derived neurosphere formation. Here, we

cultured pup-derived pNSCs with pharmacological inhibitors and siRNAs to test whether

the cell surface markers target primary pNSCs in vitro.

To confirm that inhibition of C-Kit increases brain-derived pNSCs, we cultured

pup-derived primary cells with C-Kit inhibitor at varying concentrations (Fig. 4A).

Gleevec, a C-Kit inhibitor, had a significant interaction effect on pNSCs vs. dNSCs at

various concentrations and significantly increased pNSCs over dNSCs at 4 µM and 10

µM (Fig. 4B). We confirmed that the Gleevec-mediated increase in pNSCs was C-Kit

 

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Figure 3

 

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Figure 3. FACS analysis of primary pup brain cells. A. Cells from Oct4-GFP pups

were sorted to isolate Oct4+ vs. Oct4– cells. B. pNSCs arose exclusively from the Oct4+

population, a significant increase over the ungated control (1-way ANOVA F(2,6)=12.53,

p=0.01, n=3). C. dNSCs arose exclusively from the Oct4– population (1-way ANOVA

F(2,6)=171.4, p=0.001, n=3). D. GFAP-GFP periventricular cells were sorted into

GFAP– and GFAP+. E. pNSCs were increased in the GFAP– population (1-way ANOVA

F(2,6)=11.55, p=0.01, n=3). F. dNSCs arose from the GFAP+ population (1-way

ANOVA (F2,6)=6.34, p=0.03, n=3). G. Nestin-GFP pup cells were sorted for low,

middle and high nestin expression. H. pNSCs were increased in the nestinmid population

(1-way ANOVA F(3,8)=15.0, p=0.001, n=3). I. dNSCs were significantly increased in

the nestin-high population (1-way ANOVA F(3,8)=34.04, p=0.001, n=3).

 

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Figure 4

 

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Figure 4. Inhibition of C-Kit signaling increased pup-derived pNSC neurosphere

formation. A. Primary cultures of pNSCs and dNSCs were cultured with C-Kit inhibitor

and Kit siRNA. B. Addition of C-Kit inhibitor to primary cultures increased pNSCs and

depleted dNSCs at increasing concentrations, causing a significant increase in pNSCs

over dNSCs (2-way ANOVA F(3,24)=3.55, p=0.03, n=4). C. A siRNA specific to C-Kit

similarly increased pNSC spheres in primary culture significantly over scramble control

without effecting dNSC-derived neurospheres (2-way ANOVA F(1,12)=7.91, p=0.02,

n=4). D. Addition of the C-Kit ligand SCF had a significant effect on the interaction of

pNSCs vs. dNSCs at increasing doses. (2-way ANOVA F(5,24)=3.00, p=0.03, n=3). E.

To test stem/progenitor specific effects, primary cultures were passaged into secondary

cultures in the presence of C-Kit inhibitor. F. Addition of C-Kit inhibitor to secondary

cultures of passaged pNSCs and dNSCs similarly increased pNSCs and depleted dNSCs,

leading to significantly increased pNSCs over dNSCs (2-way ANOVA F(1,11)=4.96,

p=0.05, n=4). G. C-kit siRNA significantly increased secondary pNSC neurosphere

formation without an effect on dNSCs (2-way ANOVA F(1,8)=6.26, p=0.05, n=3).

 

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specific using a Kit siRNA, which decreased C-Kit transcripts to 20% of control

(DeVeale et al., 2014). Kit siRNA at 100 nM induced a significant increase in pNSCs

over thescrambled control, without affecting dNSCs (Fig. 4C). We did not detect a

change in neurosphere size in the presence of C-kit siRNA (control pNSC spheres were

61 + 1 µm vs. kit siRNA exposed were 62 + 2 µm (mean + SEM), n=3). Primary cells

were cultured with the C-Kit ligand stem cell factor (SCF) at varying concentrations (Fig.

4D). With SCF treatment, pNSC-derived neurospheres were depleted while dNSC-

derived neurosphere were increased, the opposite result of Gleevec treatment. Next, to

confirm the specificity of C-Kit inhibition on the stem/progenitor cells rather than a

secondary effect mediated by niche cells in primary culture, we passaged primary

untreated neurospheres into secondary culture with C-Kit inhibitor (Fig. 4E). The

increase in pNSC neurospheres continued in secondary culture and a significant increase

over inhibitor-treated dNSCs in the presence of C-Kit inhibitor (Fig. 4E). Together, these

data suggest that C-Kit inhibition preferentially targets pup-derived pNSCs over dNSCs.

ErbB2 also regulates pNSC proliferation (DeVeale et al., 2014). We investigated

whether ErbB2 inhibition depleted pup-derived pNSCs (Fig. 5A), similar to its effect on

ESC-derived primitive neurospheres (DeVeale et al., 2014). At high concentrations

(13µM) ErbB2 inhibition completely abolished pNSCs and dNSCs (data not shown), as

previously reported (DeVeale et al., 2014). At lower concentrations, ErbB2 inhibition

abolished dNSCs but not pup-derived pNSC neurospheres (Fig. 5B). ErbB2 siRNA

similarly attenuated dNSC neurosphere formation while slightly increasing pNSCs,

resulting in a significant increase over dNSCs (Fig. 5C). To confirm that the ErbB2

inhibitor targeted the stem cell populations rather than niche cells in the dish, we added

 

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ErbB2 inhibitor to secondary cultures (Fig. 5D). ErbB2 inhibitor in secondary cultures

severely depleted dNSCs and significantly increased pNSCs over dNSCs (Fig. 5E),

similar to its effect on primary cultures. These in vitro experiments confirm that ErbB2

has cell-type specific effects on pNSCs and dNSCs.

Cell surface proteins that increased pup-derived pNSCs targeted adult pNSCs in

vivo

To validate the cell surface markers that we tested on pup-derived neurospheres in

vitro, we delivered the C-Kit and ErbB2 inhibitors directly into the lateral ventricles of

adult mice via cannula connected to a micro-osmotic pump to determine the effects of C-

Kit or ErbB2 inhibition on pNSCs and dNSCs in vivo (Fig 6A). Mice received drug

infusions over 3 days and were dissected immediately following for the neurosphere

Direct infusion of the C-Kit inhibitor significantly increased pNSCs to 275 + 84% (mean

+ SEM) of vehicle infused control, without a significant effect on dNSCs (Fig. 6B).

