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Regulatory Role of Fibroblast Growth Factors

on Hematopoietic Stem Cells

Joyce S. G. Yeoh

Regulatory Role of Fibroblast Growth Factors

on Hematopoietic Stem Cells

RIJKSUNIVERSITEIT GRONINGEN

Regulatory Role of Fibroblast Growth Factors on

Hematopoietic Stem Cells

Proefschrift

ter verkrijging van het doctoraat in de

Medische Wetenschappen

aan de Rijksuniversiteit Groningen

op gezag van de

Rector Magnificus, dr. F. Zwarts,

in het openbaar te verdedigen op

woensdag 14 februari 2007

om 16.15 uur

door

Joyce Siew Gaik Yeoh

geboren op 26 november 1979

te Penang, Maleisië

Promotor Prof. dr. G. de Haan

Copromotor Dr. R. van Os

Beoordelingscommissie Prof. dr. E. Vellenga

Prof. dr. P. Coffer

Prof. dr. A. Mueller

In memory of my father

And for my dearest mother

The research described in this thesis was conducted in the Department of Cell

Biology, Section Stem Cell Biology, University Medical Center Groningen,

University of Groningen, the Netherlands.

This research project was financially supported by the National Institutes of Health

and Ubbo Emmius Foundation.

The printing of this thesis was financially supported by the Groningen University

Institute for Drug Exploration (GUIDE), Faculty of Medical Sciences, University of

Groningen, Amgen B.V. Breda, Harlan Nederland and Nederlandse Aardolie

Maatschappij B.V. (NAM).

© 2006 by J. S. G. Yeoh. All rights reserved. No part of this book may be reproduced

or transmitted in any forms or by any means without permission from the author.

ISBN: 90-367-2857-6

Page layout Joyce Yeoh and Chevy Yick

Cover design Josh Harrop and Adam Gourlay, Dept. of Medical Illustration,

University of Aberdeen

Cover illustration Cellular Dreamtime. Oil canvas painting of cells based on

aboriginal dreamtime paintings.

Printed by PrintPartners Ipskamp B.V., Enschede, The Netherlands

TABLE OF CONTENTS

SCOPE OF THESIS 11

CHAPTER 1 FIBROBLAST GROWTH FACTORS AS REGULATORS OF STEM CELL

SELF RENEWAL AND AGING 17

Submitted to Mechanisms of Ageing and Development, in press

CHAPTER 2 FIBROBLAST GROWTH FACTOR-1 AND 2 PRESERVE LONG-TERM

REPOPULATING ABILITY OF HEMATOPOIETIC STEM CELLS IN SERUM-

FREE CULTURES 39

Stem Cells, 2006, 24 (6) 1564 - 1572

CHAPTER 3 EFFECTS OF FIBROBLAST GROWTH FACTOR OVEREXPRESSION AND

CELLULAR LOCALIZATION ON HEMATOPOIETIC STEM CELL

FUNCTION 67

In preparation

CHAPTER 4 MOBILIZED PERIPHERAL BLOOD STEM CELLS PROVIDE RAPID

RECONSTITUTION BUT IMPAIRED LONG-TERM ENGRAFTMENT 105

Submitted

CHAPTER 5 SUMMARIZING DISCUSSION AND FUTURE PERSPECTIVES 133

NEDERLANDSE SAMENVATTING 143

ACKNOWLEDGEMENTS 153

CURRICULUM VITAE 157

Scope of Thesis

Scope of Thesis

12

Scope of Thesis Hematopoietic stem cells (HSCs) are pluripotent cells with the ability to generate

progeny for at least eight major hematopoietic lineages; B lymphocytes, T

lymphocytes, erythrocytes, megakaryocytes/platelets, basophils, eosinophils,

granulocytes and monocytes/macrophages (Figure 1). Mature bloods cells have a

finite lifespan, ranging from a few (neutrophil) to more than 100 (erythrocytes) days

in humans. Thus, the ultimate function of HSCs is to ensure that billions of new blood

cells are produced each day over the lifespan of an organism. As a result of these

properties, HSCs are widely used to restore blood cell production in leukemia and

lymphoma patients that have received cytotoxic therapeutic agents.

HSC

Erythrocyte

Eosinophil

Basophil / Mast cell

T lymphocyte

B lymphocyte

Macrophage

Natural Killer cell

Megakaryocyte

Granulocyte

Neutrophil

Lymphoid progenitor cell

Multipotentprogenitor cell

HSC

Erythrocyte

Eosinophil

Basophil / Mast cell

T lymphocyte

B lymphocyte

Macrophage

Natural Killer cell

Megakaryocyte

Granulocyte

Neutrophil

Lymphoid progenitor cell

Multipotentprogenitor cell

Figure 1: Schematic representation of the HSC differentiation pathway. HSCs are able to self renew

and are responsible for generating a variety specialized blood cells such as erythrocytes, B

lymphocytes, T lymphocytes, granulocytes and basophils.

Scope of Thesis

13

The classical source of HSCs is bone marrow (BM). For more than 40 years clinicians

have been using HSCs isolated from BM for transplantations purposes. Following

migration from the BM, HSCs can also be isolated from the peripheral blood. Due to

its ease of collection and increased rate of engraftment, peripheral blood has become

the primary source of HSCs for medical treatments in the past 10 years. Despite its

prevalent use, few reports exist comparing the quality of blood stem cells and BM

stem cells. This knowledge may be relevant to already established clinical techniques.

The aim of many researchers is to culture and expand primitive, self renewing stem

cells isolated from multiple tissues (such as BM and brain) in order to generate

specific cell types so that they can be used to treat injury or disease. The maintenance

and expansion of stem cells in in vitro studies remains one of the greatest challenges.

Most culture conditions result in a net loss of stem cells indicating that differentiation

is favored over expansion. This is most evident in the hematopoietic system, where

stem cells are rare (< 1 per 105 BM cells) and attempts to expand numbers in culture

have been cumbersome. By using growth factors important during embryonic

development, a phase where stem cells were first generated, it may be possible to

generate and maintain stem cells in vitro. Commonalities may exist between distinct

stem cell systems functioning in different tissues so that all stem cells can be cultured

in a similar fashion with the same growth factors. Finally and most importantly, it

may be possible to genetically modify stem cells to improve their outcome, that is,

increase stem cell expansion whilst maintaining their self renewal property.

What is becoming increasingly evident is that the large family of fibroblast growth

factors (FGFs) plays an important role in regulating and maintaining stem cells. FGFs

belong to one of the largest families of polypeptide growth factors. During

embryogenesis, FGFs govern the development of organs such as the lung and limb. In

adult tissues they maintain tissue homeostasis by playing roles in mitogenesis, cellular

differentiation, cell migration and angiogenesis. This thesis focuses on the role of

FGFs in maintaining and regulating HSCs both in vitro and in vivo.

Chapter 1 provides an overview on the regulatory role of FGFs in maintaining stem

cell self renewal to counteract senescence/aging of HSCs, neural stem cells (NSCs)

and embryonic stem (ES) cells. Aging involves a slow deterioration of tissue function,

including an elimination of new growth and decreased capacity for repair. Aging is

also associated with increased cancer incidence in all tissues that contain stem cells.

In general, tissue homeostasis is lost during aging. These observations suggest a link

Scope of Thesis

14

between aging and stem cell self renewal function. We speculate that FGFs and their

receptors promote self renewal, maintenance and proliferation of HSCs, NSCs and ES

cells in order to sustain tissue homeostasis during aging.

Previous studies from our group demonstrated that the exogenous addition of FGF-1

to unfractionated bone marrow (BM) cultures in serum-free media resulted in a

sustained expansion of cells with lymphoid and myeloid repopulating capacity. FGF-1

was also shown to be involved in serially transplantable long-term repopulating

HSCs. In Chapter 2 we study the effects of exogenous addition of FGF-1, FGF-2 and

the combination of both to unfractionated C57BL/6.SJL (CD45.1) BM cells in serum-

free media. BM cells treated with FGFs for 1, 3 and 5 weeks were mixed with freshly

isolated BM cells from C57BL/6 (B6) mice and transplanted into lethally irradiated

B6 mice.

To further examine the roles of FGF in regulating and maintaining HSCs, in Chapter

3 we retrovirally overexpress FGF-1 and FGF-2 into CD45.1 5-Fluorouracil (5-FU)

BM cells. Additionally, we investigate the role of intracellular FGF localization and

its effects on hematopoietic cells. Site-directed mutagenesis was performed to create

two FGF-1 mutants. The first mutant, S130E, exchanges serine at phosphorylation site

130 for glutamic acid. This is expected to mimic the phosphorylated state of FGF-1

and is therefore constitutively exported into the cytoplasm. In the second mutant, the

serine is exchanged for alanine (S130A), thereby mimicking the unphosphorylated

state of FGF-1 and hypothesized to retain FGF-1 in the nucleus. In an attempt to

translocate FGF-2 from the cytoplasm into the nucleus, an artificial nuclear

localization signal (NLS) was inserted upstream of the FGF-2 gene (NLS/FGF-2).

Well-established in vitro assays exist for measuring the growth of precursors in the

hematopoietic system. However, currently the only conclusive assay for HSCs is to

assess their in vivo ability to give rise to the lymphoid and myeloid lineage in a

lethally irradiated host following transplantation. This assay, known as the

competitive repopulation assay, is thoroughly examined in Chapter 4. Mobilized

peripheral blood (MPB) has replaced BM cells as the primary source of hematopoietic

stem cells for clinical transplantations due to its ease of retrieval and faster

regeneration of neutrophils and platelets. Using the competitive transplantation assay,

in Chapter 4 we directly examine the quality and frequency of MPB stem cells

compared to normal BM stem cells.

Scope of Thesis

15

Finally, in Chapter 5 we briefly summarize and discuss the data obtained from

previous chapters followed by future perspectives.

16

17

CHAPTER 1

Fibroblast growth factors as regulators of stem cell

self renewal and aging

Joyce S. G. Yeoh and Gerald de Haan

Department of Cell Biology, Section Stem Cell Biology, University

Medical Centre Groningen, The Netherlands

Submitted in Mechanisms of Ageing and Development, in press

Fibroblast growth factors regulates stem cell self renewal and aging

18

Abstract Organ and tissue dysfunction which is readily observable during aging results from a

loss of cellular homeostasis and reduced stem cell self renewal. Over the past 10

years, studies have been aimed at delineating growth factors that will sustain and

promote the self renewal potential of stem cells and support the expansion of

primitive stem cells in vitro and in vivo. Recently, strong evidence is emerging

indicating that fibroblast growth factors (FGFs) play a crucial role in stem cell

maintenance. FGFs belong to a family of polypeptide growth factors that are involved

in multiple functions including cell proliferation, differentiation, survival and motility.

In this review, we discuss the regulatory role of FGFs on hematopoietic stem cells

(HSCs), neural stem cells (NSCs) and embryonic stem (ES) cells in maintaining stem

cell self renewal. These findings are useful and important to further our knowledge in

stem cell biology and for therapeutic approaches.


19

Introduction FGFs are a family of polypeptide growth factors that are involved in embryonic

development and adult tissue homeostasis. Twenty-two FGF genes have been

identified in the genome of humans and mice1. During embryogenesis, FGFs (FGF-7,

-8 and -10) govern the development of parenchymal organs, such as the lung and

limb2-4. In adults, FGFs are important in maintaining general tissue homeostasis,

including functions such as tissue repair and regeneration, metabolism and

angiogenesis5;6.

Stem cells are active during embryonic development and in most adult tissues, playing

an important role in normal homeostasis and tissue integrity. In adult tissues, stem

cells have been identified or isolated from an ever increasing array of tissues, such as

bone marrow7;8, skin9-12, intestinal epithelium13, myocardium14;15 and brain16-18.The

unique ability of tissue specific stem cells to self renew, differentiate and proliferate is

critically important to an organism during development and to maintain tissue

homeostasis. Stem cell self renewal ensures that a potentially unlimited supply of

stem cells exists, irrespective of demands put on the system by repetitive cell turn-

over during aging or stress.

However, unlimited stem cell self renewal may not exist, as stem cells are exposed to

both intrinsic and extrinsic effectors of damage. For example, the skin will age more

rapidly for an individual who has more exposure to ultraviolet rays than one who does

not. The liver of an alcoholic would have aged more rapidly than a teetotaler and

chemotherapeutic drugs used to treat cancer will have deleterious effects on HSCs.

Evidently, exposure to environmental or genetic factors induces differentiation and

apoptosis or inhibits the asymmetrical self renewal division of stem cells. Tissues in

which stem cell self renewal is inhibited would not be able to replenish the

differentiated cells, thereby impending function and integrity. Thus, this suggests that

tissue aging results from stem cell aging which in turn results from a decrease in stem

cell self renewal capacity19-22. Consequently, it has become increasingly important to

study regulators which will enhance stem cell self renewal during aging. Here, we

review recent advances in our understanding of the effects of FGFs on enhanced self

renewal and maintenance of stem cells. Our focus will be on HSC, NSCs and ES cells

as FGFs are crucial growth factors for all three stem cell systems. In addition, these

three stem cell species are by far the best characterized stem cells in in vitro and in


20

vivo models. Finally, these stem cells appear to be of vital relevance in a variety of

age-related diseases, ranging from hematopoietic disorders, cancer, cardiovascular

disease and neurodegenerative disorders.

Fibroblast Growth Factors To date, 22 FGFs have been identified. They range in molecular weight from 17 to 34

kDa and share 13-71% amino acid identity in vertebrates (reviewed by Ornitz and

Itoh, 20011). FGFs mediate their biological responses by binding with high affinity to

cell surface tyrosine kinase FGF receptors (FGFR). Four functional FGFR genes

(Fgfr1-Fgfr4) have been identified in vertebrates1. Following receptor binding, FGFs

induce dimerization and phosphorylation of specific cytoplasmic tyrosine residues.

The phosphorylation of FGFRs triggers the activation of downstream cytoplasmic

signal transduction pathways23. Furthermore, FGFs interact with low affinity to

heparan sulfate proteoglycans (HSPGs)24, which acts to stabilize FGFs and prevent

thermal denaturation and proteolysis. In addition HSPGs are required for FGFs to

activate FGFRs effectively (reviewed by Powers et al. 2000; Dailey et al. 2005 6;25).

The expression of all known FGFs in an array of mouse and human tissues and

organs, was analyzed by researchers at the Genomics Institute of the Novartis

Research Foundation (GNF) and is publicly available in the SymAtlas database

(http://symatlas.gnf.org/SymAtlas/)26. Figure 1.1 illustrates a selection of those FGFs

with the most variable expression levels across various tissues and organs. Most FGFs

are ubiquitously expressed. However, some FGFs are selectively expressed in tissues

and organs such as spleen (FGF-2), blastocysts (FGF-4), kidney (FGF-1), lung (FGF-

1), dorsal root ganglion (FGF-1 and -13), embryo (FGF-5 and -15), salivary gland

(FGF-23), pancreas (FGF-4 and -18) and pituitary (FGF-14). The complex expression

of FGFs suggests that these growth factors play an important role in maintaining

tissue homeostasis in many tissues and organs.


21

Main olfactory epithelium

Brown fatAdipose tissueAdrenal gland

BoneBone marrow

AmygdalaFrontal cortex

PreopticTrigeminal

CerebellumCerebral cortex

Dorsal root gangliaDorsal striatumHippocampusHypothalamus

Olfactory bulbSpinal cord lowerSpinal cord upper

Substantia nigraBlastocysts

Embryo day 10.5Embryo day 6.5Embryo day 7.5Embryo day 8.5Embryo day 9.5

Fertilized eggMammary gland (lact)

OvaryPlacentaProstate

TestisUmbilical cord

UterusOocyte

HeartLarge intestineSmall intestineB220+ B-cellsCD4+ T-cellsCD8+ T-cells

LiverLung

Lymph nodeSkeletal muscle

Vomeronasal organSalivary gland

TonguePancreas

PituitaryDigits

EpidermisSnout epidermis

SpleenStomachThymusThyroid

TracheaBladderKidneyRetina

Relative Expression

Fgf1 gnf1m11999_at Fgf2 gnf1m01118_at Fgf4 gnf1m27000_at



BoneBone marrow


PreopticTrigeminal









UterusOocyte


LiverLung



TonguePancreas

PituitaryDigits




Fgf5 gnf1m11382_a_at

Fgf10 gnf1m12419_at Fgf13 gnf1m00725_at Fgf14 gnf1m24788_at Fgf15 gnf1m01116_a_at

Fgf18 gnf1m27457_at



BoneBone marrow


PreopticTrigeminal









UterusOocyte


LiverLung



TonguePancreas

PituitaryDigits




Relative Expression

Relative Expression

Fg20 gnf1m27635_at Fgf23 gnf1m30275_atBrown fatAdipose tissueAdrenal glandBoneBone marrowAmygdalaFrontal cortexPreopticTrigeminalCerebellumCerebral cortexDorsal root gangliaDorsal striatumHippocampusHypothalamusMain olfactory epitheliumOlfactory bulbSpinal cord lowerSpinal cord upperSubstantia nigraBlastocystsEmbryo day 10.5Embryo day 6.5Embryo day 7.5Embryo day 8.5Embryo day 9.5Fertilized EggMammary gland (lact)OvaryPlacentaProstate

TestisUmbilical cordUterusOocyteHeartLarge intestineSmall intestineB220+ B-cellsCD4+ T-cellsCD8+ T-cellsLiverLungLymph nodeSkeletal muscleVomeronasal organSalivary glandTonguePancreasPituitaryDigitsEpidermisSnout epidermisSpleenStomachThymusThyroidTracheaBladderKidneyRetina

Legend

*900

*50,

000

*200

*85

*300

*450

*90

*9,0

00

*225

*150

*130



BoneBone marrow


PreopticTrigeminal









UterusOocyte


LiverLung



TonguePancreas

PituitaryDigits





BoneBone marrow


PreopticTrigeminal









UterusOocyte


LiverLung



TonguePancreas

PituitaryDigits




Relative Expression

Fgf1 gnf1m11999_at Fgf2 gnf1m01118_at Fgf4 gnf1m27000_at



BoneBone marrow


PreopticTrigeminal









UterusOocyte


LiverLung



TonguePancreas

PituitaryDigits





BoneBone marrow


PreopticTrigeminal









UterusOocyte


LiverLung



TonguePancreas

PituitaryDigits




Fgf5 gnf1m11382_a_at

Fgf10 gnf1m12419_at Fgf13 gnf1m00725_at Fgf14 gnf1m24788_at Fgf15 gnf1m01116_a_at

Fgf18 gnf1m27457_at



BoneBone marrow


PreopticTrigeminal









UterusOocyte


LiverLung



TonguePancreas

PituitaryDigits





BoneBone marrow


PreopticTrigeminal









UterusOocyte


LiverLung



TonguePancreas

PituitaryDigits




Relative Expression

Relative Expression

Fg20 gnf1m27635_at Fgf23 gnf1m30275_atBrown fatAdipose tissueAdrenal glandBoneBone marrowAmygdalaFrontal cortexPreopticTrigeminalCerebellumCerebral cortexDorsal root gangliaDorsal striatumHippocampusHypothalamusMain olfactory epitheliumOlfactory bulbSpinal cord lowerSpinal cord upperSubstantia nigraBlastocystsEmbryo day 10.5Embryo day 6.5Embryo day 7.5Embryo day 8.5Embryo day 9.5Fertilized EggMammary gland (lact)OvaryPlacentaProstate

TestisUmbilical cordUterusOocyteHeartLarge intestineSmall intestineB220+ B-cellsCD4+ T-cellsCD8+ T-cellsLiverLungLymph nodeSkeletal muscleVomeronasal organSalivary glandTonguePancreasPituitaryDigitsEpidermisSnout epidermisSpleenStomachThymusThyroidTracheaBladderKidneyRetina

Legend

*900

*50,

000

*200

*85

*300

*450

*90

*9,0

00

*225

*150

*130

Figure 1.1: Expression levels of FGFs in an array of tissues and organs obtained from SymAtlas

database. Only FGFs showing significant expression are shown in this figure. Further information

regarding the remaining FGFs can be found at http://symstlas.gnf.org/SymAtlas. Horizontal scales are

linear, ranging from zero to the final marked value.


22

Effects of FGFs on stem cells Hematopoietic Stem Cells

HSCs are pluripotent cells, possessing high proliferative and self renewal potential.

They ultimately regenerate and maintain all blood cell types of both the lymphoid and

myeloid lineages, producing billions of new circulating but short-lived cells each day.

In the early 1960s, Till and McCulloch introduced the first quantitative in vivo assay

for stem cells in the bone marrow8. Ever since, researchers have focused on soluble or

cell-bound growth factors that promote HSC self-renewal, maintenance, survival and

proliferation.

Hematopoiesis involves interactions between HSCs and stroma to provide a favorable

microenvironment for the development of progenitors. Stromal cells are an important

source of cytokines that regulate proliferation, differentiation and maturation of

progenitor cells27-29. As hematopoietic regulatory factors, FGFs can potentially exert

their effects at two critical aspects of hematopoiesis: the HSC proper and/or

maintenance of stem cell supporting stromal cells30.

Initial studies demonstrated that FGF-1 induced granulopoiesis31 and

megakaryocytopoiesis32;33. Recent studies have shown that both FGF-1 and FGF-2

can sustain the proliferation of hematopoietic progenitor cells, maintaining their

primitive phenotype34-36. FGF-1 was shown to be involved in the expansion of multi-

lineage, serially transplantable long-term repopulating HSCs34. Most noticeably, we

reported that the culturing of unfractionated mouse bone marrow in serum-free media

supplemented only with FGF-1 resulted in a significant and sustained expansion of

cells with both lymphoid and myeloid repopulating capacity34. Additionally, we

recently reported that culturing bone marrow stem cells in the combination of FGF-1

and FGF-2 for at least five weeks, maintained long-term repopulation (LTR)35. Using

the FGF culture system as a tool, Crcareva et al. demonstrated that FGF-1 expanded

bone marrow cells could be utilized for gene delivery to promote radioprotection and

increase long-term BM reconstitution36. FGF-1 has also been used in combination

with stem cell factor (SCF), thrombopoietin (TPO) and insulin-like growth factor 2

(IGF2) to culture BM HSCs for 10 days. Competitive repopulation analysis revealed a

~20-fold increase in long-term HSCs37.

FGF-2 has been known to enhance the colony stimulating activity of IL-3,

erythropoietin (Epo) and granulocyte macrophage colony stimulating factor (GM-


23

CSF) on hematopoietic progenitor cells derived from normal human adult peripheral

blood and murine bone marrow in vitro38;39. In addition, FGF-2 appears to have a

stimulatory role on murine pluripotent hematopoietic cells in a spleen colony-forming

unit (CFU-S) assay. Murine non-adherent bone marrow cells were infused into

lethally irradiated mice to reconstitute hematopoiesis in vivo. Preincubation of the

marrow cells with FGF-2 lead to an increase in the number of day 9 and day 12 CFU-

S39. Several reports exist demonstrating a role of other FGFs in maintaining

hematopoiesis and HSCs. For example, FGF-4 alters the hematopoietic potential of

human long-term bone marrow cultures by increasing the number of progenitors of

the cultures40.

The stimulation of both early and late hematopoiesis suggests the presence of FGFRs

on both pluripotent and lineage committed progenitors. Fgfr1 and Fgfr2 expression

was detected in murine unfractionated bone marrow cells, and in several purified

mature peripheral cell populations (megakaryocytes and platelets, macrophages,

granulocytes, T and B lymphocytes)32. Previous studies from our group have shown

that Fgfr1, -3 and -4 can be detected on a primitive mouse Lin-Sca-1+c-Kit+ HSC

population34.

Unlike systemically acting growth factors, such as Epo and granulocyte colony

stimulating factor (G-CSF), FGFs probably function locally in a paracrine or

autocrine manner. For example, it is clear that FGF-2 plays a regulatory role in early

and late hematopoiesis possibly by interacting directly with HSCs. However, as FGF-

2 lacks signal sequences it is a poorly secreted protein suggesting that it must be

produced locally by surrounding cells. Thus it is of importance to know which cells

produce the growth factor in situ as these cells may specify stem cell self renewal.

Hematopoietic stem cells are in intimate contact with the bone microenvironment

(niche) and cell-cell contact appears to be responsible for the proliferative capacity of

HSCs41.Within the niche, the bone marrow extracellular matrix is postulated to serve

as a reservoir of growth factors and hematopoietic cytokines42;43. Since FGF-2 was

found to be present in the bone marrow extracellular matrix, the most obvious source

of FGF-2 is the stromal fibroblasts44, which are able to both produce and respond to

FGF-2. FGF-2 was reported to be a potent mitogen for bone marrow stromal cells in

vitro38;45-48. Non-adherent hematopoietic progenitors can grow and differentiate over a

pre-established stromal cell layer in long-term bone marrow cultures. Under these

conditions, FGF-2 stimulated myelopoiesis by increasing the number of non-adherent


24

myeloid precursors48. Clearly, FGFs, in particular FGF-2 affects both stroma and

hematopoietic progenitor cells, indicating that it is involved in both hematopoiesis and

maintenance of the microenvironment.

It is therefore highly probable that besides acting on the stem cell itself, FGFs may

interact with stromal niche cells (Figure 1.2)29;49;50. Both mechanisms may induce the

release of other molecules important for the regulation of cell proliferation. Taken

together, these results indicate that in vitro FGFs play a variety of regulatory roles

which prolong homeostatic tissue renewal. The relevance of these in vitro data for in

vivo stem cell behavior remains to be explored. Most notably, it would be highly

relevant to assess how activities of FGFs provide a buffer against age-related cell

stress that may occur.

Figure 1.2: Mechanism of action of FGFs on HSCs. FGFs may interact directly with the HSC

promoting self renewal and/or interact with progenitor cells, releasing autocrine growth factors or other

molecules important for cell proliferation. FGFs may also interact directly with stromal cells within the

stem cell niche, releasing secondary growth factors necessary for the expansion of HSCs. Both methods

are plausible as receptors for FGFs have been found on HSCs and stromal cells. Red diamonds

represent the FGF ligand, green rectangles represent FGFR and question marks indicate that the

existence of FGF/FGFR complex remains speculative.


25

Neural stem cells

Neurogenesis occurs throughout vertebrate life in the subgranular zone of the

hippocampal dentate gyrus and in the telencephalic subventricular zone (SVZ).

Although neurogenesis persists in the adult, its rate declines with age in rats51, mice52,

monkeys53 and humans54;55. The functional consequence of age-associated reduction

in neurogenesis is not clear. However, restoring neurogenesis may be a strategy for

preventing neural stem cell aging.

Like many other stem cells, neural stem cells (NSCs) possess three cardinal

properties: self renewal, extensive proliferation and the ability to generate functional

end-stage cells such as neurons, glial cells and oligodendrocytes (see review by Gage,

2000; Alvarez-Buylla et al., 2001; Weiss et al., 1996; Seaberg et al., 2003 and Rao,

199956-60). NSCs appear to be present in the SVZ in all vertebrate species tested. They

can be selectively cultured from the central nervous system using only two growth

factors; epidermal growth factor (EGF) and FGF-218;61;62. For example, FGF-2 and

EGF have been used alone and in combination to isolate and maintain stem cells of

the adult SVZ, the spinal cord, adult striatum and hippocampus18;63-69.

In the presence of EGF and/or FGF-2, cultured NSCs in suspension give rise to

clonally expanded aggregates called neurospheres. Cultured neurospheres will

generate neurons and glia when plated without growth factor on adhesive substrates18.

Neurospheres have been derived from adult striatum, hippocampus, mesencephalon,

SVZ and spinal cord18;67;68;70;71. It is important to realize that not all the NSC progeny

in neurospheres are stem cells. Rather, a heterogenous population of cells exists in

which only 10% to 50% of the progeny retain stem cell features and the remaining

cells undergo spontaneous differentiation. Quite similar to the situation in

hematopoiesis, this brings up the concept of a stem cell niche. The cluster of a

heterogeneous population of cells may aid the survival of NSCs in vitro or enable

growth factors such as FGF-2 to act on differentiated/accessory cells, resulting in the

release of additional growth factors which regulate NSC self renewal and

proliferation. Following in vitro culturing differentiating/differentiated cells rapidly

die and only surviving NSCs that retain long-term, self-renewal capacity produce new

neurospheres72. Neurospheres may be cultured for extended periods of time,

representing a renewable source of NSCs that may facilitate neurogenesis. It is

therefore a promising cellular source for biotherapies of neurodegenerative diseases.


26

There is also ample evidence that FGFs are relevant for in vivo neurogenesis. The

intraventricular delivery of FGF-2 increased cell proliferation within the adult

SVZ73;74, however it no longer promoted the generation of pyramidal neurons in the

cerebral cortex75. Further studies report that subcutaneous injections of FGF-2

enhanced dentate neurogenesis in both neonatal and adult brain76;77.

Intracerebroventricular infusion of FGF-2 has been shown to upregulate dentate

neurogenesis in the aged brain78. FGF-2 knock-out mice are viable, fertile and

phenotypically indistinguishable from wild-type. However they do display a reduction

in neuronal density in the motor cortex, neuronal deficiency in the cervical spinal cord

and ectopic neurons in the hippocampal commissure79;80. With these phenotypes, it is

not surprising that FGF-2 plays such a crucial role in neurogenesis and NSCs

regulation. Mice deficient for FGF-481, FGF-882-85, FGF-986 and FGF-1087 are lethal.

Taken together, it is evident that FGF-2 plays a key role in regulating the

proliferation, differentiation and survival of NSCs in vitro and in vivo. Despite a

reduction in basal neurogenesis in dentate gyrus and SVZ the aged mouse retains the

ability to respond to neurogenesis-stimulating effects of growth factors, such as FGF-

278. These observations suggest that a decrease in levels of stem/progenitor cell

proliferation factors, such as FGF-2, in the microenvironment of the subgranular zone

are one of the potential causes of age-related decreases in neurogenesis. Increasing the

concentration of FGF-2 and perhaps other growth factors may decrease the process of

stem cell aging and enhance neurogenesis.

Embryonic stem cells

Embryonic stem (ES) cells are derived from totipotent cells of the inner cell mass of

the early mammalian embryo and are capable of seemingly unlimited and

undifferentiated proliferation in vitro88;89. For unknown reasons they are refractory to

the senescence program that halts proliferation in adult cells. Potentially, the high

levels of telomerase in ES cells contribute to this characteristic indefinite self renewal

potential. As a result, ES cells have much greater developmental potential than adult

stem cells. ES cell lines have been derived from mouse (mES) cells and human (hES).

The factors and culture condition that regulate and mediate self-renewal of mouse and

hES cells appear to be different, although their isolation conditions are similar.

Similar to mES cells, hES cells were initially isolated by culturing inner cell mass

cells on fibroblast feeder layers in medium containing serum90;91. In contrast to mES


27

cells92;93, leukemia inhibiting factor (LIF) does not sustain and support hES cells90;94.

It is now widely accepted that the exogenous addition of FGF-2 to hES cells promote

hES cell self-renewal and the capacity to differentiate into a large number of somatic

cell types95;96. The addition of FGF-2 to medium containing a commercially available

serum replacement enables the clonal culture of hES cells on fibroblasts95. Recently, it

was reported that high doses of FGF-2 are adequate to maintain hES cells over 30

passages under feeder-free and serum-free growth conditions97. Wang et al. showed

that incubation of conditioned media from feeders with a neutralizing antibody against

FGF-2 abrogates the capacity of conditioned media to support hES cells, suggesting

that indeed FGF-2 is an essential factor produced by feeder cells97. The recent

observation that high dose FGF-2 (40ng/ml) can sustain hES cells has been

independently reported, thereby corroborating this important insight98;99. It has

recently been demonstrated that elevated levels of FGF-2, FGF-11, FGF-13 and all

four FGFRs are expressed in undifferentiated hES cells100-105. Correlating with

reported data, SymAtlas database analysis (Figure 1) demonstrates that most FGFs

(15 out of 22) are expressed in blastocysts and fertilized egg of the mouse genome.

The mechanism of action for FGF-2 in maintaining self-renewal of hES cells is still

not understood. Because expression of all four FGFRs was observed in cultured hES

cells106;107, one can speculate that FGF-2 may stimulate undifferentiated hES cell

proliferation directly. Alternatively, FGF-2 may block the differentiation of hES cells,

as FGF-2 has been shown to inhibit maturation of oligodendrocyte precursors108;109.

Together, it is now clear that FGF signaling is unconditionally required for the

sustained self-renewal and pluripotency of hES cells. This knowledge may serve as a

base for future developments in using FGFs to culture undifferentiated hES cells for

cell-based therapies to counteract the process of age-related diseases.

Concluding Remarks and Future Perspectives The behavior of stem cells is carefully regulated to meet the demands of normal

homeostasis of the organism, ensuring that a balance between proliferation, survival

and differentiation exists. For many tissues, this balance is impeded during aging.

Data presented in this review describe the role of FGFs and their receptors in

promoting self-renewal, maintenance and proliferation of HSCs, ES cells and NSCs.