ErbB2 inhibitor infusion significantly increased pNSCs to 278 + 49% (mean + SEM) of

control, without affecting dNSCs (Fig. 6C). Therefore, both cell surface marker targeting

drugs identified in culture on pup-derived neurospheres induced significant increases in

the pNSC population when infused into the brains of adult mice in vivo. C-Kit and ErbB2

inhibitors constitute novel mechanisms to enhance neurogenesis in the adult brain.

 

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Figure 5

 

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Figure 5. ErbB2 inhibition increased pup-derived pNSC neurospheres. A. Primary

cultures of pNSCs and dNSCs were cultured with ErbB2 inhibitor and ErbB2 siRNA. B.

Addition of ErbB2 inhibitor to primary cultures attenuated dNSC neurosphere formation,

while pNSC neurospheres were selectively increased at a low concentration (2-way

ANOVA F(3,19)=5.05, p=0.01, n=3). C. ErbB2 siRNA had a similar effect in primary

culture and decreased dNSCs while increasing pNSCs (2-way ANOVA F(1,12)=7.03,

p=0.02, n=4). D. To test stem/progenitor specific effects, primary cultures were passaged

into secondary cultures in the presence of ErbB2 inhibitor. E. In secondary cultures of

passaged pNSC and dNSC spheres, dNSCs spheres were severely attenuated with little

effect on pNSCs, leading to a significant increase in pNSCs over dNSCs (2-way ANOVA

F(1,9)=8.69, p=0.02, n=3). F. ErbB2 siRNA increased both secondary dNSC and pNSC

neurosphere formation (2-way ANOVA F(1,10)=6.02, p=0.03, n=4).

 

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Figure 6

 

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Figure 6. C-Kit and ErbB2 inhibitors delivered into the lateral ventricle of adult

CD1 mice increased pNSC neurosphere formation. A. Pharmacological inhibitors

were infused directly into the lateral ventricle of adult mice, then these mice were

assayed for pNSC and dNSC number. B. C-Kit infusion significantly increased pNSC

number relative to vehicle control without effecting dNSC number (2-way ANOVA

F(1,20)=5.12 p=0.04, n=6). C. ErbB2 infusion significantly increased pNSC number

relative to vehicle control without affecting dNSC number (2-way ANOVA

F(1,16)=4.81, p=0.04, n=5).

 

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Discussion

pNSCs located in the periventricular region of the mouse brain are at the top of

the NSC hierarchy (Tropepe et al., 2001; Hitoshi et al., 2004; Smukler et al., 2006;

Sachewsky et al., 2014). Here, we show that P7 pup-derived pNSCs are more numerous

than adult pNSCs but otherwise comparable. P7-derived pNSCs self-renewed at the same

frequency, were multipotential, and had similar gene expression by qPCR. Pup pNSCs

were Oct4+, Sox2+ and LIF-R+ similar to adult pNSCs (Sachewsky et al., 2014), but in

contrast were ß-catenin+. In addition, pup pNSC-derived neurospheres arose from the

Oct4+, GFAP–, nestinmid population after FACS. The increased abundance of pup pNSCs

makes them an ideal population to test the functions of specific cell surface markers. We

show that cell surface proteins identified on in vitro ESC-derived primitive neurospheres

and validated using pup-derived NSCs modeled pNSCs in the adult mouse brain.

We targeted endogenous pNSCs in order to develop strategies to activate the

earliest cell in the NSC lineage. pNSCs give rise to dNSCs and are Oct4-expressing as

shown by qPCR, immunohistochemistry, Oct4-driven antibiotic resistance, flow sorting

and the ability to integrate into the inner cell mass in morula aggregation experiment

(Sachewsky et al., 2014). Expanding pNSCs at the top of the neural hierarchy could lead

to larger increases in dNSCs and downstream progeny. These data suggest that pNSCs

have potential to contribute to endogenous repair strategies. Infusion of the

pharmacological inhibitors directly into the brains of adult mice induced an

approximately 3-fold increase in pNSCs over vehicle infused controls, without affecting

dNSCs. The 3-fold increase in pNSCs when C-Kit inhibitor or ErbB2 inhibitor were

infused in vivo suggests that niche cells may be maintaining pNSCs in a quiescent state

 

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and pharmacological inhibition can induce pNSCs to proliferate in vivo or prime cells to

proliferate when placed in culture. This may explain the larger effect observed after in

vivo delivery as the inhibitors are acting on pNSCs when they are within a restrictive

environment, rather than when added in vitro where the cells have already been removed

from their niche. C-Kit ligands are overexpressed following brain injury (Sun et al.,

2006), and ErbB2 signaling maintains cells in an undifferentiated state (Schmid et al.,

2003). Inhibiting them may modulate endogenous NSC activation. These studies suggest

that the balance of cell surface signaling may regulate NSC quiescence and activation,

and overcoming quiescence could enable pNSCs to contribute to endogenous repair. Our

data suggest that C-Kit and ErbB2 signaling could act as barriers to pNSC activation after

injury, and hold promise as valuable targets to modulate NSC-induced regeneration.

C-Kit is expressed at high levels in the neural tube and ventricular regions during

embryonic development and its expression continues in the periventricular region,

olfactory bulbs, cerebral cortex and hippocampus in the adult mouse brain (Keshet et al.,

1991; Motro et al., 1991; Jin et al., 2002). NSCs and their progeny also express the C-Kit

receptor in culture (Erlandsson et al., 2004). We did not detect a change in neurosphere

size in the presence of C-Kit siRNA suggesting that C-Kit does not exert a proliferation

effect on the progeny of pNSCs. Previous work showed that C-Kit does not exert a

survival effect (DeVeale et al., 2014). DeVeale et al. suggested that C-Kit inhibition

might repress a quiescence-inducing signal (2014), which is consistent with the present

report where we observed increased pNSC proliferative effects after the inhibitors were

delivered in vivo, where we predict that pNSCs are predominantly quiescent under

baseline conditions (chapter 3).

 

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ErbB2 inhibition reduced proliferative capacity of ESC-derived primitive

neurospheres when used at a high dose (13µM) (DeVeale et al., 2014). We observed that

low dose (1.3µM) ErbB2 inhibition increased pup-derived pNSCs when delivered in vitro

and increased adult-derived pNSCs approximately 3-fold when delivered in vivo, without

affecting dNSCs in vivo. ErbB2 expression is present in the brain at E13 and becomes

restricted during development so that by postnatal day 7 it is predominantly restricted to

the periventricular regions and persists into the adult (Fox and Kornblum, 2005).