Many studies have shown striking similarities with respect to FGF biology shared


28

between these three stem cell species. However, the molecular mechanisms by which

the effects of FGFs occur in all three types of stem cells remains unknown.

Although most stem cell studies that have been carried out were restricted to the use

of FGF-2. In total, 22 FGFs exist (not including spliced forms). Why has FGF-2

historically been the most commonly used growth factor? Given their pleiotropic

effects and sequence similarities it seems reasonable to argue that at least some other

members of the FGF family will possess interesting stem cell stimulating activity.

Clearly the next challenge will be to examine the effects of the remaining FGFs in

well characterized stem cell systems. Only then would we be able to begin to

understand the complete role of FGFs in regulating stem cell self renewal behavior

and its impact on cellular aging, tissue aging and indeed organismal aging.


29

Acknowledgements This work was supported by the National Institutes of Health (R01HL073710) and by

the Ubbo Emmius Foundation


30

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38

39

CHAPTER 2

Fibroblast growth factor-1 and 2 preserve long-term

repopulating ability of hematopoietic stem cells in

serum-free cultures

Joyce S. G. Yeoh1, Ronald van Os1, Ellen Weersing1,

Albertina Ausema1, Bert Dontje1, Edo Vellenga2, Gerald

de Haan1

1 Department of Cell Biology, Section Stem Cell Biology, University

Medical Centre Groningen, The Netherlands 2 Department of Hematology, University Medical Centre Groningen, The

Netherlands

Stem Cells 2006; 24 (6): 1564 – 1572

Fibroblast growth factors preserve stem cell functioning

40

Abstract In this study we demonstrate that extended culture of unfractionated mouse bone

marrow (BM) cells, in serum-free medium, supplemented only with Fibroblast

Growth Factor (FGF)-1, FGF-2 or FGF-1+2 preserves long-term repopulating

hematopoietic stem cells (HSCs). Using competitive repopulation assays, high levels

of stem cell activity were detectable at 1, 3 and 5 weeks after initiation of culture.

FGFs as single growth factors failed to support cultures of highly purified Lin-Sca-

1+c-Kit+ (LSK) cells. However, co-cultures of purified CD45.1 LSK cells with whole

BM CD45.2 cells provided high levels of CD45.1 chimerism after transplant, showing

that HSC activity originated from LSK cells. Subsequently, we tested reconstituting

potential of cells cultured in FGF-1+2 with the addition of early acting stimulatory

molecules, stem cell factor + interleukin-11 + Flt3 ligand. The addition of these

growth factors resulted in a strong mitogenic response, inducing rapid differentiation

and thereby completely overriding FGF-dependent stem cell conservation.

Importantly, although HSC activity is typically rapidly lost after short term culture in

vitro, our current protocol allows us to sustain stem cell repopulation potential for

periods up to 5 weeks.


41

Introduction Hematopoietic stem cells (HSCs) play a vital role in establishing and maintaining

hematopoiesis throughout life. Key to all stem cell transplantation therapies is the

unique property of HSCs to undergo self-renewal and to functionally repopulate the

tissue of origin when transplanted into a myeloablated recipient.

It has been shown that HSCs can undergo a large number of self-renewing divisions

in vivo, where the actual number of HSCs can increase. For example, in mice, it has

been shown that a single stem cell can regenerate and maintain the entire

lymphohematopoietic system after transplantation into an irradiated or

immunocompromised host1-3.

Recent evidence suggests that signaling molecules involved in embryonic

development, when the hematopoietic system is first formed, play an important role in

regulating stem cell self renewal. These include Wnt, Bmp and Shh family members4-

9. In a recent study we showed that fibroblast growth factor (FGF)-1 is also a potent

stimulator of stem cell activity in vitro10. In addition, we showed that all long term

repopulating hematopoietic stem cell activity is contained in the lineage-depleted,

FGF receptor (FGFR)-positive cell population in mouse bone marrow (BM).

FGF-1 belongs to the family of FGFs of which to date, 22 FGFs and 4 FGF receptors

have been identified in vertebrate genomes11. All FGFs have a high affinity for

heparin and for cell surface heparan sulfate proteoglycan (HSPG)12. This complex

formation is crucial for high affinity binding of FGF to its receptors. Multiple

pleiotropic and overlapping activities of FGF family members have been reported. A

large body of evidence from human disorders and gene knockout studies shows that

FGF pathways are required for vertebrate and invertebrate development13-18. FGFs are

also prominent in the development of the limbs19-21, nervous system22;23, and

angiogenesis24;25. Additionally, several members of the FGF family are potent

inducers of mesodermal differentiation in early embryos26. Interestingly, FGF-2 has

been identified as a strong stimulator of human embryonic stem cell (hESC) self-

renewal. The addition of FGF-2 to serum-free medium allows the clonal culture of

hESCs27. Recently, it has been reported that hESC culture can be simplified by using

high doses of FGF-2 which are adequate to maintain hESCs long-term under feeder-

free and serum-free growth conditions28-30. Interestingly, neural stem cells grown in


42

three dimensional sphere-like structures in vitro also require the presence of FGF-

231;32.

The role of FGFs for in vitro maintenance of hematopoietic stem cells has remained

largely unexplored. In the present study, we compared the growth of HSCs in serum-

free medium supplemented with FGF-1 and/or FGF-2. We show that long-term

repopulating stem cells can be conserved in vitro for periods up to 5 weeks.


43

Materials and Methods Mice

Female C57BL/6 SJL CD45.1 mice, originally obtained from the Jackson Laboratory

(Bar Habor, ME, http://www.jax.org) and bred in our local animal facility were used

as a donor source of HSCs. C57BL/6-Tg(ACTB-EGFP)10sb/J transgenic green

fluorescent protein (GFP) mice originally purchased from The Jackson Laboratory

were bred in our local animal facility and also used in certain experiments. Wild type

female C57BL/6 mice were purchased from Harlan (Horst, The Netherlands,

www.harlaneurope.com) and maintained under clean conventional conditions in the

animal facilities of the Central Animal Facilities, University of Groningen (The

Netherlands). Mice were fed ad libitum with food pellets and acidified tap water (pH

= 2.8). All animal procedures were approved by the local animal ethics committee of

the University Medical Centre Groningen.

Hematopoietic Cells

Mice were sacrificed by cervical dislocation and BM cells were obtained by crushing

both femora. Marrow cells were resuspended in α-minimum essential medium (α-

MEM; Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com) supplemented with

2% fetal calf serum (FCS; Gibco-BRL). The cell suspensions were filtered through a

100μM cell strainer (BD Falcon, Two Oak Park, MA http://www.bdbioscience.com)

to remove debris. Cells were counted on a Coulter Counter Model Z2 (Coulter

Electronics, Hialeah, FL, http://www.beckmancoulter).

Stem Cell Expansion Culture System

Unfractionated C57BL/6.SJL CD45.1 BM cells were cultured at 5 x 106 cells per well

in a 6 well plate (Corning Incorporated, Corning, NY, http://www.corning.com) in

StemSpan serum free medium (Stem Cell Technologies; Vancouver, Canada,

http://www.stemcell.com) in the presence of 10ng/ml recombinant human FGF-1

(Gibco, Grand Island, NY, http://www.invitrogen.com), or with 10ng/ml FGF-2

(Sigma-Aldrich, St Louis, http://www.sigmaaldrich.com), or a combination of both

cytokines at 10ng/ml each. Culture media was also supplemented with 10μg/ml

heparin (H3149 Sigma-Aldrich). In some experiments, unfractionated C57BL/6.SJL

5.1 cells isolated and cultured with StemSpan serum-free medium, 10μg/ml heparin


44

and FGF-1+2 were treated with a cocktail of hematopoietic growth factors (GFs). A

cocktail of SCF (300ng/ml) (Amgen, Thousand Oaks, CA), interleukin (IL)-11

(20ng/ml) (R&D Systems, Minneapolis, http://www.rndsystems.com) and Flt3 ligand

(Flt3L) (1ng/ml) (Immunex, Seattle, http://immunex.com) was added to the cultures

for 1, 3 and 5 weeks. Non-adherent cells were harvested weekly, counted to

determine growth kinetics and re-introduced into the expansion culture and fresh GFs

were added to the culture. At 1, 3 and 5 weeks of culture, non-adherent and adherent

cells were harvested and counted in preparation for cell analysis and in vivo

transplantation assay into lethally irradiated C57BL/6 mice.

Isolation of Lin-Sca-1+c-Kit+ Cells

Freshly isolated C57BL/6 BM cells were stained with biotinylated lineage-specific

antibodies (Mouse Lineage Panel, containing anti-CD45R, anti-CD11b, anti-TER119,

anti-Gr-1 and anti-CD3e (BD Pharmingen, San Diego,

http://www.bdbiosciences.com/pharmingen), FITC-anti-Sca-1 and APC-anti-c-kit

(BD Pharmingen). Lin-Sca-1+c-Kit+ cells were stained as described33. Cells were

either analyzed on the FACS Calibur (Becton, Dickinson and Company, San Jose,

CA, http:www.bd.com) or sorted by a MoFlow cell sorter (DakoCytomation, Fort

Collins, CO, http://www.dako.com).

Cell Analysis

FGF-expanded cells were spun for cytospin preparation. Cytospin preparations were

stained with May-Grünwald-Giemsa. Cytospots were washed with distilled water and

allowed to air dry before analysis under a microscope.

Levels of chimerism were determined by detecting the presence of GFP or CD45.1

and CD45.2 positive cells in transplanted mice. To detect CD45.1 and CD45.2

positive cells, cells were stained with anti-CD45.2 (FITC) and CD45.1

(phycoerythrin) antibodies (BD Pharmingen) for 30 minutes and analyzed on a flow

cytometer (FACS Calibur; Becton, Dickinson and Company).

Cobblestone Area Forming Cell Assays

Cobblestone area forming cell (CAFC) assays were performed as described34 to assess

the number of hematopoietic progenitor cells (day 7 CAFCs) or more primitive stem

cells (day 35 CAFCs) in the FGF expanded cultures. Adherent and non-adherent cells


45

were collected and seeded in limiting dilution in 96-well plates (Corning Incoporated)

containing a pre-established fetal bone marrow-derived-1 stromal layer. The cells

were cultured in Iscove’s modified Dulbecco’s modified Eagle’s Medium (Gibco-

BRL, Paisley, Scotland, http://www.gibcobrl.com) supplemented with 20% horse

serum at 34oC in a 10% CO2 incubator. On a weekly basis, wells were scored and half

the volume of the medium was changed. Wells were scored positive if cobblestone

areas were present. P values were used to test the statistical significance of different

groups. The student’s t-test was used assuming unequal variances of the two

variables. The Poisson-based limiting dilution analysis calculation was used with a

95% confidence interval to determine significant differences at p < .05. As previously

reported, quantification of CAFCs at days 7 and 35 was performed by using

maximum likelihood ratio method35.

In Vivo Transplantation Assays

Female C57BL/6 mice were used to provide competitor cells and as recipient mice.

BM cells were obtained by flushing the femoral content 3 times with α-MEM

supplemented with 2% FCS. Recipient mice were irradiated with 9.5Gy γ-rays

(0.7026Gy/minute) in a IBL 637 Cesium 137 source (CIS bio-international, Gif-sur-

Yvette, France, http://www.cisbiointernational.fr), 24 hours prior to transplantation.

For competitive repopulation determination, varying doses of cultured BM cells were

mixed with a constant number of BM competitor cells. Thus, recipient mice were

intravenously transplanted with different dilutions of expanded stem cells, with or

without 2 x 105 life-sparing C57BL/6 BM competitor cells. Each transplant group

consisted of 6 recipients. After transplantation, blood samples (60μl) were taken

monthly from the retro-orbital sinus for flow cytometer analysis. At the time of

sacrifice, chimerism in BM samples was analyzed by fluorescence-activated cell

sorting (FACS) in the same manner. For each recipient, the competitive repopulating

index (CRI) was determined. CRI is a relative measure of the competitive ability of

cultured cells in comparison with that of fresh BM cells. The CRI was calculated by

using the following formula:

ted transplancells competitor tocells cultured of Rationcirculatio in the cells competitor tocells cultured of RatioCRI =


46

A CRI value of 1 indicates by definition that cultured cells and competitor cells have

equal competitive ability. The repopulation ability of our cells can also be measured

in repopulation units (RU). The RU takes into account the total number of cells

generated. Each RU is equivalent to the repopulation function of 100 000 competitor

BM cells. The RU was calculated using the following formula:

( ) RU competitor of No. x ncirculatio in the cells competitor tocells cultured of Ratio RU =

( ) ( )( )cells ted transplanofNumber

per well cells cultured ofnumber Total x RU ofNumber RU/well =


47

Results

Cell Growth Kinetics and Cell Morphology

We used FGF-1 and FGF-2 separately and the combination of both FGFs as the only

stimulus in serum-free media to culture CD45.1 BM cells for a period up to 5 weeks.

One and 3 weeks following initiation of culture, a decrease in the number of cells was

observed. The number of cells per well had dropped dramatically from 5 x 106 to an

average of approximately 5 x 105 cells for all FGF conditions (Figure 2.1A). Five

weeks after the initiation of culture, cells treated with FGF-1 and/or FGF-2 had

increased close to the input cell number, whereas cells cultured only in serum-free

media remained low throughout the 5-week culture period at 1 x 105 (Figure 2.1A).

As a first screen test, prior to in vivo assays, in vitro CAFC assays were setup. CAFC

subsets were quantified in cells harvested from the FGF-1+2 expansion cultures at 3

and 5 weeks. The absolute number of day 7 CAFCs that were harvested from 3-week

cultures had increased 1.5- fold (Figure 2.1B). Interestingly, a substantial 26-fold

expansion of day 7 CAFCs was observed at the 5 week culture time point. In contrast,

day 35 CAFCs at week 3 cultures, were slightly lower than input, whereas a 3.5-fold

expansion was apparent after 5 weeks of culture (Figure 2.1B).

May-Grunwald-Giemsa staining of the starting cell population (Figure 2.1C) and of

FGF-1+2-treated cells showed an accumulation of macrophages and blast-like cells

after 3 and 5 weeks of culture (Figure 2.1D and 2.1E). The presence of blast-like cells

and extensive CAFC activity indicated the possible existence of immature cells with

stem cell properties in FGF stimulated cultures.


48

1

10

100

1000

10000

input Week 3 Week 51

10

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1000

10000

100000

input Week 3 Week 5

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Weeks in culture

A.

Day 0 Week 3 Week 5

C. D. E.

Num

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ls/w

ell (

x106

)

B.

Num

ber o

f C

AFC

/wel

l

CAFC Day 35: FGF-1+2 culturesCAFC Day 7: FGF-1+2 cultures

No FGFs

FGF-1

FGF-2

FGF-1+2

P<0.05P<0.05

N.S.N.S.

1

10

100

1000

10000

input Week 3 Week 51

10

100

1000

10000

100000

input Week 3 Week 5

0

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Weeks in culture

A.

Day 0 Week 3 Week 5

C. D. E.

Num

ber o

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ell (

x106

)

B.

Num

ber o

f C

AFC

/wel

l

CAFC Day 35: FGF-1+2 culturesCAFC Day 7: FGF-1+2 cultures

No FGFs

FGF-1

FGF-2

FGF-1+2

P<0.05P<0.05

N.S.N.S.

Figure 2.1: Cell growth, morphology and phenotype of cells. (A): Growth kinetics of unfractionated

bone marrow (BM) cells in culture for 5 weeks. These are 4 representative cultures out of 30 cultures.

Some were performed in 25cm2 flasks; the remaining in 6-well plates. Cells were cultured in serum-

free medium, supplemented with FGF-1, or FGF-2, or FGF-1+2 in the presence of 10µg/ml heparin.

(B): Unfractionated BM cells and cells cultured for 3 and 5 weeks with FGF-1+2, were placed in

limiting dilutions in a 96-well plate and absolute numbers of day 7 and 35 CAFCs were compared with

input cells. The input values refer to freshly isolated untreated bone marrow cells. Day 7 and 35 CAFC

activity was higher for cells after a culturing period of 5 weeks with FGF-1+2; p-values were calculated

using Student’s t test and Poisson-based limiting dilution analysis was used to determine the CAFC

frequency. (C): May-Grünwald-Giemsa staining was performed on BM cells cultured in the presence of

FGF-1+2 at day 0 of initiation of culture. (D): May-Grünwald-Giemsa staining was performed on BM

cells cultured in the presence of FGF-1+2 at 3 weeks after initiation of culture. (E): May-Grünwald-

Giemsa staining was performed on BM cells cultured in the presence of FGF-1+2 at 5 weeks after

initiation of culture; N.S., not significant.


49

Long-Term Competitive In Vivo Reconstitution of FGF Expanded Cells

To assess whether cells cultured with FGF-1, FGF-2 or FGF-1+2 contained stem cell

activity, we competitively transplanted congenic B6.CD45.1 or transgenic B6.GFP+

FGF expanded cells with CD45.2 BM cells at 1, 3 and 5 weeks after the initiation of

culture. In each group of transplants, 6 recipient mice were transplanted. Animals

receiving 1 week cultured cells were transplanted with 2.5 x 105 cultured cells and 2.5

x 105 B6 CD45.2 BM cells. As shown in Figure 2.2A and Table 2.1, after 1 week of

culturing, the CRI of FGF-1+2 cultured cells, was approximately 50, 18 weeks after

transplant. Interestingly, CRI levels of FGF-1+2 cultured cells increased with time,

suggesting engraftment of relatively more cells with long-term repopulation ability.

Average chimerism levels after 18 weeks were 96% (Table 2.1). After 3 weeks of

culturing, recipient mice received 1.8 x 105, 2.2 x 105, and 3.5 x 105 FGF-1, FGF-2

and FGF-1+2 cultured cells, respectively, together with 2 x 105 B6 CD45.2

competitor cells. Chimerism levels of ~80% were achieved, corresponding to a CRI

level of approximately 5, 16 weeks post-transplant. No significant differences were

observed between the FGF groups (Figure 2.2B and Table 2.1). As expected, cell

cultured for 3 and 5 weeks in serum-free medium without any supplements had no

long-term reconstituting activity. Remarkably, 5 weeks after culturing 1.2 x 106 FGF-

2 and 1.1 x 106 FGF-1+2 cells still outcompeted 2 x 105 freshly isolated BM cells,

although CRI values dropped significantly compared to cells cultured for 1 or 3 weeks

(Figure 2.2C; Table 2.1). Low CRI values of 0.5 ± 0.2 for FGF-1 cultured cells were

obtained, suggesting that these cells provide little competitive repopulation ability

(Figure 2.2C) even though 1.62 x 106 cells were transplanted with 2 x 105 B6 CD45.2

BM cells. The reconstitution activity of FGF cultured cells as indicated by the total

number of repopulation units/well (RU) correlates with the CRI values (Figure 2.2D;

Table 2.1). A 5-fold increase in RU was observed with cells treated with FGF-1+2 for

1 week. After 5 weeks of culturing, both FGF-2 and FGF-1+2 cultured cells had a 1.5-

fold increase in RU compared to input cells (Figure 2.2D; Table 2.1).


50

00.5

11.5

22.5

33.5

0 5 10 15 20

0

2

4

6

8

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12

0 5 10 15 20

0102030405060708090

0 5 10 15 20

Weeks post-transplant

Ave

rage

CR

IA

vera

ge C

RI

Ave

rage

CR

I

A.

C.

B.

1 week

3 week

5 week

FGF-1FGF-2

No FGFsFGF-1+2

D.

FGF-1+2FGF-2

FGF-1No FGFs

1 3 5

RU

/wel

l

Weeks in culture

00.5

11.5

22.5

33.5

0 5 10 15 20

0

2

4

6

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0102030405060708090

0 5 10 15 20

Weeks post-transplant

Ave

rage

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IA

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ge C

RI

Ave

rage

CR

I

A.

C.

B.

1 week

3 week

5 week

FGF-1FGF-2

No FGFsFGF-1+2

FGF-1FGF-2

No FGFsFGF-1+2

D.

FGF-1+2FGF-2

FGF-1No FGFs

1 3 5

RU

/wel

l

Weeks in culture

FGF-1+2FGF-2

FGF-1No FGFs

1 3 5

RU

/wel

l

Weeks in culture

Figure 2.2: In vivo competitive transplantation assay of FGF cultured cells. (A): B6 CD45.1 cells

cultured in serum-free medium alone, or in the presence of FGF-1, FGF-2 or both FGF-1+2 were

transplanted into lethally irradiated B6 CD45.2 recipient mice after 1 week of culture. (B): B6 CD45.1

cells cultured in serum-free medium alone, or in the presence of FGF-1, FGF-2 or both FGF-1+2 were

transplanted into lethally irradiated B6 CD45.2 recipient mice after 3 weeks of culture. (C): B6 CD45.1

cells cultured in serum-free medium alone, or in the presence of FGF-1, FGF-2 or both FGF-1+2 were

transplanted into lethally irradiated B6 CD45.2 recipient mice after 5 weeks of culture. For week 1

culture, mice were transplanted with 2.5 x 105 cultured cells and 2.5 x 105 B6 CD45.2 bone marrow

(BM) cells. Recipients receiving 3 weeks cultured cells were transplanted with 1.8 x 105 FGF-1, 2.2 x

105 FGF-2, 3.5 x 105 FGF-1+2 and 7.5 x 104 no FGF treated cells with 2 x 105 B6 CD45.2 BM cells. 5

weeks after culturing 1.6 x 106 FGF-1, 1.2 x 106 FGF-2, 1.1 x 106 FGF-1+2 and 4.8 x 105 no FGF

treated cells were transplanted into recipient mice with 2 x 105 B6 CD45.2 BM cells. Average CRI ±


51

SD was calculated in each group consisting of 6 mice. (D): The absolute number of repopulating units

of cultured cells was compared to input cells. Each RU is equivalent to the repopulation function of

100,000 competitor bone marrow cells. Therefore, at initiation of culture, 5 x 106 whole BM cells

contain 50 RU. Following 1 week of culturing in FGF-1+2, 253 RU were generated. Both FGF-2 and

FGF-1+2 cultured cells after 5 weeks produced 75 RUs. Abbreviations: CRI, competitive repopulation

index; FGF, fibroblast growth factor; RU, repopulation unit.

Table 2.1 In vivo reconstitution of FGF expanded cells

The data presented in this table were used to generate Figures 2.1 and 2.2. All mice were transplanted

in competition with 2 x 105 B6 CD45.2 BM cells. N.D. indicates that the experiment was not

performed.

* Each RU is equivalent to the repopulation function of 100,000 competitor bone marrow cells.

Therefore, at day 0, 5 x 106 whole BM cells contain 50 RU

Abbreviations: CRI, competitive repopulation index; FGF, fibroblast growth factor; N.D., experiment

not performed; RU, repopulation unit.

Radioprotection and Long-Term In vivo Reconstitution of FGF-Expanded Cells

To test the ability of expanded cells in a more clinically relevant model, 1 x 104 and 5

x 104 FGF-1+2 expanded cells were transplanted in lethally irradiated C57BL/6 mice

without competitor cells. After 3 weeks of culture, 1 x 104 and 5 x 104 cells were able

to stably engraft into most recipient mice providing long-term repopulation.

Transplantation of 1 x 104 cells provided radioprotection to 60% of animals (Figure

2.3A). This value increased to 80% when 5 x 104 FGF-1+2 expanded cells were

transplanted (Figure 2.3A). The survival rate was higher when culture time was

extended to 5 weeks with 1 x 104 expanded cells providing radioprotection to 80% of

animals (Figure 2.3A). A further increase in survival was observed with 5 x 104

expanded cells providing radioprotection to 100% of the mice (Figure 2.3A). In all

cases a radioprotection endpoint of 4 weeks after transplant was used. Animals that

died of hematopoietic failure, died within 14 days post-transplant. Transplantation of

1 x 104 cells cultured for 3 weeks resulted in an average chimerism of 50%, whereas 5

x 104 cells engrafted with an average chimerism of 75%, 28 weeks after transplant

(Figure 2.3B). Chimerism results from transplants carried out with cells from 5 week


52

cultures are shown in Figure 2.3C. With 1 x 104 FGF-1+2 expanded cells, engraftment

levels steadily increased, stabilized after 8 weeks and 26 weeks after transplant donor

contribution was more than 90%. Mice transplanted with 5 x 104 cells showed an

average level of chimerism of more than 95%.

0

20

40

60

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0

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0 5 10 15 20 25 30

0

20

40

60

80

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3 5

A.

B.

C.

% C

D4 5

.1 C

h im

eris

m

Weeks after transplant

% S

urvi

val

Weeks in culture


% C

D4 5

.1 C

h im

eris

m

5 x 104 cells

1 x 104 cells

5 x 104 cells

1 x 104 cells

3 week culture

5 week culture

5 x 104 cells

1 x 104 cells

0

20

40

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0 5 10 15 20 25 30

0

20

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3 5

A.

B.

C.

% C

D4 5

.1 C

h im

eris

m


% S

urvi

val

Weeks in culture


% C

D4 5

.1 C

h im

eris

m

5 x 104 cells

1 x 104 cells

5 x 104 cells

1 x 104 cells

3 week culture

5 week culture

5 x 104 cells

1 x 104 cells

5 x 104 cells

1 x 104 cells

Figure 2.3: Radioprotective potential of fibroblast growth factor (FGF)-expanded cells. (A): Survival

rate of mice transplanted only with expanded cells. (B): Unfractionated B6 CD45.1 bone marrow (BM)

cells were cultured in FGF-1+2 for 3 weeks. (C): Unfractionated B6 CD45.1 bone marrow (BM) cells

were cultured in FGF-1+2 for 5 weeks. Recipient CD45.2 C57BL/6 mice were lethally irradiated and

transplanted with 1 x 104 or 5 x 104 FGF-1+2 cultured cells without life sparing competitor cells.

Analysis of chimerism was performed and average results are shown.


53

Growing of Lin-Sca-1+c-Kit+ Cells in Cocultures

In our 3 and 5 week FGF cultures described above, the percentage of LSK cells was

analyzed prior to transplantation. After 3 weeks of culturing LSK frequencies of FGF-

1 cultured cells was 0.7% whilst FGF-2 and FGF-1+2 cells were 1.3%. LSK

frequencies increased after 5 weeks of culturing to 0.9% for FGF-1 cells and 2.8% for

FGF-2 and FGF-1+2 cells (data not shown). Despite the high percentage of LSK cells

in these cultures compared to normal BM cells which have a LSK frequency of

∼0.2%, we were not successful at culturing purified LSK HSCs or bulk Lin+ and Lin-

cells in serum-free medium supplemented with FGF-1+2 (data not shown). Thus, we

speculated that either the stem cell growth in unfractionated BM cultures did not

originate from LSK cells, or alternatively, that an accessory population of cells

contained within the bone marrow was required. To test this hypothesis, we sorted

LSK cells from B6 CD45.1 mice and co-cultured 7 x 103 and 5 x 104 of these cells in

the presence of 5 x 106 CD45.2 unfractionated BM cells. FACS analysis showed that

5 x 106 unfractionated CD45.2 BM cells contained ~5,500 LSK cells. Purified stem

cells and whole BM were cocultured for 5 weeks. After culture, all cells were

harvested and 2 x 105 cells were transplanted into lethally irradiated recipients without

competitors. The percentage of white blood cells originating from the purified LSK

CD45.1+ fraction or from the CD45.2+ unfractionated cells was assessed in the

recipients. If only LSK cells were responsible for the FGF stimulated stem cell

activity in unfractionated BM, we would expect chimerism levels of the sorted 7,000

CD45.1 LSK cells to reach or come close to 60% (7,000 CD45.1 LSK cells + 5500

LSK CD45.2 cells: 7,000/(7,000+5,500) = 56%). Strikingly, 16 weeks post-transplant,

chimerism levels had increased to 51%, 52% and 64%, implying that indeed all FGF

induced stem cell activity is derived from the LSK population, (Figure 2.4A).

Chimerism levels in recipients transplanted with 5 x 104 CD45.1 LSK cultured in 5 x

106 CD45.2 unfractionated BM ranged from 70% to 99%, 16 weeks after transplant

(Figure 2.4B). Engraftment was seen in all mice transplanted. The estimated expected

level of chimerism in these animals was 50,000/(50,000+5,500) = 90%, clearly well in

range with the experimental findings. These results suggest that FGFs may be acting

both on LSK cells and on other cell types indirectly affecting LSK cells to induce

stem cell activity in vitro.


54

0

20

40

60

80

100

0 5 10 15 20 25

0

20

40

60

80

100

0 5 10 15 20 25

A.

B.

Expected

Expected%

CD

45.1

Chi

mer

ism



% C

D45

.1 C

him

eris

m

0

20

40

60

80

100

0 5 10 15 20 25

0

20

40

60

80

100

0 5 10 15 20 25

A.

B.

Expected

Expected%

CD

45.1

Chi

mer

ism



% C

D45

.1 C

him

eris

m

Figure 2.4: Highly purified CD45.1 Lin-Sca-1+c-Kit+ (LSK) cells co-cultured with unfractionated

CD45.2 bone marrow (BM) cells. (A): Seven thousand B6 CD45.1 LSK cells were cocultured with 5 x

106 B6 CD45.2 whole bone marrow cells (estimated to contain 5500 CD45.2 LSK cells) for 5 weeks in

serum-free medium supplemented with fibroblast growth factor (FGF)-1+2. Subsequently, 2 x 105

cultured cells were transplanted into B6 CD45.2 mice without additional competitor cells. At start of

the culture, the percentage of CD45.1 LSK cells in the co-culture was 7,000/(7,000+5500) = 56%. 12

weeks after transplant, chimerism levels of all mice steadily increased to an average of 57%. (B): Five

week coculture of 50,000 B6 CD45.1 LSK and 5 x 106 B6 CD45.2 whole BM cells was set-up and

similarly transplanted. The percentage of CD45.1 LSK cells in this culture was 50,000/(50,000+5,500)

= 90%. Chimerism levels in transplanted recipients rapidly increased reaching 83%. Chimerism levels

for each transplanted recipient are denoted by different symbols.


55

Effect of SCF, IL-11 and Flt3L on FGF-1+2 Induced Clonogenic Activity In Vitro

Although single LSK cells cultured in FGF-1+2 did not divide, they did remain

visible for 7 days (data not shown). Thus it appeared that the addition of FGF-1+2 to

cell cultures prolonged the lifespan of the cells, but did not induce a strong enough

mitogenic signal. More classical hematopoietic GFs such as SCF, IL-11 and Flt3L,

have a much stronger proliferating effect and are shown to maintain stem cells in

short-term cultures36. Therefore we cultured cells with a cocktail of SCF, IL-11 and

Flt3L with or without the addition of FGF-1+2. After 5 weeks of culturing, cell

numbers of GF treated cells had exponentially increased from 5 x 106 to 7 x 108,

similar to GF + FGF-1+2 cultured cells (Figure 2.5A; Table 2.2). We next tested

whether FGFs would be able to maintain stemness of cells when used in combination

with SCF, IL-11 and Flt3L, which provides stronger mitogenic signals.

CAFC assays were carried out to determine clonogenic activity of cells treated with a

cocktail of growth factors. We observed a significant (p < .05) increase in day 7

CAFC activity after 3 weeks of culturing in GFs (Figure 2.5B). CAFC day 7 numbers

for cells cultured in the presence of GFs and GF + FGF-1+2, increased 222- and 273-

fold, respectively, over the input value, whereas cells treated with FGF-1+2 had a

modest 1.5-fold increase over the input value. A contrasting pattern was observed

when primitive day 35 CAFC numbers were evaluated (Figure 2.5B). Day 35 CAFCs

of 3 week GFs alone, or GF + FGF-1+2 cultures were below the detection level (less

than 1 CAFC per 1x106). Nevertheless, FGF-1+2 cultured cells were able to maintain

day 35 CAFC activity.

Effect of GFs on FGF-1+2 Induced Stem Cell Activity In Vivo

Finally, we determined whether cells cultured in FGF-1+2 with or without SCF, IL-11

and Flt3L provided engraftment in vivo. To this end, varying cell doses ranging from

1.8 x 105 to 2 x 106 B6 CD45.1 cultured cells were transplanted in competition with 2

x 105 freshly isolated B6 CD45.2 BM cells and compared with results for transplanted

FGF-1+2 cells (Figure 2.2). Although highly elevated CRI levels were observed 16

weeks after transplant, when FGF-1+2 treated cells were cultured for 1 week, the CRI

had dropped to 1 for cells cultured with GFs alone or GF+FGF-1+2 treated cells

(Figure 2.5C; Table 2.2). Continued culturing of cells for 3 and 5 weeks decreased

CRI values for all conditions. All competitive repopulation ability was lost after 3


56

weeks of culturing or cells treated with GF alone, or with the addition of GF + FGF-

1+2 (Figure 2.5C; Table 2.2).