Reduced ErbB2 activity led to differentiation of E9.5 derived radial glial cells into

astrocytes in culture (Schmid et al., 2003), which is consistent with the loss of dNSCs in

our culture experiments. The loss of dNSCs in vitro but not in vivo in response to ErbB2

inhibition suggests that the drug can target pNSCs in the brain without a detrimental

effect on dNSCs. Conversely, we found that pNSCs were not lost or induced to

differentiate, but increased. This supports ErbB2 inhibition as a potentially useful

mechanism to stimulate endogenous regeneration by activating the upstream pNSC,

leading to a greater expansion of the downstream dNSCs and improved ability to

generate neuronal and glial cells lost to injury.

The cell surface receptor inhibitors add to the differences between the two NSC

populations. pNSCs and dNSCs are both multipotential, but vastly differ in frequency,

differentiation profiles, gene and protein expression. pNSCs are Oct4+, Sox2+, LIF-R+, ß-

catenin+ and have higher expression of stem cell markers and lower expression of neural

commitment markers than dNSCs, regardless of age of origin. Whether derived from

postnatal day 7 or adult brain, pNSCs self renewed at a low frequency suggesting that

they undergo asymmetric division as a singly passaged neurosphere only gave rise to one

 

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new neurosphere, whereas dNSCs expand upon passaging suggesting symmetric

expansion divisions (Reynolds and Weiss, 1992; Morshead et al., 1994; Chiasson et al.,

1999). pNSCs generated fewer astrocytes than dNSCs, more oligodendrocytes from the

pup brain and more neurons from the adult brain compared to dNSCs. Together, these

findings support that pNSCs and dNSCs are distinct cell populations present in the

developing and adult mouse brain, with pNSCs able to generate dNSCs as previously

reported (Sachewsky et al., 2014).

Many neurodegenerative diseases and injuries lead to a loss of neurons and

demyelination. pNSCs generate equal proportions of neurons, astrocytes and

oligodendrocytes when differentiated in culture, unlike dNSCs that generate

predominantly astrocytes in vitro. The differentiation profiles suggest that pNSCs might

be the preferential population to target to generate non-astrocyte cell types if pNSCs

exhibit this characteristic in vivo. In this study, we demonstrate that a short infusion of

pharmacological inhibitors can increase the number of pNSCs and this could be used in

conjunction with a cue for differentiation to produce a 2-step strategy in the future.

Demyelinating diseases would benefit from the activation of an upstream NSC with the

capability of producing oligodendrocytes in the brain.

We compared pup and adult pNSCs and concluded that despite the large difference

in abundance, the populations are comparable. The perinatal peak suggests that pNSCs

might have a specific role in postnatal development, and the increased propensity to

differentiate into oligodendrocytes in culture suggests that pNSCs may contribute to the

wave of oligodendrogenesis postnatally. Flow cytometry for Oct4-GFP primary

periventricular cells suggested that approximately 0.08% of P7 pup-derived cells are

 

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Oct4+, similar to the number of Oct4+ cells reported in the adult periventricular region

(Sachewsky et al., 2014). This observation suggests two opposing hypotheses. First, that

the increased number of P7 pup pNSC-derived neurospheres may arise from a mixed

population of stem and progenitors that self-renew only perinatally (Seaberg et al., 2005;

Clarke and van der Kooy, 2011). Second, that all pup pNSCs survive but become

predominantly quiescent during development, and thus persist into the adult but most do

not generate neurospheres in our assay. These hypotheses could explain the consistent

number of Oct4+ cells but substantial decrease in pNSC-derived neurospheres in our adult

cell cultures. A long-term lineage tracing experiment initiated immediately after birth

might help elucidate pup pNSC fates.

Many cell surface markers were identified in the ESC-derived mass spectrometry-

based screen (DeVeale et al., 2014). We can continue to take advantage of the abundance

of pNSCs in the pup to screen for signaling molecules targeting adult pNSCs. This will

contribute to the development of pharmacological tools to target and activate NSCs in the

adult brain to aid in endogenous recovery in response to brain injury or disease.

 

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Chapter 5

General Discussion

 

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Implications for the NSC lineage

The discovery of adult-derived pNSCs suggests the presence of a reserve NSC

population at the top of the adult NSC hierarchy. Adult pNSCs are Oct4+, quiescent,

upstream of dNSCs, and activated to proliferate following ablation of the downstream

neural lineage (Fig 1A). pNSCs reside in the periventricular zone in close proximity to

blood vessels (chapter 4), and it remains unknown whether they contact the CSF (Fig

1B). pNSCs can be targeted and activated using pharmacological manipulations and cell

specific markers, which can be utilized to enrich pNSCs to better study them and to

develop future therapies to promote endogenous regeneration. The unique characteristics

of pNSCs, including that they express Oct4 and act as an adult reserve stem cell pool,

suggests that they may be the ideal population to target to induce adult regeneration.

Oct4CKO transgenic mice implicated that the dNSC population is unperturbed by

pNSC loss of function under baseline conditions. However, pNSCs were responsible for

repopulating dNSCs after dNSC and downstream progenitor ablation. I propose that

pNSCs do not maintain dNSCs under baseline conditions in the adult mouse brain, at

least in our Oct4CKO mouse strain. In addition, pNSC-derived neurospheres increased

after a stroke lesion in advance of an increase in dNSC-derived neurospheres (chapter 2).

This posits an interesting lineage whereby pNSCs act as a reserve stem cell population

upstream of dNSCs that are only activated in ablation/injury situations.

The detection of Oct4 in adult somatic stem cells has been widely challenged

(Lengner et al., 2008; Liedtke et al., 2008; Zangrossi et al., 2007). Oct4 is the hallmark

gene of pluripotency and as such is traditionally exclusive to ESCs. However, we

demonstrated Oct4 expression in pNSCs both in vitro and in vivo in a variety of ways to

 

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quantify RNA, protein and contribution to chimeric blastocysts. We performed numerous

experiments to validate our report of Oct4-expressing pNSCs. In addition, our

experiments always employed the correct controls, including ESCs as positive control

and dNSCs as negative controls, in contrast to the criticism by Lengner et al. that controls

are absent in reports of somatic Oct4 expression (2008). Oct4CKO mice that did not give

rise to pNSC-derived neurospheres and had reduced ability to repopulate the neural

lineage after AraC/GCV provide functional data that Oct4 plays a critical role in pNSCs.