1

10

100

1000

0 1 2 3 4 5 6

Num

ber o

f cel

ls/w

ell (

x106

)

1

10

100

1

10

100

1000

input FGF 1+2 GF only GF+FGF

1

10

100

1000

10000

100000

1000000


CR

IA.

B.

Num

ber o

f C

AFC

/wel

l

CAFC Day 7: 3 week cultured cells CAFC Day 35: 3 week cultured cells

C.

Weeks in culture

N.D. N.D.

P<0.05

N.S.N.S.

GF (SCF+IL-11+Flt3L) treated cells

GF + FGF-1+2 treated cells

1 3 5GF

GF+FGF-1+2FGF-1+2

Weeks in culture

1

10

100

1000

0 1 2 3 4 5 6

Num

ber o

f cel

ls/w

ell (

x106

)

1

10

100

1

10

100

1000


1

10

100

1000

10000

100000

1000000


CR

IA.

B.

Num

ber o

f C

AFC

/wel

l

CAFC Day 7: 3 week cultured cells CAFC Day 35: 3 week cultured cells

C.

Weeks in culture

N.D. N.D.

P<0.05

N.S.N.S.





1 3 51 3 5GF

GF+FGF-1+2FGF-1+2

Weeks in culture Figure 2.5: Effects of GF treatment on FGF-cultured cells. (A): Growth kinetics of unfractionated bone

marrow (BM) cells cultured with a cocktail of GFs (SCF, IL-11 and Flt3L) in the presence or absence

of FGF-1+2. Cell growth exponentially increased from 5 x 106 to 7 x 108. (B): Day 7 and 35 CAFC

content of unfractionated BM cells and cells cultured for 3 weeks with FGF-1+2, GFs (SCF, IL-11 and

Flt3L) alone and GFs + FGF-1+2. No day 35 CAFC activity was observed for cells cultured with GFs

alone and GFs + FGF-1+2; p-values were calculated using Student’s t test, and Poisson-based limiting

dilution analysis was used to determine the CAFC frequency. (C): CRI ± SD values (shown in Table 2)

for mice transplanted with varying cell doses of FGF-1+2, GF and GF + FGF-1+2 cultured cells in

competition with 2 x 105 B6.CD45.2 cells. Data are the same as in Figure 2 for FGF-1+2 and are only

included for comparison. Abbreviations: CAFC, cobblestone area-forming cell; CRI, competitive


57

repopulation index; FGF, fibroblast growth factor; Flt3L, Flt3 ligand; GF, growth factor; IL,

interleukin; N.D., not detected; N.S., not significant; SCF, stem cell factor.

Table 2.2: Effect of the addition of stem cell factor, interleukin-11 and Flt3 ligand on FGF-1+2

induced stem cell activity

The data on this table were used to generate Figure 2.5. Results shown for FGF-1+2 condition are also

presented in Table 1 and are added for comparison. All recipients were transplanted in competition

with 2 x 105 B6 CD45.2 bone marrow (BM) cells.

* Each RU is equivalent to the repopulation function of 100,000 competitor bone marrow cells.

Therefore, at day 0, 5 x 106 whole BM cells contain 50 RU

Abbreviations: CRI, competitive repopulation index; FGF, fibroblast growth factor; GF, growth factor;

RU, repopulation unit.


58

Discussion In the present study, we tested the potential of different FGFs to support HSC growth

in serum-free medium. In all 3 FGF conditions, FGF-1, FGF-2 and FGF-1+2, similar

trends in overall cell growth and cell morphology were observed. Although not

evident from the growth and morphology of the cultured cells, the addition of FGF-1

and/or -2 to serum-free medium proved to be an effective culture condition to support

primitive HSCs. Our in vitro CAFC data and in vivo reconstitution results clearly

document that bona fide long-term repopulating stem cells can be preserved in vitro

for up to 5 weeks when FGFs are added to the medium (Figure 2.1 and 2.2).

To test the repopulating potential of FGF-treated stem cells in a clinically relevant

model, cultured cells were transplanted into lethally irradiated mice without

competitor cells. Importantly, BM cells cultured for 3 and 5 weeks in the presence of

only FGF-1+2 were able to provide radioprotection and reconstitution.

Thus, our data clearly document that FGFs (of which we tested 2 out of a family of

22) can be added to a growing list of signaling proteins that act on primitive stem

cells. Interestingly, whereas “classical” hematopoietic growth factors turn out to have

limited potential in sustaining and expanding HSCs37-40 the effect of growth factors

and morphogens that historically have been associated with embryonic development

(Wnt, BMP, FGF, Shh, and insulin-like GF [IGF]-2) may be more powerful4;7;41;42.

Recently it was reported that FGF-2 allows the clonal growth of hESCs in medium

containing serum replacement27. In addition, ESCs are known to express multiple

FGF receptors43. Xu et al, documented that FGF-2 synergizes with Noggin to suppress

BMP signaling and thus sustain undifferentiated proliferation of hESCs in the absence

of fibroblasts or conditioned medium29. It is also interesting to note that during

development TGF-β/BMP, IGF, Notch receptors and Wnts may be influenced and

dependent on heterologous factors such as FGFs44. Lately , it was reported that cross-

talk between the Notch and FGFR signaling pathways may be an important auto-

regulatory mechanism involved in the regulation of cell growth45. Also, FGFs and

Wnts have been shown to interact in a variety of developmental systems including

tracheal development in Drosophila, mesoderm induction in Xenopus, and brain,

tooth, and kidney development in other vertebrates46. FGF signaling maintains the

proliferation of multipotent neural stem cells and also affects subsequent lineage

commitment during neural differentiation. It was shown that these FGF effects are


59

mediated by β-catenin47. Collectively, these data imply an important physiological

role for FGF in stem cell fate decisions. The specific biological response, such as

proliferation, apoptosis, and differentiation, that a cell will deliver in response to FGF

signals, will depend on the interaction with many other factors44.

We were unable to culture purified LSK cells with only FGFs, whilst recently Zhang

and Lodish were able to culture purified BM side population cells with a greater than

8-fold increase in repopulating HSCs when grown in low levels of SCF,

thrombopoietin (TPO), IGF-2 and FGF-1 in serum-free medium for 10 days48. In our

purified cell culture system, such cross-talk with other signaling networks was not

possible, suggesting that the combination of SCF, TPO, IGF-2 and FGF-1 are better

suited for the expansion of a purified population of HSCs. The results of Zhang and

Lodish and our results highlight the importance of FGF in an in vitro culture system to

maintain HSCs, however our findings also suggests that the effect of FGFs on stem

cells requires other stimuli. The addition of SCF, IL-11 and Flt3L increased the

proliferation of stem cells. We tested whether FGFs were able to maintain the

primitiveness of stem cells, thereby negating the potential differentiation effect of

these GFs when placed in combination. Thus, GF + FGF-1+2 cultured cells would

have been expected to provide competitive reconstitution whereas GF-only cultures

would have been expected to provide no or very little reconstitution. Unfortunately,

this was evidently not the case (Figure 2.5). The optimal combination of GFs required

for maintaining primitive HSC activity remains to be discovered.

Our studies were not aimed to delineate the molecular consequences of incubating

BM cells with FGFs. Consequently, we can only speculate on how FGFs maintain

stem cells in whole BM cultures. It has been shown that receptors for FGF-1 are

present on primitive hematopoietic cell subsets10. Therefore, we speculate that FGFs

maintain stem cells at least partially by acting directly on FGFRs expressed on the

stem cells. Additionally, other non-LSK cell types normally present in the stem cell

niche may carry FGFRs and therefore be responsive to FGFs and may play an

important role in the maintenance of stem cells. In the intact animal, stem cells are

found in close association with their cellular microenvironments49-51. These

observations suggest both the existence of stem cell niches6;52-54 and the notion that in

vivo stem cell regulatory mechanisms are likely to require cell-cell contact or short-

range interactions55, providing further evidence that additional stimuli may be

required for a desired effect. The niche is composed of stem cells and a diverse


60

variety of neighboring hematopoietic and non-hematopoietic cell types such as

osteoblasts and endothelial cells. These act to secrete and organize a rich milieu of

extracellular matrix and other factors that allow stem cells to manifest their unique

intrinsic properties56. We speculate that for HSC to be properly maintained and

amplified in vitro, the whole BM in co-culture must act as a niche, facilitating stem

cells to expand.

It is tempting to postulate that many stem cell expansion studies have not been so

successful, because cultures almost invariably were initiated with purified cells. As

shown in our purification studies, disruption of HSC from their niche is likely to have

detrimental effects on their subsequent developmental potential. Our study represents

a clear example of an in vitro system using FGFs, capable of supporting primitive

HSCs for an extended period of time. Future studies will be aimed at creating a niche

for stem cells in vitro, while stimulating their proliferation.


61

Acknowledgements We wish to thank Geert Mesander and Henk Moes for their assistance with cell

sorting and the animal facility staff for taking care of the mice. This work was

supported by grants from the Dutch Cancer Society (RUG 2000 – 2182) and (RUG

2000 – 2183), National Institutes of Health (R01-HL073710), European Union (EU-

LSHC-CT-2004-503436), and the Ubbo Emmius Foundation.


62

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36. Uchida N, Dykstra B, Lyons KJ, Leung FY, Eaves CJ. Different in vivo repopulating activities of purified hematopoietic stem cells before and after being stimulated to divide in vitro with the same kinetics. Exp.Hematol. 2003;31:1338-1347.

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47. Israsena N, Hu M, Fu W, Kan L, Kessler JA. The presence of FGF2 signaling determines whether beta-catenin exerts effects on proliferation or neuronal differentiation of neural stem cells. Dev.Biol. 2004;268:220-231.

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66

67

CHAPTER 3

Effects of fibroblast growth factor overexpression and

cellular localization on hematopoietic stem cell

function

Joyce S. G. Yeoh, Ellen Weersing, Bert Dontje, Leonid

Bystrykh, Ronald van Os, Gerald de Haan



In preparation

Fibroblast growth factor overexpression

68

Abstract Exogenous addition of Fibroblast Growth Factor (FGF)-1 and FGF-2 maintains and

expands long-term repopulating hematopoietic stem cells (HSCs) in vitro. These

proteins are also highly expressed by Lin-Sca-1+c-Kit+ (LSK) cells. In this study we

retrovirally transduced post 5-fluorouracil (5-FU) bone marrow (BM) cells with

retroviral vectors which express wild-type (WT) FGF-1 and WT FGF-2 to examine

their cell intrinsic role in hematopoietic cell expansion. In addition, we examine the

role of nucleocytoplasmic trafficking of FGFs in hematopoietic cells by

overexpressing two mutant isoforms of FGF-1 in which a serine residue at the

phosphorylation site is exchanged for glutamic acid (S130E) or alanine (S130A).

S130E mimics the phosphorylated state of FGF-1 and should hypothetically be

constitutively exported to the cytoplasm whilst S130A should remain in the nucleus.

A third mutant was created by inserting an artificial nuclear localization signal (NLS)

upstream of FGF-2. Using fluorescence microscopy, we demonstrate that FGF-1,

FGF-2 and S130E predominantly localizes to the cytoplasm whilst 2% of S130A and

11% of NLS/FGF-2 overexpressing cells shown nuclear localization of FGF-1 and

FGF-2 respectively. We present evidence demonstrating that in an in vivo competitive

repopulation assay, stem cells expressing S130A engrafted into secondary recipients

with superior kinetics compared to WT cells, suggesting that nuclear localization of

FGF-1 may improve hematopoietic stem cell functioning.


69

Introduction A small population of HSCs plays a pivotal role in the lifelong maintenance of

hematopoiesis. A critical property of HSCs is their ability to undergo self-renewal.

The relative inability to expand HSCs ex vivo imposes substantial limitations on the

current use of HSC transplantation. While studies have shown that some self renewal

is clearly possible in vitro1-3, the magnitude of expansion obtained for human and

murine HSCs is modest4-7.

In studies aimed to maintain HSCs in vitro, recent attention has been focused on the

large family of FGFs which are involved in embryonic development and adult tissue

homeostasis. To date, there are 22 members of the FGF family and only four distinct

FGF receptors (FGFRs) (reviewed by Ornitz and Itoh 2001)8. There is a growing body

of evidence demonstrating the role of FGFs in hematopoiesis in general and HSCs in

particular. For example, previous in vitro studies showed impaired hematopoietic

development in FGFR-/- embryoid bodies (EB), such that the number of blast colonies,

primitive erythroid and myeloid progenitors were greatly reduced9. FGFs, in

particular FGF-2, have been shown to sustain the proliferation of hematopoietic

progenitor cells, maintaining their primitive phenotype10;11. FGF-1 induces

granulopoiesis12 and megakaryocytopoiesis13;14. Recently, our group showed that

Fgfr-1, -3 and -4 are expressed by mouse primitive hematopoietic cell subsets and that

FGF-1 was involved in the expansion of multi-lineage (lymphoid and myeloid), long-

term (LT) repopulating HSCs 15. Additionally, we recently reported that

unfractionated bone marrow cells could be cultured in the combination of FGF-1 and

FGF-2 for up to five weeks without loss of stem cell repopulation activity. We

showed that this originated from FGF cultured HSCs16.

Several lines of evidence exist indicating that nuclear localization of FGFs may be

required for the mitogenic effect in certain conditions, in different cells types. For

example, radiolabeled exogenous FGF-1 localized to the nuclear fraction and was

shown to stimulate DNA synthesis and cell proliferation in cells containing receptors

for FGF-117. In glioma cells and in primary cultures of human astrocytes, cell

proliferation rate and nuclear association of FGF-2 was reported to change in

parallel18. These observations support the notion that nuclear translocation of FGFs

could be related to mitogenesis in different cells.


70

Studies have shown that the translocation of FGF-1 from the cytosol to the nucleus

requires tyrosine kinase and phosphatidylinositol 3-kinase (PI3K) activity and that

phosphorylation of FGF-1 occurs in the nucleus by protein kinase C (PKC) at the only

functional phosphorylation site (Serine 130) 19-22. In contrast to FGF-1, FGF-2

contains both autocrine and intracrine effects resulting from the existence of different

isoforms. For example, human FGF-2 contains five different forms; a low molecular

mass form (18kDa), which acts as an autocrine/paracrine factor and four high

molecular mass forms (21-22, 22.5, 24 and 34kDa) which are intracrine effectors.

These four high molecular mass forms are generated by differential initiation of

translation sites from an upstream CUG codon. They contain at least two short N-

terminal extensions in which the NLS is located23;24. Only N-terminally extended

forms initiated at upstream CUG codons are translocated to the nucleus while the

normal 18 kDa AUG-initiated form is confined to the cytoplasm25-28. When the

intracrine FGF-2 forms were expressed in NIH 3T3 cells, high proliferation rates and

growth in soft agar were observed29 and stimulated cell growth under low-serum

conditions was evident30;31

In previous studies, our goal was to maintain and expand HSCs in vitro by

exogenously adding FGF-1 and FGF-2 to serum-free media15;16. In the current study

we focused on retroviral overexpression of FGF-1 and FGF-2 on HSCs to promote

expansion and maintenance of hematopoietic cells in both in vitro and in vivo studies.

Parallel to this, we examined the effects of nuclear localized FGFs and their ability to

provide long-term (LT) repopulation. To clarify the role of differential localization of

FGFs in maintaining hematopoietic cells, we created two FGF-1 mutants. Firstly, a

serine residue at the phosphorylation site (amino acid 130) was exchanged with

glutamic acid to mimic phosphorylated FGF-1, which is expected to be constitutively

transported to the cytosol (S130E)22. In the second mutant the same serine residue was

exchanged for alanine, which is expected to remain in the nucleus (S130A)22. A third

mutant for FGF-2 was also created whereby an upstream nuclear localization signal

(NLS) was inserted into the FGF-2 coding sequence.

Using retroviral overexpression, long-term in vivo repopulation and principles of

limiting dilution assays to quantitatively determine hematopoietic cell expansion for

both WT FGF-1 and FGF-2 and their mutant forms, we demonstrate that in in vitro

assay, endogenous FGFs confer increased mitogenic activity to BM cells. However, it

does not provide enhanced long-term repopulation in primary and secondary in vivo


71

repopulating assays. Fluorescence microscopy images indicated that FGF-1, FGF-2

and S130E were preferentially located in the cytoplasm. In S130A and NLS/FGF-2

overexpressing cells, only 2% of FGF-1 and 11% of FGF-2 positive cells showed

nuclear FGF activity, respectively. S130A mutants transplanted competitively

engrafted into secondary recipients with improved efficiency compared to WT cells,

indicating that targeting FGF-1 to the nucleus may provide improved stem cell

quality.