While the function of Oct4 remains unclear in pNSCs, be it survival, proliferation, self-

renewal or multipotency, these data suggest that Oct4 is not merely expressed but in fact

essential to pNSC function.

pNSCs, as adult somatic cells expressing Oct4, may have interesting implications

for their contribution to the adult neural lineage as an Oct4+ cell was previously not

reported in the adult mouse brain. pNSCs are quiescent under baseline but activated

following injury/ablation, which suggests that they have potential to contribute to

endogenous repopulation if targeted pharmacologically. We have yet to confirm that

pNSCs are multipotent in vivo and instead tested the intrinsic potential of the cells with

our cell culture techniques (chapter 2). To address the ability of pNSCs to differentiate in

vivo we transplanted pNSC-derived colonies into the brain and observed labeled cells

migrating along the RMS to the olfactory bulb where they adopted a neuronal-like

morphology (chapter 2). It will be interesting to determine whether pNSCs give rise

directly to astrocytes and oligodendrocytes in vivo, and this might be achieved with

lineage tracing in the adult or in the pup brain when these cell types are being born.

Neurons, astrocytes and oligodendrocytes are generated in the adult brain (Doetsch and

 

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Figure 1 A.

B.

 

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Figure 1. The NSC lineage and niche supported in this thesis. A. NSC lineage where

pNSCs are upstream of dNSCs in the adult mouse brain. B. NSC niche with pNSCs,

which appear to reside within close proximity of blood vessels but it remains unknown

whether they contact the lateral ventricle. TA – transit amplifying cell, N – neuroblasts,

E – ependymal cells, A – astrocytes.

 

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Alvarez-Buylla, 1996; Nait-Oumesmar et al., 1999; Menn et al., 2006), and if pNSCs

contribute to these newborn cell types in vivo they would be a valuable target to increase

neurogenesis or gliogenesis. Since pNSCs become activated following injury they may

contribute to increase the speed of regeneration of downstream progenitors.

Repopulation of dNSCs after ablation

A kill paradigm with two thymidine-H3 ablations two days apart led to two

conclusions important to this thesis (Morshead et al., 1994). First, it only took two days

for 50% of dNSCs to be activated to repopulate their downstream progeny (Morshead et

al., 1994). That the majority of dNSCs become activated within 2 days of ablation fits

with the findings in this thesis. We observed a strong dNSC-derived neurosphere ablation

with 7 days of AraC treatment, fitting with the initial report of quick dNSC activation

(Morshead et al., 1994). What remains unknown, and of great interest, is how quickly

pNSCs become activated to repopulate their downstream progeny (dNSCs). In particular,

we determined the repopulation potential of pNSCs in Oct4CKO after ablation, as

supported with AraC delivered to H2B-GFP mice, but it remains unclear how long

pNSCs take to activate/proliferate since if the AraC was still present it would have

targeted the pNSCs. While we would not expect AraC, an antimitotic drug, to directly

target a quiescent cell population, the finding that pNSCs are becoming activated but not

ablated by AraC suggests a method of activation other than proliferation or at least a

delay in the proliferation until the toxic signal is gone.

A second intriguing conclusion was that the number of proliferating cells never

recovered back to baseline numbers (Morshead et al., 1994). It was originally predicted

 

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that the incomplete recovery was due to the second kill targeting dNSCs as they became

activated to repopulate the lineage after the first kill. Interestingly, although we now

know that pNSCs are upstream of dNSCs in the adult mouse brain, it remains unknown

why dNSC-derived neurospheres never recovered to baseline levels in this Morshead et

al., experiment or the AraC and AraC/GCV experiments reported in this thesis. Multiple

reasons could explain why the dNSCs never fully recover, for example the possibility that

pNSCs are also targeted by AraC, thus their population has reduced ability to contribute

to repopulation. Although other quiescent NSC populations have been reported to not be

targeted by AraC (Codega et al., 2014; Mich et al., 2014), I presume that if AraC is given

for a long enough period of time or administered in pulses, that an ablation paradigm

could be developed to activate and target quiescent cells as they occasionally proliferate.

This inability to recover the stem cell population to baseline numbers has also been

reported in the hematopoietic system following serial transplantation (Harrison et al.,

1978). Possibly, cells may limit their proliferation to prevent against replication-

dependent mutations (reviewed in (Wang and Dick, 2005; Orford and Scadden, 2008)).

Therefore, it remains interesting to determine the cause of the limited regenerative

capacity.

Other quiescent NSC hypotheses

As a result of the absence of selective cell markers and the limitations of the

retrospective nature of cell culture techniques, a unified NSC lineage remains unknown.

We report a pNSC that is Oct4+, GFAP–, nestinlow, Sox2+ and has a very slow cell cycle

time of approximately 3-5 months. Other groups have reported other quiescent NSCs

 

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with an array of cell markers in the periventricular region. It remains unclear whether

these quiescent cell populations overlap, to what extent, and how they fit together into a

cohesive NSC lineage. In Figure 2, I present a proposed unified hypothesis of the NSC

lineage taking into recent reports in the field (Codega et al., 2014; Mich et al., 2014).

A population of quiescent (q)NSCs was proposed by Fiona Doetsch’s group that

are GFAP+, CD133+, EGF-R– (Codega et al., 2014). It was proposed that qNSCs can

become activated (a)NSCs and cycle between quiescence and proliferation by

upregulating nestin and EGF-R expression (Pastrana et al., 2009; Codega et al., 2014).

Both qNSCs and aNSCs reside in the center of pinwheels in close proximity to each other

and in contact with the ventricle (Pastrana et al., 2009; Codega et al., 2014). These

qNSCs are much more abundant and have shorter cell cycle time than pNSCs. qNSCs

were suggested to comprise 25-30% of the periventricular GFAP+ cell population (2.3-

2.8% total periventricular zone population) while aNSCs comprise 20-25% of GFAP+

cells (1.9-2.3% total periventricular zone population) (Codega et al., 2014). In addition, a

major difference is that qNSCs are GFAP+ while pNSCs are GFAP–. Potentially, qNSCs

are a subpopulation of dNSCs that are upstream of the aNSCs, which I propose is more

similar to a TA cell (Fig. 2). Interestingly, a similarity between qNSCs and pNSCs is

their low nestin expression (chapter 4), which reflects their distinction from the

proliferating adult aNSCs. In addition, qNSCs were reported to express Sox2 but lower

levels than aNSCs, with approximately 60% of qNSCs described as Sox2dim based on

flow cytometry (Codega et al., 2014), and pNSCs have lower Sox2 expression than

dNSCs by qPCR (chapter 2 and 4). qNSCs gave rise to fewer colonies in the presence of

EFH as compared to aNSCs, and it was concluded that quiescent stem cells cannot

 

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proliferate to generate colonies in culture (Codega et al., 2014). This poses an interesting

question for pNSCs as we report that pNSC-derived neurospheres are quite rare, but may

reflect a reduced ability to proliferate to generate neurospheres. In addition, qNSCs were

reported to be quiescent based on BrdU retention (Codega et al., 2014), but a large caveat

of BrdU retention models is that cells must replicate their DNA to incorporate BrdU. This

model biases towards cells that are cycling when the initial population is labeled. In

chapter 3, we took advantage of the H2B-GFP mouse model for unbiased label retention

studies due to the replication-independent initial labeling and identified label-retaining

cells after 1 year of chase that were pNSC neurosphere initiating cells.