72

Materials and Methods Animals

Eight to twelve week old female B6.SJL-PtprcaPep3b/BoyJ (CD45.1) mice, bred at

our local animal facility, were used as a donor source of hematopoietic stem cells for

in vitro cultures and transplantations. Female C57BL/6 (CD45.2) (B6) mice were used

as recipients and were purchased from Harlan (Horst, The Netherlands). All animals

were maintained under clean conventional conditions in the animal facilities of the

Central Animal Facilities, University Medical Centre Groningen (The Netherlands).

Mice were fed ad libitum with food pellets and acidified tap water (pH = 2.8). All

animal procedures were approved by the local animal ethics committee of the

University Medical Centre Groningen.

DNA constructs, expression vectors and retroviral vectors

The MIEV vector (kindly provided by Prof. C. Jordan, University of Rochester)

contains an internal ribosomal entry site (IRES) sequence, ψ packaging signal and the

reporter gene for enhanced green fluorescent protein (eGFP). The empty MIEV

vector served as a control and was the backbone from which all other vectors were

made.

FGF-1 was initially cloned into pCR4 vector following SalI end modification by using

the following primers; 5’-CTACCACCGCTGCTTGC-3’ (forward), 5’-

GTCGACCAAAATAGAGAACACTCAG-3’ (reverse) (fragment size 529bp). The

FGF-1 coding sequence was cut with EcoRI/SalI and inserted into MunI/SalI site of

the MIEV vector in between the pPGK promoter and IRES. We refer to this vector as

WT FGF-1. A retroviral vector encoding for WT FGF-2 was created identically. The

primers used were; 5’-CCCCAAGAGCTGCCACAG-3’ (forward) and 5’-

TCAGTGACAGTGTCAAAAGTGAGTC-3’ (reverse) (fragment size 531bp).

Site directed mutagenesis was used to create two mutant isoforms of FGF-1. At the

phosphorylation site, the serine at amino acid 130 was exchanged for either glutamic

acid (S130E) or alanine (S130A). The initial FGF-1 cloned in pCR4 was used to

create the two mutants by amplifying the whole fragment with the following primers;

S130E, 5’- CAAGAAGAACGGGgagTGTAAGCGCGGTCC-3’ (forward) and 5’-

GGACCGCGCTTACACTCCCCGTTCTTCTTG-3’ (reverse); S130A, 5’-

CAAGAAGAACGGGgccTGTAAGCGCGGTCC-3’ (forward) and 5’-


73

GGACCGCGCTTACAggcCCCGTTCTTCTTG-3’ (reverse). Lower case letters

indicates the place of mismatch. Both mutants were subcloned from pCR4 vector at

EcoRI/SalI site into MunI/SalI of MIEV vector.

Contrary to the human FGF-2 gene, in the mouse genome Fgf-2 does not contain an

upstream NLS. The 5’ UTR NLS was artificially created based on homology to the

human FGF-2 coding sequence using these primers; 5’-

ACGGACTGGGAGGCTGGCAG-3’ (forward) and 5’-

CGAGGTGATGCCGCTGGCAG-3’ (reverse) (fragment size 157bp). Following

successful sequencing results, an EcoRV and ATG site was inserted into the upstream

sequence with the following primers; 5’-

gatatcACGatggTACGGACTGGGAGGCTGGCAG-3’ (forward) and 5’-

CGAGGTGATGCCGCTGGCAG-3’ (reverse) (fragment size 140bp). Following

cloning into pCR4 vector, the 5’UTR NLS was excised from the vector at

EcoRV/NcoI site and ligated into the original FGF-2 pCR4 vector at SmaI/NcoI site.

The ligated 5’UTR NLS/FGF-2 was removed from pCR4 vector with EcoRI/SalI and

subcloned into MIEV at MunI/SalI site creating the NLS/FGF-2 retroviral vector.

Retroviral overexpression of FGFs in primary BM cells

Bone marrow cells were obtained from CD45.1 mice injected i.p. with 150mg/kg 5-

Fluorouracil (5-FU, Pharmachemie Haarlem, The Netherlands), 4 days prior to BM

isolation. Bone marrow cells were harvested by flushing the femoral content with

StemSpan (Stem Cell Technologies, Vancouver, BC, Canada). Cells were cultured for

48 hours in StemSpan supplemented with 10% fetal calf serum (FCS; GibcoBRL,

Invitrogen, CA), 300ng/ml Stem Cell Factor (SCF; Amgen, Thousand Oaks, CA,

USA), 20ng/ml Interleukin-11 (IL-11; R&D Systems, Minneapolis, MN, USA),

1ng/ml Flt3 Ligand (Flt3L; Amgen, Thousand Oaks, CA, USA), penicillin and

streptomycin (Invitrogen, Breda, The Netherlands).

Twenty-four hours prior to transfection, 3 x 105 ecotropic Phoenix packaging

cells/well (ATCC-LGC Promochem, Middlesex, United Kingdom) were seeded onto

6-well plates. Using Fugene 6 (Roche, Basel, Switzerland), plasmid DNA (1µg) was

transfected on to pre-seeded ecotropic phoenix packaging cells. Virus-containing

supernatants from transfected ecotropic Phoenix packaging cells were harvested 24

hours after transfection and seeded onto retronectin (Takara, Kyoto, Japan) coated 6-

well plates. Plates containing viral supernatant were centrifuged for 1 hour at


74

2200rpm at room temperature and then incubated at 37oC with 5% CO2 for 4 hours.

The viral supernatant was then removed from the well prior to the incubation of 7.5 x

105 cultured BM cells with 4µg polybrene (Sigma, St Louis, MO, USA). The whole

procedure was repeated 48 hours after the initial transfection and 2µg of polybrene

was added. Four days after transduction, the transduction efficiency was determined

by flow cytometry (FACS Calibur, Becton Dickinson, Palo Alto, CA).

In vitro proliferation of BM cells

Four days following transduction, Green Fluorescent Protein (GFP+) cells were sorted

by a MoFlow cell sorter (DakoCytomation, Fort Collins, CO). 6 x 105 to 1 x 106 GFP+

cells were placed in 6-well plate in StemSpan supplemented with 10% fetal calf

serum, 300ng/ml SCF, 20ng/ml IL-11, 1ng/ml Flt3L, penicillin and streptomycin. On

a weekly basis, cells were counted, cultures were depopulated and passaged.

In vitro hematopoietic cell assays

Cobblestone Area Forming Cell Assays (CAFC) were performed as previously

described16;32 to assess the number of hematopoietic progenitor cells (CAFC day 7) or

more primitive stem cells (CAFC day 28-35) among the transduced BM cells.

Colony Forming Unit Granulocyte-Macrophage (CFU-GM) was determined using

standard methylcellulose cultures (0.8% methylcellulose, 30% FCS in α-MEM).

Transduced BM cells were added to methylcellulose cultures supplemented with

100ng/ml SCF and 10ng/ml recombinant mouse granulocyte-macrophage colony

stimulating factor (GM-CSF; Behringwerke, Marburg, Germany). Cultures were

cultured at 37oC with 5% CO2 and scored after 6-7 days.

In vitro proliferation of BM cells co-cultured on a stromal layer

Following retroviral transduction, 5,000 or 10,000 GFP+ cells were sorted and co-

cultured with Fetal Bone Marrow Derived (FBMD)-1 stromal layer in a culture flask.

Cells were cultured in Iscoves modified DMEM (IMDM; GibcoBRL, Paisley,

Scotland) supplemented with 20% horse serum (GibcoBRL, Paisley, Scotland),

pencillin/streptomycine (Gibco, Paisley, Scotland) with β-mercaptoethanol (Merck

Schuchardt, Hohenbrunn, Germany) and hydrocortisone (Sigma, St Louis, MO). Nine


75

and 14 days after initiation of culture, all non-adherent cells were removed, counted

and placed in CFU-GM and CAFC assays.

Sorting of hematopoietic cell populations

Hematopoietic cells were stained and sorted for populations of cells that differentially

expressed Lin-, Sca-1 and c-Kit using the MoFlow cell sorter as previously

described16;33. BM cells were stained with biotinylated lineage-specific antibodies

Mouse Lineage Panel, containing anti-CD45R, anti-CD11b, anti-TER119, anti-Gr-1

and anti-CD3e (BD Pharmingen, San Diego, CA), FITC-anti-Sca-1 and APC-anti-c-

kit (BD Pharmingen, San Diego, CA). Biotinylated antibodies were visualized with

streptavidin-PE (Pharmingen, San Diego, CA). Four different populations were

sorted, Lin-Sca-1-c-Kit- (L-S-K-), Lin-Sca-1+c-Kit- (L-S+K-), Lin-Sca-1-c-Kit+ (L-S-K+)

and Lin-Sca-1+c-Kit+ (L-S+K+).

Reverse Transcriptase (RT-PCR) and Quantitative PCR (Q-PCR)

Total RNA was prepared from approximately 1 x 106 to 3 x 106 sorted GFP+ cells

using RNeasy Mini kit (Qiagen). M-MLV reverse transcriptase (Invitrogen, Breda,

The Netherlands) was used to synthesize cDNA. Expression of FGF-1 and FGF-2

transcripts was assessed by RT-PCR with the following primers; FGF-1, 5’-

CGGCTCGCAGACACCAAATGAGG-3’ (forward) and 5’-

GTCGACCAAAATAGAGAACACTCAG-3’ (reverse) (fragment size 238bp); FGF-

2, 5’–CCCCAAGAGCTGCCACAG-3’ (forward) and 5’-

TCAGTGACAGTGTCAAAAGTGAGTC-3’ (reverse) (fragment size 531bp). The

cDNA products were quantified using SYBR Green (Bio-rad) in a 96-well microtiter

plates in an iCycler thermal cycler (Bio-rad, Hercules, CA, USA). Primers used for

quantifying Fgf-1 and Fgf-2 expression were as follows; Fgf-1, 5’-

CGGCTCGCAGACACCAAATGAGG-3’ (forward), 5’-

CCATAGTGAGTCCGAGGACCGC-3’ (reverse) (fragment size 155bp) and Fgf-2,

5’- CGACCCACACGTCAAACTACAACTC-3’ (forward), 5’-

GAAGCCAGCAGCCGTCCATC-3’ (reverse) (fragment size 113bp).

FGF expression levels were compared with expression of housekeeping genes Gapdh

and Actin using relative quantification ΔΔCT technique34. This value was then

corrected for the initial number of cells used.


76

Western blotting

Cell lysates were prepared using the acid-acetone precipitation method. 3 x 106

transduced cells were pelleted and washed with 0.1M hydrochloric acid (MERCK

KGaA, Darmstadt, Germany), followed by a 60 minute incubation at -20oC. Samples

were centrifuged for 30 minutes at maximum speed at 4oC, washed in 70% ethanol

and resuspended in chilled (-20oC) acetone. All acetone was removed and the pellets

were left to dry at 37oC for 1-5 minutes. The pellets were redissolved in sample buffer

at 60oC. Lysates were then boiled for 5 minutes. Proteins were separated by 12%

SDS-PAGE and transferred to nitrocellulose membrane. Following overnight

blocking in 5% skim milk, membranes were probed overnight with 1:1000 dilution of

rabbit anti-FGF-1 (Sigma, Saint Louis, MO, USA) and rabbit anti-FGF-2 (Santa Cruz

Biotechnology, Santa Cruz, California, USA). Membranes were washed and

incubated with anti-rabbit IgG Horseradish peroxidase secondary antibody

(Amersham Biosciences, Buckinghamshire, UK). ECL western blotting detection

reagents (Amersham, Biosciences, Buckinghamshire, UK) were used to develop the

membranes. Equal loading of membranes were verified with commassie staining of

SDS-PAGE gels.

Immunocytochemistry

Transduced GFP+ cells were spotted on to cytospin preparations following one week

of culturing in culture conditions described above. Cells were fixed with 4%

paraformaldehyde and permeabilized with 0.3% Triton X-100/PBS. FGF-1 expression

was detected using an anti-FGF-1 (C-19) goat polyclonal IgG antibody (1:20 dilution)

(Santa Cruz Biotechnology, Santa Cruz, California, USA) followed by a Donkey

Anti-Goat IgG-Cy3 (1:1500 dilution) (Jackson Immunoresearch Laboratories, West

Grove, PA, USA) secondary antibody. FGF-2 expression was detected using an anti-

FGF-2 (147) rabbit polyclonal IgG antibody (1:20 dilution) (Santa Cruz

Biotechnology, Santa Cruz, California, USA) followed by a Goat Anti-Rabbit IgG-

Cy3 (1:1500 dilution) (Santa Cruz Biotechnology, Santa Cruz, California, USA)

secondary antibody. DAPI (0.2µg/ml) (Sigma, St Louis, Missouri, USA) was used to

stain the nuclei. Images were taken with Leica DM6000B (Wetzlar, Germany,

www.leica-microsystems.com).


77

Primary transplantations of transduced cells

Following transduction, 3 x 106 CD45.1 BM cells were transplanted into lethally

irradiated (9 Gy, IBL 637137Cs-source 0.7026Gy/min, CIS Biointernational, Gif-sur-

Yvette, France) CD45.2 recipients. GFP+ cells were not selected, thus transplants

consisted of a mixture of both transduced and non-transduced cells. Each transplant

group consisted of 10 recipients, and two independent experiments were performed.

Following transplantation, blood samples (60μl) were taken monthly to determine

donor chimerism. Levels of chimerism were determined by detecting the presence of

CD45.1+ and GFP+ cells in transplanted mice. To detect CD45.1+ cells, cells were

stained with anti-CD45.1 (PE) antibody (BD Pharmingen, San Diego, CA) for 30

minutes and analyzed on a flow cytometer (FACS Calibur; Becton Dickinson

Biosciences, San Jose, CA).

Secondary transplantations of transduced cells

Four months after primary transplantation, five recipients from each group were

randomly selected, sacrificed and BM cells were isolated from two hind legs.

Unfractionated BM cells were transplanted into lethally irradiated (9Gy) CD45.2

secondary recipients in three limiting dilutions (1:1, 1:2 and 1:4) with a fixed number

of 4 x 105 CD45.2 competitor cells. Blood analysis was carried out each month to

determine original donor CD45.1+GFP+ chimerism levels. Each transplanted group

consisted of 5 recipients and two independent experiments were performed.

Transplantation of cultured transduced cells

Ten days after cells were plated onto a FBMD-1 stromal layer, all cells (including

stromal cells) were removed and CD45.1+GFP+ cells were selected by FACS. Lethally

irradiated (9Gy) B6 recipients were transplanted with CD45.1+GFP+ cells in

competition with freshly isolated B6 BM cells. Recipients received; (i) 5 x 105 control

cells + 5 x 105 competitor cells, (ii) 2.8 x 105 WT FGF-1 cells + 8.5 x 105 competitor

cells, (iii) 1.5 x 105 S130E cells + 4.4 x 105 competitor cells and (iv) 2.5 x 105 S130A

cells + 7.5 x 105 competitor cells. On a monthly basis, blood was analyzed for

CD45.1+GFP+ chimerism.


78

Statistical Analysis

P-values were calculated using the Mann-Whitney test or the student’s t-test

(assuming unequal variances of the two variables) to test the statistical significance (p

< 0.05) of different groups. Quantification of CAFC at day 7, 28 and 35 was

performed by using maximum likelihood ratio method35. The Poisson-based limiting

dilution analysis calculation was used with a 95% confidence interval (CI) to

determine significant differences at p < 0.05.


79

Results Relative expression of FGF-1 and FGF-2 in HSCs

To quantify the normal expression levels of FGF-1 and FGF-2 in hematopoietic stem

cells, freshly isolated BM cells were stained with lineage (Lin) specific markers and

hematopoietic stem cell markers, Sca-1 and c-Kit. As shown in Figure 3.1A, 5% Lin-

cells were separated into four populations; Sca-1-c-Kit-, Sca-1+c-Kit-, Sca-1-c-Kit+ and

Sca-1+c-Kit+. The levels of expression of FGF-1 and FGF-2 relative to Sca-1-c-Kit- is

shown in Figure 3.1B and 3.1C. Both FGFs are expressed in all four populations.

Interestingly, the highest expression of FGF-1 and FGF-2 was in the Lin-Sca-1+c-Kit+

population, a population which is highly enriched for primitive hematopoietic stem

cells36;37. This further confirmed our hypothesis that FGFs may play an important role

in HSCs regulation.

0

2

4

6

8

10

12

14

0

5

10

15

20

25

30

35

40FGF1 FGF2

Sca- c

-Kit-

Sca

+ c-K

it-

Sca

- c-K

it+

Sca

+ c-K

it+

Rel

ativ

e ex

pres

sion

Sca

- c-K

it-

Sca

+ c-K

it-

Sca

- c-K

it+

Sca

+ c-K

it+

Rel

ativ

e ex

pres

sion

A.

B. C.

Line

age

c-K

it

Sca-1

5%

2.2%44%

38% 12%

FSC

0

2

4

6

8

10

12

14

0

5

10

15

20

25

30

35

40FGF1 FGF2

Sca- c

-Kit-

Sca

+ c-K

it-

Sca

- c-K

it+

Sca

+ c-K

it+

Rel

ativ

e ex

pres

sion

Sca

- c-K

it-

Sca

+ c-K

it-

Sca

- c-K

it+

Sca

+ c-K

it+

Rel

ativ

e ex

pres

sion

0

2

4

6

8

10

12

14

0

5

10

15

20

25

30

35

40FGF1 FGF2

Sca- c

-Kit-

Sca

+ c-K

it-

Sca

- c-K

it+

Sca

+ c-K

it+

Rel

ativ

e ex

pres

sion

Sca

- c-K

it-

Sca

+ c-K

it-

Sca

- c-K

it+

Sca

+ c-K

it+

Rel

ativ

e ex

pres

sion

A.

B. C.

Line

age

c-K

it

Sca-1

5%

2.2%44%

38% 12%

FSC

Line

age

c-K

it

Sca-1

5%

2.2%44%

38% 12%

FSC

Figure 3.1: Relative expression of FGF-1 and FGF-2 in Lin- Sca-1 and c-Kit populations. (A): FACs

plot of Lineage (Lin-), Sca-1 and c-Kit gates from B6 BM cells. Lineage- cells were sorted into four

populations, Sca-1-c-Kit-, Sca-1+c-Kit-, Sca-1-c-Kit+ and Sca-1+c-Kit+. (B): Relative expression of FGF-

1 by Q-PCR in the four populations of Sca-1 and c-Kit relative to Sca-1-c-Kit- BM cells. (C): Relative

expression of FGF-2 by Q-PCR in Sca-1 and c-Kit populations relative to Sca-1-c-Kit- BM cells.


80

Expression of FGF-1 and FGF-2 in BM cells

A schematic representation of the control, WT FGF-1 and WT FGF-2 retroviral

vectors used to transduced 5-FU treated BM cells is shown in Figure 3.2A. The

control vector expressing only eGFP was used as a control in all assays. Bone marrow

cells harvested from 5-FU treated CD45.1 donor mice were transduced with control,

WT FGF-1 and WT FGF-2 retroviral vectors. Transduction efficiencies of 30-40%

were always achieved (data not shown). Following retroviral transduction, GFP+ cells

were selected and cultured for a total of 33 days in StemSpan supplemented with

10%FCS, IL-11, SCF and Flt3L. On a weekly basis, cells were counted for growth

kinetic analysis. Cell growth in control, FGF-1 and FGF-2 overexpressing cells was

comparable (Figure 3.2B). FGF-1 overexpressing cells maintained slightly higher but

not statistically significant cell numbers throughout the 33 days of culturing, peaking

at day 26 (Figure 3.2B). At day 26, a 2.6-fold and 16-fold increase (although not

statistically significant p > 0.05) in FGF-1 overexpressing BM cells was observed

when compared to FGF-2 and control cells respectively (Figure 3.2B). Twelve days

into the culture period the expression level of FGF-1 and FGF-2 in BM transduced

cells was assessed by RT-PCR (Figure 3.2C). The expression of FGF-1 was only

detectable in FGF-1 overexpressing BM cells and FGF-2 expression was detected

only in FGF-2 overexpressing BM cells (Figure 3.2C). Although both FGF-1 and

FGF-2 expression were detected by Q-PCR (Figure 3.1B), their expression in LSK

cells by RT-PCR were below detection levels, possibly due to the use of different

primers. Protein levels in FGF-1 and FGF-2 overexpressing BM cells correlated with

RT-PCR results (Figure 3.2D). The relative expression of both FGFs in transduced

BM cells was also examined by Q-PCR. As expected, high expression levels of FGF-

1 and FGF-2 were detected in FGF-1 and FGF-2 overexpressing BM cells

respectively (Figure 3.2E). It should be pointed out that expression of FGF-1 and

FGF-2 in control cells was extremely low with CT value of 33 whilst both FGF-1 and

FGF-2 displayed CT values of ~17.


81

FGF1 17KDa

FGF2 18KDa

Control FGF

A.

B.

FGF-1238bp

GAPDH 451bp

FGF-2 531bp

WT

FGF1

Con

trol

LSK

WT

FGF2

WT

FGF2

Con

trol

LSK

WT

FGF1

C. D.

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

1.00E+11

0 5 10 15 20 25 30 35106

107

108

109

1010

1011

Cum

ulat

ive

cell

grow

th

Days in culture

ControlWT FGF-1WT FGF-2

E.

Rel

ativ

e ex

pres

sion

0

10000

20000

30000

40000

50000

60000

70000

80000

Control FGF-1 FGF-2

500 1K 1.5K 2K 2.5K 3.5K3K 4K

Control

WT FGF-1

WT FGF-2

EGFPLTR Ψ pPGK IRES LTR

EGFPIRES LTRFGF1

EGFPIRES LTRFGF2

LTR Ψ pPGK

LTR Ψ pPGK

FGF1 17KDa

FGF2 18KDa

Control FGF

FGF1 17KDa

FGF2 18KDa

Control FGF

A.

B.

FGF-1238bp

GAPDH 451bp

FGF-2 531bp

WT

FGF1

Con

trol

LSK

WT

FGF2

WT

FGF2

Con

trol

LSK

WT

FGF1

FGF-1238bp

GAPDH 451bp

FGF-2 531bp

WT

FGF1

Con

trol

LSK

WT

FGF2

WT

FGF2

Con

trol

LSK

WT

FGF1

C. D.

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

1.00E+11

0 5 10 15 20 25 30 35106

107

108

109

1010

1011

Cum

ulat

ive

cell

grow

th

Days in culture


1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

1.00E+11

0 5 10 15 20 25 30 351.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

1.00E+11

0 5 10 15 20 25 30 35106

107

108

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1011

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grow

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Days in culture



E.

Rel

ativ

e ex

pres

sion

0

10000

20000

30000

40000

50000

60000

70000

80000

Control FGF-1 FGF-2

500 1K 1.5K 2K 2.5K 3.5K3K 4K

Control

WT FGF-1

WT FGF-2


EGFPIRES LTRFGF1

EGFPIRES LTRFGF2

LTR Ψ pPGK

LTR Ψ pPGK

500 1K 1.5K 2K 2.5K 3.5K3K 4K500 1K 1.5K 2K 2.5K 3.5K3K 4K

Control

WT FGF-1

WT FGF-2


EGFPIRES LTRFGF1

EGFPIRES LTRFGF2

LTR Ψ pPGK

LTR Ψ pPGK

Figure 3.2: Overexpression of WT FGF-1 and WT FGF-2 in 5-FU bone marrow cells. (A): Schematic

representation of control, WT FGF-1 and WT FGF-2 vectors used to retrovirally transduce 5-FU BM

cells. (B): Cumulative cell growth curve of 5-FU BM cells retrovirally transduced with control, WT

FGF-1 and WT FGF-2. Following retroviral transduction, GFP+ cells were selected and cultured for 33

days in StemSpan containing 10%FCS, IL-11, SCF and Flt3L. On a weekly basis cells were counted

and passaged. (C): RT-PCR analysis for FGF-1 and FGF-2 expression in cells transduced with control

vector, WT FGF-1 and WT FGF-2 after 12 days of culturing. As a further control RNA from non-

transduced LSK cells was included. (D): FGF-1 and FGF-2 protein expression in control, WT FGF-1

and WT FGF-2 transduced cells after 12 days of culturing. (E): Using Q-PCR, the relative expression

of FGF-1 and FGF-2 in WT FGF-1 and WT FGF-2 transduced cells relative to control transduced cells,

was determined five days after initiation of culture respectively. CT value of 33 was recorded for

control cells, whilst both FGF-1 and FGF-2 contained CT values of ~17.


82

Subcellular localization of FGF-1 and FGF-2

A schematic representation of S130E, S130A and NLS/FGF-2 retroviral vectors is

shown in Figure 3.3A. Phosphorylation of FGF-1 by protein kinase C occurs at serine

13022. Phosphorylation at this site should initiate the translocation of FGF-1 to the

cytosol from the nucleus. Mutating the serine in the phosphorylation site to a

negatively charged glutamic acid (E) (S130E) should mimic the constitutively

phosphorylated state of FGF-1. Substituting serine to the uncharged alanine (A)

(S130A) should mimic the unphosphorylated state of FGF-1 and causing the growth

factor to remain in the nucleus21. Similar successful strategies had previously been

used with other proteins38-40. The artificial addition of NLS upstream of FGF-2 coding

sequence is expected to translocate FGF-2 from the cytosol into the nucleus.

The subcellular localization of FGF-1 and FGF-2 in transduced BM cells was

examined using fluorescence microscopy. We were not able to detect GFP and FGF

proteins in all spotted cells. Localization patterns of FGF-1 and FGF-2 were therefore

only quantified in GFP+ and FGF double positive cells. FGF-1 and FGF-2 were not

detected in control cells (Figure 3.3B and 3.3F respectively), correlating with RT-

PCR and western blot data in Figure 3.2C-E. Fluorescent images of BM cells

overexpressing WT FGF-1 and WT FGF-2 indicated that both FGF-1 and FGF-2 were

localized in the cytoplasm in all cells in which FGF expression could be detected

(Figure 3.3C and 3.3G respectively).

The S130E mutant mimics phosphorylated FGF-1. Thus, we hypothesize that it

should be constitutively transported to the cytosol. Overlay images of FGF-1 positive

cells indicate that the protein is indeed predominantly localized in the cytoplasm

(Figure 3.3D). The S130A mutant lacks a phosphorylation site. Therefore, FGF-1 was

expected to be localized in the nucleus. In FGF-1 positive cells (n = 100), 2% of cells

showed nuclear staining (Figure 3.3Ei) and the remaining 98% showed cytoplasmic

staining (Figure 3.3Eii). Similar to S130A mutant, in NLS/FGF-2 overexpressing

cells, FGF-2 was expected to be present in the nucleus of cells. In 11% (n = 100) of

FGF-2 positive cells, FGF-2 was indeed localized in the nucleus (Figure 3.3H). In

remaining cells, FGF-2 was found in the cytoplasm (results not shown). Although

only a small percentage of FGF-1 and FGF-2 were localized to the nucleus, the

localization pattern was distinct when compared to WT FGF-1, WT FGF-2 and

S130E, indicating that nuclear localization did occur.


83

Our findings indicate that FGF-1 and FGF-2 are localized in the cytoplasm of BM

cells overexpressing WT FGFs. The localization distribution of FGF-1 and FGF-2 in

S130A and NLS/FGF-2 mutants respectively, suggests that the transport of FGF-1

and FGF-2 to and from the nucleus may be a dynamic process. FGFs enter the

cytoplasm following FGFR binding. Their NLS will transport FGFs into the nucleus

and upon phosphorylation, FGFs exit the nucleus into the cytoplasm. Cellular stress

will transport FGFs out of the cell41.

Growth kinetics of S130 and NLS/FGF2 mutants

Overexpression of both S130 mutants showed similar cell growth kinetics when

compared to the control vector in BM cells (Figure 3.4A). However, WT FGF-1

appears to have a slight increase in cell numbers suggesting that the change in

phosphorylation status of FGF-1 may not affect the mitogenic effect of BM cells. It

should be noted that growth kinetics of WT FGF-1 and control BM cells are the same

data as shown in Figure 3.2B.

It is often thought that nuclear localization of FGFs would increase its mitogenic

effect17;18;29. Bone marrow cells overexpressing NLS/FGF-2 displayed an increase in

cell growth during the culture period compared to WT FGF-2 and control BM cells.

Thirty-three days after initiation of culture there was a 7-fold increase in NLS/FGF-2

overexpressing cells when compared to WT FGF-2 overexpressing BM cells

respectively (Figure 3.4B). This suggests that NLS/FGF-2 increases the mitogenic

activity of BM cells. It should be noted that growth kinetics of WT FGF-2 and control

BM cells are the same data as shown in Figure 3.2B.


84

A.S130E/S130A

NLS/FGF-2

500 1K 1.5K 2K 2.5K 3.5K3K 4K

EGFPIRES LTRFGF1

EGFPIRES LTRFGF2

S130

LTR Ψ pPGK

NLSLTR Ψ pPGK

Control

WT FGF-1

S130E

S130A

WT FGF-2

NLS/FGF-2

100%Cytoplasmic(n = 100)

2%Nuclear(n = 100)



Control


11%Nuclear(n = 100)

B.

C.

D.

E. (i)

E. (ii)

F.

G.

H.

Anti FGF-1 and FGF-2Cy3 GFP Overlay

A.S130E/S130A

NLS/FGF-2

500 1K 1.5K 2K 2.5K 3.5K3K 4K

EGFPIRES LTRFGF1

EGFPIRES LTRFGF2

S130

LTR Ψ pPGK

NLSLTR Ψ pPGK

A.S130E/S130A

NLS/FGF-2

500 1K 1.5K 2K 2.5K 3.5K3K 4K500 1K 1.5K 2K 2.5K 3.5K3K 4K

EGFPIRES LTRFGF1

EGFPIRES LTRFGF2

S130

LTR Ψ pPGK

NLSLTR Ψ pPGK

Control

WT FGF-1

S130E

S130A

WT FGF-2

NLS/FGF-2


2%Nuclear(n = 100)



Control


11%Nuclear(n = 100)

B.

C.

D.

E. (i)

E. (ii)

F.

G.

H.


Control

WT FGF-1

S130E

S130A

WT FGF-2

NLS/FGF-2


2%Nuclear(n = 100)



Control


11%Nuclear(n = 100)

B.

C.

D.

E. (i)

E. (ii)

F.

G.

H.


Figure 3.3: Overexpression and subcellular localization of S130 mutants and NLS/FGF2 in 5-FU bone

marrow cells. (A): Schematic representation of S130 and NLS/FGF-2 retroviral vectors. At the

phosphorylation site of FGF-1 serine 130 is replaced with glutamic acid (E) (S130E) or alanine (A)

(S130A). Subcellular localization of FGF-1 and FGF-2 in transduced BM cells. Fluorescent microscope

images of BM cells transduced with (B): control vector stained with anti-FGF-1, (C): WT FGF-1, (D):

S130E, (Ei): S130A with cytoplasmic localization, (Eii): S130A with nuclear localization, (F): control

vector stained with anti-FGF-2, (G): WT FGF-2 and (H): NLS/FGF-2. Localization pattern was

quantified in 100 GFP+ cells expressing FGF-1 or FGF-2.


85

A.

B.

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

1.00E+11

0 5 10 15 20 25 30 35106

107

108

109

1010

1011

Cum

ulat

ive

cell

grow

th

Days in culture

ControlWT FGF-1

S130AS130E

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

0 5 10 15 20 25 30 35106

107

108

109

1010

Cum

ulat

ive

cell

grow

th

Days in culture

Control

WT FGF-2NLS/FGF-2

A.

B.

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

1.00E+11

0 5 10 15 20 25 30 35106

107

108

109

1010

1011

Cum

ulat

ive

cell

grow

th

Days in culture

ControlWT FGF-1

S130AS130E

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

1.00E+11

0 5 10 15 20 25 30 351.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

1.00E+11

0 5 10 15 20 25 30 35106

107

108

109

1010

1011

Cum

ulat

ive

cell

grow

th

Days in culture

ControlWT FGF-1

S130AS130E

ControlWT FGF-1

S130AS130E

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

0 5 10 15 20 25 30 35106

107

108

109

1010

Cum

ulat

ive

cell

grow

th

Days in culture

Control

WT FGF-2NLS/FGF-2

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

0 5 10 15 20 25 30 351.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

0 5 10 15 20 25 30 35106

107

108

109

1010

Cum

ulat

ive

cell

grow

th

Days in culture

Control

WT FGF-2NLS/FGF-2

Control

WT FGF-2NLS/FGF-2

Figure 3.4: Cell growth of 5-FU BM cells overexpressing S130 mutants and NLS/FGF-2. (A):

Cumulative cell growth of 5-FU BM cells transduced with control, WT FGF-1, S130E and S130A

vectors. Following transduction GFP+ cells were selected and cultured for a total of 33 days in

StemSpan supplemented with 10% FCS, IL-11, SCF and Flt3L. Growth curves for control and WT

FGF-1 cells are the same as those in Figure 3.1 and were used for comparison purposes. (B):

Cumulative cell growth of 5-FU BM cells transduced with control, WT FGF-2 and NLS/FGF-2.