Another group recently reported a NSC classification system termed pre-

GEPCOT and GEPCOT cells (Mich et al., 2014). Contrary to the dogma of stem cell

hierarchies, pre-GEPCOT cells were more abundant than GEPCOT cells (Mich et al.,

2014). Pre-GEPCOT cells are classified as GlasthighEGFRlowPlexinB2low, and I propose

that these cells fall within the dNSC population (Fig. 2). Pre-GEPCOT cells form

neurospheres at low frequency and approximately 3% incorporate BrdU after a 24 h pulse

(Mich et al., 2014), which extrapolates to a cell cycle time of 33 days. GEPCOT cells are

GlastmidEGFRhighPlexinB2high and a cell cycle time of 6 h (based on our calculation from

their BrdU pulse data), which suggests they may be more similar to a transit amplifying

cell than a dNSC (Fig. 2).

In Figure 2, I present a possible hypothesis of a unified NSC lineage. In this

lineage I propose that pre GEPCOT and qNSCs are subpopulations of dNSCs.

Interestingly, pre-GEPCOT and qNSC cell cycle times (33 and 5-6 days, respectively,

 

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Figure 2

 

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Figure 2. Unified theory of the NSC lineage. pNSCs are the only NSC that are Oct4+

and GFAP–, they are the most rare, slowest proliferating and have lowest neurosphere

forming ability. I present a hypothesis integrating our data on pNSCs, recent reports of

Codega et al., and Mich et al., (2014), and past reports of dNSCs/type B cells. We used

data presented within these reports as best possible to generate data for comparisons

across the cell populations to generate a proposed NSC lineage. % pop = % of population

within the periventricular region. % NIC = % of neurosphere initiating cells in the

neurosphere assay (all cultured in EFH except for pNSCs, which are cultured in LIF).

GFAP: Codega et al., Fig. 3A, C; Mich Fig. 4A. % pop: Codega et al., Fig. 3A, B; Mich

et al., Fig. 1B, 3E. cell cycle: Codega et al., Fig. 3E (qNSCs were 0.8% labeled after 1h

BrdU pulse = 100% labeled after 5.2 days), Fig. 3E (aNSCs were 35.5% labeled after 1h

BrdU pulse = 100% labeled after 3h);Michet al., Fig. 1D (GEPCOTS were 35% labeled

after 2h BrdU pulse = 100% after 6h), Fig. 4C (pre-GEPCOTS were 3% labeled after 24h

BrdU pulse = 100% labeled after 33 days). % NIC: Codega et al., Fig. 5K; Mich et al.,

Fig. 1C, 4B.

 

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based on my calculations from their BrdU pulse data) average to approximately the cell

cycle time previously reported for dNSCs (15 days, Morshead et al., 1998). In addition,

their neurosphere initiating cell (NIC) frequencies (0.06% and 1%) also approximately

average to the NIC of dNSCs (0.6%). I believe these data together with GFAP expression

maintained in the populations suggest they could be subpopulations of dNSCs.

The relationship of each cell population within the periventricular region and

whether this is a traditional hierarchy of distinct cell fates or whether some populations

could be described as different fates of the same cell type remains to be consolidated. In

addition, I propose that aNSCs may be either dNSCs or TA cells and that GEPCOT cells

are TA cells. I suggest this based on their cell cycle time, reduced GFAP expression and

high nestin expression. However, TA cells do not form neurospheres and this is in

contrast to the reports of aNSCs and GEPCOT cells. Therefore these criteria remain to be

confirmed, but due to the low size restrictions used in Mich et al., and Codega et al., I

suggest that aNSCs and GEPCOT cells are in fact TA cell populations (2014). Further

classification of the adult NSC lineage remains of exceptional interest to investigate these

cell populations in a single study to test a cohesive NSC lineage.

Reports suggesting that ependymal cells become activated following injury to

repopulate the NSC lineage debate these SEZ quiescent NSC hierarchies (Devaraju et al.,

2013). Although previous work has excluded the possibility that ependymal cells are

NSCs (Chiasson et al., 2009), it was proposed that ependymal cells activate following

injury to act as NSCs. FoxJ1 protein is expressed in motile cilia and was used as an

marker exclusive to ependymal cells for lineage tracing (Devaraju et al., 2013). However,

upon closer inspection of the data in this report, I propose that FoxJ1 was not exclusive to

 

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ependymal cells and appears to be expressed in cells that resemble dNSCs (removed from

the ventricle and extending a single process into the CSF). Therefore, the specificity of

FoxJ1 is called into question, and I propose that the lineage tracing that identified labeled

ependymal cells activated following injury and generating new neurons in the olfactory

bulb (Devaraju et al., 2013) is in fact indicative of accidently labeled dNSCs. Therefore,

ependymal cells are not NSCs and do not have neurogenic potential, thus are not included

in the proposed NSC lineage in Figure 2.

Great progress is being made to identify and classify NSCs and determine their

proliferative capacity in the adult mouse brain. I propose that pNSCs either proliferate to

give rise to dNSCs or transition from their quiescent state to a less quiescent, GFAP+

state to comprise dNSCs. Classic stem cell hierarchies are based on the dogma that cells

exist in distinct cell states. However, less distinct cell states are being considered and

challenging this dogma and suggesting that cells might transition between states and can

exist as intermediates between tiers in the hierarchies. Despite this trend, distinct cell

states are still advantageous, as specific cell surface markers are needed to classify cells

and determine their contribution to tissue homeostasis and repair.

Are all neurospheres NSC-derived?

The neurosphere assay is essential to the NSC field. Neurospheres are derived

from NSCs cultured at low cell density, generating a clonal colony that enables testing of

the stem cell criteria of self-renewal and multipotency. However, despite it’s widespread

use, debate over its ability to exclude progenitors exists (reviewed in (Pastrana et al.,

2011)). Neurospheres are measured with a strict size requirement, based on the premise

 

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that NSCs have increased potential to proliferate and generate a large colony compared to

progenitors (Morshead et al., 1994). In the brain, TA cells proliferate faster than dNSCs

since their role is to amplify the NPC pool. However, TA cells are not the source of

neurospheres in culture as shown by a double kill with thymidine as a single dose did not

ablate neurospheres but a double dose, that targets the dNSCs pulled into division,

depletes neurospheres (Morshead et al., 1994). TA cells do not generate neurosphere to

meet the size cutoff of 100 µm and the small neurospheres do not self-renew. Therefore,

despite the concern over TA-derived neurospheres, proper assay execution including

strict size criteria and passaging the neurospheres multiple times excludes any non NSC-

derived neurospheres (Reynolds and Rietze, 2005). In addition, only large neurospheres

are multipotent and provides further evidence that they are NSC-derived (Chiasson et al.,

1999).

Many of the limitations of the neurosphere assay are associated with the lack of

clonality of the neurospheres that arise from improper cell culture technique. This

interferes with the ability to test self-renewal and multipotency, and even gene expression

or any other characteristic that neurospheres can be assayed. The gold standard to ensure

clonality is to plate less than one cell per well and thereby guarantee that the starting

population is a single stem cell. However, plating less than one cell per well isolates the

cell from any signaling factors released from neighboring niche cells that play a role in its

survival or proliferation (reviewed in (Pastrana et al., 2011)). Very rare cell populations

cannot be reasonably studied in this system due to physical limitations in the number of

cells that can be plated and counted. These limitations lead to insufficient numbers of

cells studied and increase the risk of a sampling error that could mislead experimental

 

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conclusions. With increasing stem cell rarity the sampling risks outweigh the risk of non-

clonality, especially when cultured at low density. Clonality can be achieved without

single cell plating as long as cells are plated at 10 cells/µl (Coles-Takabe et al., 2008), as

performed in all experiments in this thesis. Therefore, even though single cell cultures are

often considered ideal, in the case of very rare cell populations the cons outweigh the

pros and single cell culture is not necessarily feasible.

How many pNSCs are really in the brain?

Calculating the number of pNSCs and even dNSCs in the adult niche is a difficult

task. Many of the methods we use to quantify a cell population involve processing tissue,

which leads to cell death and thus interferes with our ability to draw an accurate

conclusion. The initial experiments on pNSCs calculated the abundance of pNSCs in the

adult mouse brain based on the frequency of pNSC-derived neurosphere formation in the

neurosphere assay. This calculation suggested that pNSCs are extremely rare in the adult

brain, and that only 5 pNSC exist per brain. While pNSCs are evidently rare in the brain,

I believe that they are more numerous than 5 per brain.

The neurosphere assay involves many opportunities for cell death. It is extremely

difficult to estimate the amount of cell death that occurs during tissue dissection in

preparation for cell culture. Cell death occurs during the dissection, during exposure to

enzymes, during trituration to dissociate the tissue, during the time required to prepare

and plate the cells for culture, and finally during the culture period. These processes

likely produce a lot of cell death, but it is difficult to estimate the number of cells lost to

calculate the proportion of the starting population that survives into culture. Even if we

 

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did estimate the amount of cell death, certain cell populations could die preferentially

over other populations and thus provide limited insight. Flow cytometry analysis of

primary dissections of the periventricular region from Oct4-GFP transgenic mice

suggested that 0.08% of the sorted cell population was Oct4+, which suggests that there

are 80 Oct4+ cells in the mouse brain, and perhaps all of these are pNSCs. This number

can be reconciled with the estimates from the neurosphere assay because pNSC

quiescence may hinder proliferation and thus neurosphere generation in culture. Possibly,

the increase in pNSC-derived neurospheres we reported after pharmacological

manipulaton with C-kit inhibitor and ErbB2 inhibitor in vivo (chapter 4) might reflect

activation of quiescent pNSCs to increase neurosphere formation. Together, these data

suggest an increased abundance of pNSCs compared to previous estimates.

It is not surprising that the number of pNSCs remains unclear as reports vary on

the number of dNSCs present in the adult mouse brain. If we use the neurosphere assay

as a direct readout of the number of NSCs present in vivo we calculate that approximately

600 dNSCs are present in the adult mouse brain (assuming 30 dNSC-derived

neurospheres per 5000 cells and 100 000 cells per dissection). This number is lower than

other estimates of the number of dNSCs. Morshead et al. estimated that there are 1200

dNSCs, which equivocates to 0.2-0.4% of SE cells (1998). On the other hand, Mirzadeh

et al. reported that 6225 B1 cells are present on the lateral wall of one hemisphere lateral

ventricle, suggesting that there are 12 450 B1 cells per brain (2008). Further, Mirzadeh et

al., suggest that 31% of all cells contacting the lateral ventricle are B1 cells. These

numbers contrast with each other and offer a large range for the number of dNSCs

present in the adult mouse brain. Therefore it should be expected that there might be a

 

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range of abundances for pNSCs in the adult mouse brain, which future studies will

address.

Cell cycle times

Estimating the cell cycle time of pNSCs is a difficult task that is amplified by

variation amongst the cell cycle time of dNSCs and other NPCs. The H2B-GFP label

retention experiment suggested that pNSCs divide very slowly, and that the rate of

proliferation of pNSCs is 6-fold slower than dNSCs. From two methods of calculating the

pNSC cell cycle time, we calculated that pNSCs proliferate once approximately every 3-5

month. The 3 month cell cycle time is dependent on the 6-fold difference in proliferation

rate and previous experiments that proposed that dNSCs proliferate once every 15 days

(Morshead et al., 1998). This previous calculation arose from the observation that dNSCs

divide 1:1 to repopulate constitutively proliferating cells, and since the constitutively

proliferating cells survive in the niche for 15 days dNSCs likely divide once in that time

frame. Therefore taking the 15-day dNSC cell cycle time and the 6-fold difference in

proliferation, we calculated that pNSCs divide once every 3 months. However, we also

calculated pNSC cell cycle time based on the assumption that labeled neurosphere

detection is a sufficient readout of the in vivo cell divisions as well as other assumptions

set out in chapter 3. In this calculation, we set the y intercept to 50% GFP dilution, to

represent one cell cycle and calculated the time this takes to occur. In this instance, we

independently estimate the cell cycle time of dNSCs as 24 days and pNSCs as 5.1

months. Therefore, we suggest that pNSCs divide approximately once every 3-5 months.

 

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The H2B-GFP mouse model assumes that H2B protein only turns over during

mitosis (Kanda et al., 1998), but if H2B protein turns over independent of mitosis, it

would result in even longer cell cycle times than we calculated. In addition, a caveat to

the H2B-GFP model is if one cell type preferentially maintains the GFP label through

asymmetric histone segregation (Lark et al., 1966; Potten et al., 1978; Potten et al., 2002;

Karpowicz et al., 2005). The “immortal strand hypothesis” suggests that an upstream,

quiescent stem cell will retain the original DNA strand and that the more-restricted

daughter cell will inherit the new strand (Cairns, 1975). Thus if the original strand is

associated with and retains labeled histones it might remain GFP+ despite undergoing

divisions. However, the immortal strand hypothesis remains controversial in many stem

cell systems and it has not been reported yet to include asymmetric H2B segregation

(reviewed in (Wilson et al., 2008)). If pNSCs retained labeled histones it would negate

our cell cycle estimates but still support our conclusion that pNSCs are an upstream cell

population.

Comparing pNSCs to other stem cell hierarchies

The hematopoietic system serves as a great model system because our HSC

knowledge is years ahead of the NSC field. HSCs are well characterized with extensive

cell surface marker classifications to distinguish each cell type. Many groups have set out

to characterize NSCs with specific cell markers, however the NSC field is still striving

for a collective cell classification system. Since the HSC field is more developed than the

NSC field, it is intriguing to draw comparisons between quiescent (q)HSCs and pNSCs.

 

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The frequency of pNSCs is similar to qHSCs. Within the periventricular tissue,

0.005% of cells are pNSC neurosphere initiating cells and 0.08% are Oct4+ cells by flow

analysis (chapter 2). One estimate of the frequency of long-term reconstituting HSCs

(Thy-1loLin–Sca-1+) estimated that they comprise 0.02-0.05% of bone marrow cells

(Smith et al., 1991), while another estimate suggested that they comprise 0.00125% of

the bone marrow population (Wilson et al., 2007). It is interesting that on a percentage

basis, the populations are comparable and the numbers of cells in the brain vs. blood are

also similar. There are approximately 1x108 cells in the mouse brain (Williams, 2000) and

5x108 cells in the mouse bone marrow (Colvin et al., 2004). Therefore, pNSCs and

qHSCs could be considered comparable with respect to their rarity and quiescent nature.

Similar to pNSCs, dormant HSCs that retain H2B-GFP labeling are activated to

proliferate and dilute their label after exposure to fluorouracil (5-FU) indicating that they

are activated by ablation of their downstream progenitors (Wilson et al., 2008).

Interestingly, dormant HSCs are activated quickly after this treatment, with

approximately 40% of dHSCs undergoing mitosis 2 days after 5-FU treatment (Wilson et

al., 2008). This is very similar to the 2-day activation time reported in dNSCs (Morshead

et al., 1994). In addition, the quick repopulation of dNSCs following AraC and

AraC/GCV ablations (chapter 2 and 4) suggests that pNSCs may be activated to

proliferate quickly after downstream lineage ablation. How quickly pNSCs are activated

remains to be determined, and will be discussed in the future directions.

In addition to the commonalities of long cell cycle time and rarity between these

two primitive cell populations, qHSCs and pNSCs share other cell characteristics. Of

note, primitive HSCs are negative for the traditional HSC marker CD34 (Anjos-Afonso et

 

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al., 2013), which could be considered similar to pNSCs not expressing GFAP. CD34–

qHSCs were proposed to not contribute to baseline hematopoiesis and instead act as a

reserve pool for injury recovery where they upregulate CD34 as they become activated

(Wilson et al., 2007). In addition, qHSCs have lower frequency of colony formation but

are responsible for repopulating the entire HSC lineage following transplant into an

irradiated recipient mouse (Wilson et al., 2007). Therefore, similarities can be derived

between pNSCs and qHSCs but the pNSC field is in its infancy and more research is

needed to determine if they are the neural equivalent of cells at the top of the HSC

hierarchy.

Why target pNSCs for regeneration?

In chapter 4, I describe selective pNSC markers as well as methods to

pharmacologically target and activate pNSCs. The cell specific markers will improve our

ability to enrich for pNSCs and thus study them in the future. The collective

characterization suggests that pNSCs are Oct4+, GFAP–, LIF-response, nestinlow, and they

are activated by C-kit and ErbB2 inhibition. The identification of pNSC-specific cell

markers is valuable for the development of targeted therapies and pNSCs may be the only

NSC with an exclusive cell marker.

Despite the presence of NSCs, the brain exhibits limited endogenous repair to

counteract neurodegenerative disease or recover after injury. The ability to endogenously

activate pNSCs with pharmacological manipulation might lead to future regenerative

therapies focused on pNSC-driven regeneration. Pharmacological compounds to activate

endogenous NSCs and guide their differentiation could improve brain recovery/repair

 

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(reviewed in (Miller and Kaplan, 2012)). Initiating regeneration by activating pNSCs to

lead to large expansion of dNSCs, and thus their progeny, could lead to greater

therapeutic benefit than regenerative methods focused on the downstream progenitors. In

fact a two-step strategy is being developed to target NPCs whereby metformin is

administered to promote NPC expansion followed by cyclosporin A to promote migration

away from the niche and differentiation into newborn neurons (Hunt et al., 2010;

Erlandsson et al., 2011; Wang et al., 2012). A similar approach could be developed to

target pNSCs to promote their contribution to endogenous regeneration after injury. The

larger expansion of progenitors induced from the top of the lineage could then be guided

with other pharmacological methods to differentiate into the cell types needed. In

addition, pNSCs give rise to a different cell type distribution when differentiated in

culture compared to dNSCs. pNSCs give rise to increased neurons and oligodendrocytes

at the expense of astrocytes. Since most endogenous recovery would benefit more from

newborn neurons or oligodendrocytes to remyelinate these neurons, pNSCs might be the

better cell population to target.

Are pNSCs present in the human brain?

It remains unknown whether pNSCs exist in the human brain. Although one

would hypothesize that a NSC present in the mouse is present in all mammals, one reason

that they may not exist in humans is because human ESCs are very different from mouse

ESCs. Human ESCs have different colony morphology and are not LIF-dependent

(Dahéron et al., 2004), which posits an interesting question whether they give rise to LIF-

dependent NSCs. Human ESCs are more similar to mouse epiblast stem cells. It is

 

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unknown whether a LIF-dependent NSC is even present in human embryonic

development. Early attempts to derive pNSCs from human ESCs in culture suggest that

ESCs were primed to a neural identity in maintenance conditions (Chaddah et al., 2012).

The generation of naïve human ESCs which are LIF-dependent (Hanna et al., 2010;

Gafni et al., 2013), suggests that pNSCs may be derived from human ESCs in the future.

Therefore, while pNSCs in the human brain were outside of the scope of this thesis, it

remains interesting to determine if they exist.

Conclusions

This thesis proposes a novel NSC lineage whereby a pNSC at the top of the NSC

lineage acts as an adult reserve stem cell pool to repopulate the lineage. This pNSC is

best characterized as Oct4+, GFAP–, LIF-R+ and is rare in the adult mouse brain. pNSCs

give rise to dNSCs embryonically, but then dNSCs appear able to maintain their own

population and the downstream lineage, except in response to antimitotic ablation of the

dNSC pool itself. pNSCs are distinct from dNSCs and have unique cell surface markers

and gene expression profiles that can be utilized to independently target and activate

them within their niche.

Wholemount imaging suggest that pNSCs reside within the subependyma, similar

to dNSCs, and that pNSCs may be located within close proximity to blood vessels. It

remains unknown if pNSCs contact CSF in the lateral ventricle and whether they reside

within pinwheels next to dNSCs. pNSCs reside upstream of dNSCs in the NSC lineage

and give rise to dNSCs, which generate TA cells and then neuroblasts. pNSCs represent a

novel finding in the NSC lineage and hold great implications for the NSC field.

 

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Future Directions

The discovery that pNSCs persist in the adult mouse brain brings with it a

plethora of new research questions to be addressed. In this thesis, I report that pNSCs are

Oct4+, repopulate dNSCs in an ablation model, and that pNSCs can be selectively

targeted and activated within the mouse brain. However, many more experiments remain

to determine the role of pNSCs throughout development and under baseline in the adult

mouse brain.

We have begun to address the role of pNSCs in the adult brain, but their role

between E8.5 (when they were previously thought to disappear) and the adult remains

completely unknown. We identified a peak in the abundance of pNSCs perinatally but do

not have any information as to whether this peak in abundance has a specific function.

Finally, we observed that pNSCs are required for repopulation of dNSCs after ablation in

the adult mouse brain, but we do not know their role under baseline conditions. All three

of these questions could be addressed with a lineage tracing experiment. Fortunately, due

to unique expression of Oct4 in pNSCs, an Oct4CreERT2 mouse strain can be used to

address all these questions. One issue that may arise around identifying the contribution

of pNSCs embryonically with Oct4CreERT2 is that other cells in the embryo continue to

express Oct4 at this time and it will require a careful teasing apart how early we can label

pNSCs without labeling other cells. In addition to the lineage tracing, the Oct4CreERT2

mouse can be used for a diptheria toxin receptor (DTR) ablation to target pNSCs

specifically. This would be different than the ablations presented in this thesis because it

would kill pNSCs entirely, rather than remove Oct4 function as in our Oct4CKO mouse.

The Oct4CreERT2 mouse strain could be used to address the NSC lineage proposed in

 

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Figure 2. Labeling pNSCs followed by subsequent analysis at various survival times

might speak towards a cohesive NSC lineage hypothesis.

We demonstrated that pNSC-derived neurospheres are multipotential and the

pNSC-derived colonies transplanted into the brain gave rise to neurons. However, it

remains to be determined whether pNSCs give rise to these differentiated cell types

directly, whether they give rise to unipotential progenitors, or whether they always pass

through a dNSC intermediate. This could be determined in vitro by culturing and

differentiating pNSC-derived neurospheres from GFAP-tk mice in the presence of GCV.

While we predict that astrocytes cannot be generated in the presence of GCV, it could

still be determined if pNSCs can give rise to neurons or oligodendrocytes without the

ability to pass through the dNSC fate. In addition, culturing dNSCs in the presence of

EGF in culture biases the progeny towards a glial fate, while lower concentrations

increases neural differentiation (Burrows et al., 1997). Therefore, it remains possible that

the different percentage of differentiated progeny from dNSC and pNSC may be

influenced by the cell culture conditions in addition to the cell type. Thus it would also be

interesting to attempt to culture pNSCs in the presence of both LIF and EFH to determine

a) if pNSC-derived neurospheres can still be derived and b) if they can, if they generate

the same distribution of differentiated cells as neurospheres from LIF alone conditions.

Further characterization of the differentiation profile of pNSCs remains to be interesting.

Although the number of pNSCs present in the adult mouse brain can be predicted

and debated based on evidence in this thesis, it remains to be confirmed. We reported an

increased abundance of pNSC-derived neurospheres from the postnatal day 7 pup brain,

but the fate of these cells remains unknown. It remains unclear whether these increased

 

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pNSCs are true stem cells or whether they could apoptose or differentiate. Potentially

they remain in the adult brain but as quiescent pNSCs that are not identified in the

neurosphere assay. The abundance of pNSCs remains closely linked with whether a

subpopulation of quiescent pNSCs is present but do not form neurospheres. Interestingly,

the rapid repopulation of dNSCs after AraC treatment might suggest that quiescent

pNSCs are becoming activated to proliferate. This would be interesting to test and might

be elucidated with short AraC infusions and survival times to determine how quickly

pNSCs can be activated. Preliminary data suggests that pNSC-derived neurospheres are

increased after AraC infusion. This is contradictory to what we might expect since if

pNSCs were proliferating to increase their population in order to give rise to more pNSC-

derived neurospheres, AraC would kill them. An alternate hypothesis, suggests that

pNSCs might be activated in response to an ablation of their downstream progenitors and

exit quiescent, thus form neurospheres in culture. Preliminary data suggests that pNSC-

derived neurosphere abundance is increased as early as 24 hours after the initiation of

AraC infusion, which might suggest that activation of quiescent cells is the more likely

explanation. An increase as a result of proliferation would take a longer amount of time

and expose pNSCs to AraC-induced death. This would require that pNSCs become

activated in the brain when exposed to AraC but do not begin to proliferate until after

they are removed from the AraC exposure.

Finally, the role of Oct4 in pNSCs remains elusive. Oct4CKO mice suggest that

pNSCs require Oct4 expression, but whether this is for survival, self-renewal,

maintenance or multipotency remains unknown. Since Oct4 is not widely believed to be

expressed in the adult brain, it remains interesting to see what it’s role could be. In

 

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addition, the Oct4CreERT2 mouse strain could be used for lineage tracing to elucidate

whether pNSCs persist in Oct4CKO mice but do not generate pNSC-derived neurospheres

or contribute to dNSCs repopulation, or whether they are absent. Whether Oct4 activates

similar or different signaling pathways as it does in ESCs would shed light on whether its

role in pNSCs is in fact related to pNSC potential.

In conclusion, this thesis begins to characterize pNSCs in the mouse brain, but

their full role and potential remains to be elucidated.

 

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