Growth curves for control and WT FGF-2 are the same as those in Figure 3.1 and were used for

comparison purposes.

Co-culturing of transduced cells on a stromal layer

Previous studies in our group demonstrated that we could culture purified LSK cells

in the presence of unfractionated BM16. Based upon these results we sorted 5,000 or

10,000 GFP+ cells transduced with control, WT FGF-1, S130E and S130A onto

FBMD-1 stromal layer (Figure 3.5A). Cultures were maintained for a maximum of 14

days. At each data point (day 9 and day 14), non-adherent cells were removed,

analyzed and discarded. Analysis of growth kinetics revealed that control and WT


86

FGF-1 transduced BM cells displayed a slight increase (~ 1.3 fold) in cell numbers

compared to both S130 mutants (Figure 3.5B). The co-culturing of transduced BM

cells on a stromal layer results in the widespread presence of cobblestones, as can

easily be seen from images of co-cultures, taken eleven days after initiation (Figure

3.5C-F). More cobblestones were observed in co-cultures with WT FGF-1 and S130A

at day 11 (Figure 3.5D and 3.5F). S130E co-cultures contained more non-adherent

cells than the other co-cultures (Figure 3.5E).

5-FU treated Retroviral transduced cells

Sort GFP+ cells5000 or 10 000 GFP+ cells onFBMD stromal layer

A.

B.

Control d11 WT FGF-1 d11 S130E d11 S130A d11C. D. E. F.

0.00E+00

2.00E+06

4.00E+06

6.00E+06

8.00E+06

1.00E+07

1.20E+07

0 5 10 15Days after sort

Cum

ulat

ive

Cel

l Num

ber

0

2 x 106

4 x 106

6 x 106

8 x 106

10 x 106

12 x 106

SS

ControlWT FGF-1S130AS130E

5-FU treated Retroviral transduced cells

Sort GFP+ cells5000 or 10 000 GFP+ cells onFBMD stromal layer

A.

B.

Control d11 WT FGF-1 d11 S130E d11 S130A d11C. D. E. F.Control d11 WT FGF-1 d11 S130E d11 S130A d11C. D. E. F.

0.00E+00

2.00E+06

4.00E+06

6.00E+06

8.00E+06

1.00E+07

1.20E+07


Cum

ulat

ive

Cel

l Num

ber

0

2 x 106

4 x 106

6 x 106

8 x 106

10 x 106

12 x 106

SS


0.00E+00

2.00E+06

4.00E+06

6.00E+06

8.00E+06

1.00E+07

1.20E+07

0 5 10 150.00E+00

2.00E+06

4.00E+06

6.00E+06

8.00E+06

1.00E+07

1.20E+07


Cum

ulat

ive

Cel

l Num

ber

0

2 x 106

4 x 106

6 x 106

8 x 106

10 x 106

12 x 106

SS

ControlWT FGF-1S130AS130ESS


Figure 3.5: Co-culturing of control, WT FGF-1, S130E and S130A cells with FBMD-1 stromal cells.

(A): Schematic representation of co-culturing procedure. BM cells were retrovirally transduced with

control, WT FGF-1, S130E and S130A vectors. Following transduction, 5,000 or 10,000 GFP+ cells

were selected and co-culture in a culture flask containing pre-seeded FBMD-1 stromal cells. Cells were

cultured in IMDM supplemented with 20% horse serum, hydrocortisone and penicillin/streptomycin

containing β-mercaptoethanol. (B): Cumulative cell growth of co-cultured cells. Images of co-cultured

cells 11 days after initiation of culture transduced with (C): control, (D): WT FGF-1, (E): S130E and

(F): S130A.


87

Co-cultured cell do not provide long-term repopulation

Nine and 14 days after initiation of co-cultures, non-adherent cells were placed in

CFU-GM assay. The number of CFU/GM per 105 cells is shown in Figure 3.6A.

CFU-GMs were not detected for control cells and no significant differences were

observed between WT FGF-1 and S130 mutants after nine days of culturing (Figure

3.6A). After 14 days of culturing, there was an increase in CFU-GM for S130A

transduced cells over the control, WT FGF-1 and S130E transduced cells (Figure

3.6A). In addition, 14 days after initiation of culture, non-adherent cells were placed

in a CAFC assay. S130A cells contained higher CAFC day 7 activity, correlating with

CFU-GM data (Figure 3.6B). S130E transduced cells were observed to have

extremely high CAFC day 28 activity (30/105, 17-61; 95% confidence interval) whilst

CAFC day 28 activity was not detected for the other groups (Figure 3.6B).

Ten days after co-culturing of GFP+ cells with the stromal layer, the whole culture

was harvested and CD45.1+GFP+ cells were sorted. CD45.1+GFP+ cells were

transplanted in competition with freshly isolated B6 BM cells. Disappointingly,

CD45.1+GFP+ cells co-cultured cells failed to provide engraftment 18 weeks after

transplant (Figure 3.6C). It should be noted that the calculated chimerism levels have

not been corrected for the different number of CD45.1+GFP+ cell transplanted. In

conclusion, these results demonstrate that co-culturing of retrovirally transduced BM

cells is ineffective.

Primary transplantation of WT FGF-1 and S130 mutants

Bone marrow cells from CD45.1 donor mice were isolated four days after 5-FU

administration and retrovirally transduced with control vector and vectors encoding

WT FGF-1, S130E and S130A. Following transduction, cells were assayed for CAFC

content and transplanted into primary recipients (Figure 3.7A). Transduction

efficiencies of ~50% were achieved (data not shown). Unsorted transduced BM cells

were placed in a 4-fold limiting dilution in in vitro CAFC assays. No significant

differences in CAFC day 7 activity were observed between the different cell sources

(Figure 3.7B). Low CAFC day 28 activity was observed for all cells, however control

cells contained higher CAFC day 28 activity compared to WT FGF-1, S130E and

S130A (Figure 3.7B). Following transduction, 3 x 106 non-fractionated BM cells were

transplanted into primary lethally irradiated recipients. Donor chimerism analysis of

CD45.1+GFP+ cells showed no differences in engraftment between control, S130E


88

and S130A overexpressing cells (Figure 3.7C). Engraftment of FGF-1 overexpressing

cells throughout the transplant period was lower compared to control and S130 cells.

A ~2.5-fold decrease in engraftment was observed, 24 weeks after transplant (Figure

3.7C). Donor chimerism levels of S130E and S130A transplanted animals were

significantly higher than control and WT FGF-1 chimerism levels (p < 0.05) only at

20 weeks post transplant (Figure 3.7C).

A.

0

5

10

15

20

25

9 14

Days after sorting on to FBMDs

GM

/105

ControlWT FGF-1S130ES130A

B.

0

10

20

30

40

50

60

70

CA

FC/1

05 ce

lls

0

500

1000

1500

Day 7 Day 28


N.D. N.D. N.D.CA

FC/1

05 ce

lls

0

1

MIEV FGF1 S130E S130A

Don

or c

him

eris

mC

D45

.1+ G

FP+

C.

A.

0

5

10

15

20

25

9 14

Days after sorting on to FBMDs

GM

/105



B.

0

10

20

30

40

50

60

70

CA

FC/1

05 ce

lls

0

500

1000

1500

Day 7 Day 28


N.D. N.D. N.D.CA

FC/1

05 ce

lls

0

10

20

30

40

50

60

70

CA

FC/1

05 ce

lls

0

500

1000

1500

Day 7 Day 28



N.D. N.D. N.D.CA

FC/1

05 ce

lls

0

1


Don

or c

him

eris

mC

D45

.1+ G

FP+

C.

0

1


Don

or c

him

eris

mC

D45

.1+ G

FP+

C.

Figure 3.6: In vitro assays and in vivo repopulating assay of co-cultured cells. (A): CFU-GM analysis

of control, WT FGF-1, S130E and S130A overexpressing cells, 9 and 14 days after initiation of culture.

(B): Day 7 and day 35 CAFC activity of co-cultured cells 14 days after initiation of culture. (C):

Average CD45.1+GFP+ donor chimerism levels 18 weeks post transplant from recipients transplanted

with co-cultured control, WT FGF-1, S130E and S130A cells 10 days after initiation of culture.


89

5-FU treated

Retroviral transducedcells

CD45.1 9Gy 3 x 106

cells

20 weeks

1:11:21:4

9Gy

CAFC

A.

B.

C. D.

CA

FC/1

05ce

lls

0

5000

10000

15000

20000

25000

30000

35000

Day 7 Day 280

2

4

6

8

10

12

14

16

ControlWT FGF-1

S130AS130E

0

0.1

0.2

0.3

0.4

%LS

K

Control WT FGF-1

S130E S130A B6 BM0

10

20

30

40

50

60

4 8 12 16 20 24Don

or c

him

eris

mG

FP+ Control

WT FGF-1 S130AS130E

Weeks post transplant

CAF

C/1

05ce

lls

5-FU treated

Retroviral transducedcells

CD45.1 9Gy 3 x 106

cells

20 weeks

1:11:21:4

9Gy1:11:21:4

9Gy

CAFC

A.

B.

C. D.

CA

FC/1

05ce

lls

0

5000

10000

15000

20000

25000

30000

35000

Day 7 Day 280

2

4

6

8

10

12

14

16

ControlWT FGF-1

S130AS130E

ControlWT FGF-1

S130AS130E

0

0.1

0.2

0.3

0.4

%LS

K

Control WT FGF-1

S130E S130A B6 BM0

0.1

0.2

0.3

0.4

%LS

K

Control WT FGF-1

S130E S130A B6 BM0

10

20

30

40

50

60

4 8 12 16 20 24Don

or c

him

eris

mG

FP+ Control

WT FGF-1 S130AS130E


0

10

20

30

40

50

60

4 8 12 16 20 24Don

or c

him

eris

mG

FP+ Control

WT FGF-1 S130AS130EControl

WT FGF-1 S130AS130E


CAF

C/1

05ce

lls

Figure 3.7: Transplantation of WT FGF-1, S130E and S130A overexpressing cells into primary and

secondary recipients. (A): Schematic representation of transplantation strategy. 5-FU treated BM cells

were retrovirally transduced with control, WT FGF-1, WT FGF-2, S130E, S130A and NLS/FGF-2

vectors. Following transduction, 3 x 106 unsorted BM cells were transplanted into lethally irradiated

(9Gy) primary B6 recipients. Parallel to this, transduced cells were analyzed for CAFC. Twenty weeks

after transplant, BM cells were harvested from primary recipients and transplanted in limiting dilutions

(1:1, 1:2 and 1:4) with 4 x 105 competitors into lethally irradiated secondary recipients. (B): Day 7 and

day 28 CAFC activity of control, WT FGF-1, S130E and S130A overexpressing cells. (C): Average

CD45.1+GFP+ donor chimerism levels of primary recipients (n=10 per group) transplanted with

control, WT FGF-1, S130E and S130A overexpressing cells. Chimerism in blood was analyzed on a

monthly basis. (D): LSK analysis of BM harvested from primary recipients 20 weeks after transplant.


90

Secondary transplantation of WT FGF-1 and S130 mutants

Twenty weeks after transplant, BM was harvested from primary recipient mice and

analyzed for Lin-Sca-1+c-Kit+ (LSK) expression. In a normal C57BL/6 mouse, BM

cells contained 0.22 ± 0.05% LSK cells (Figure 3.7D). The percentages of LSK cells

in control, WT FGF-1 and S130A transplanted mice were comparable to that of B6

BM (Figure 3.7D). A small increase (1.45-fold) in the percentage of LSK cells was

observed in mice transplanted with S130E overexpressing BM cells (Figure 3.7D).

Secondary recipients were transplanted with 2 x 105 unfractionated donor BM cells in

competition with a fixed dose of 4 x 105 freshly isolated B6 CD45.2 BM cells (Figure

3.7A). Recipients were analyzed for the presence of CD45.1+GFP+ cells on a monthly

basis. As shown in Figure 3.8A, 24 weeks after transplant mice transplanted with

control, WT FGF-1 and S130E retrovirally transduced cells displayed CD45.1+GFP+

chimerism levels of ~ 8-10%. In contrast, S130A transduced cells displayed high

donor derived contribution in the peripheral blood. Chimerism levels of ~40% were

reached (Figure 3.8A). Individual donor chimerism levels of S130A secondary

recipients demonstrate that the effect brought about by S130A was delayed, with

chimerism levels only beginning to increase 12 weeks after transplant (Figure 3.8B).

FACS plots (inset) illustrate the increase in GFP+ cells from 4 weeks to 24 weeks after

transplant (Figure 3.8B). Comparison of S130A transduced cells (GFP+) over non-

transduced cells (GFP-), revealed a significant (p < 0.05) 10-fold increase in GFP+

cells compared to control, WT FGF-1 and S130E transduced cells, 24 weeks after

transplant (Figure 3.8C). These results suggest that transport of FGF-1 into the

nucleus may have an intracrine function maintaining stem cell quality.


91

C.

0

2

4

6

8

10

12

0 5 10 15 20 25

ControlWT FGF-1

S130AS130E

CD

45.1

+ GFP

+vs

CD

45.1

+ GFP

-


0

10

20

30

40

50

60

0 5 10 15 20 25

B.

Indi

vidu

al C

D45

.1+ G

FP+

chim

eris

m


GFP

PE

GFP

PE

A.

Don

or c

him

eris

mC

D45

.1+ G

FP+

0

10

20

30

40

50

Control WT FGF-1 S130E S130A

2 x 105 donor cells + 4 x 105 competitor cells

C.

0

2

4

6

8

10

12

0 5 10 15 20 25

ControlWT FGF-1

S130AS130E

CD

45.1

+ GFP

+vs

CD

45.1

+ GFP

-


C.

0

2

4

6

8

10

12

0 5 10 15 20 25

ControlWT FGF-1

S130AS130E

ControlWT FGF-1

S130AS130E

CD

45.1

+ GFP

+vs

CD

45.1

+ GFP

-


0

10

20

30

40

50

60

0 5 10 15 20 25

B.

Indi

vidu

al C

D45

.1+ G

FP+

chim

eris

m


GFP

PE

GFP

PE

0

10

20

30

40

50

60

0 5 10 15 20 25

B.

Indi

vidu

al C

D45

.1+ G

FP+

chim

eris

m


GFP

PE

GFP

PE

GFP

PE

GFP

PE

A.

Don

or c

him

eris

mC

D45

.1+ G

FP+

0

10

20

30

40

50



A.

Don

or c

him

eris

mC

D45

.1+ G

FP+

0

10

20

30

40

50

Control WT FGF-1 S130E S130A0

10

20

30

40

50



Figure 3.8: S130A increases donor chimerism levels in secondary recipients transplanted with 2 x 105

S130A overexpressing cells and 4 x 105 competitor cells. (A): Average donor chimerism

(CD45.1+GFP+) levels 24 weeks post transplant of secondary transplant recipients. (n=5 per group).

(B): Individual donor chimerism of secondary recipients transplanted with S130A transduced cells.

FACS plots highlight the increase in GFP+ cells during the transplant period. (C): Ratio of transduced

GFP+ cells and non-transduced GFP- cells.

Primary transplantation of WT FGF-2 and NLS/FGF-2

Similar to WT FGF-1 and S130 mutants, 5-FU treated BM cells were retrovirally

transduced with control, WT FGF-2 and NLS/FGF-2. Transduction efficiencies of


92

~50% were achieved (data not shown). Following transduction, unfractionated cells

were assayed for CAFC content in 3-fold limiting dilutions. CAFC day 7 activity for

control transduced cells was 3- and 2-fold higher than WT FGF-2 and NLS/FGF-2

transduced cells respectively (Figure 3.9A), indicating that control cells contain more

progenitors. Low CAFC day 35 activity was observed for all three groups (Figure

3.9A).

Lethally irradiated mice were transplanted with 3 x 106 unsorted transduced BM cells.

Monthly donor chimerism analysis (CD45.1+GFP+) showed that WT FGF-2 and

NLS/FGF-2 transduced cells had comparable chimerism levels (~16% ± 5, 20 weeks

after transplant) whilst control transduced cells maintain significantly higher

chimerism levels (29% ± 6, 20 weeks after transplant) during the 20 week

transplantation period (p < 0.05) (Figure 3.9B).

Secondary transplantation of WT FGF-2 and NLS/FGF-2

Unfractionated bone marrow from primary recipient mice 20 weeks post-transplant

were harvested and analyzed for the percentage of LSK cells. Similar to normal B6

BM cells (0.22 ± 0.05%), WT FGF-2 and NLS/FGF-2 BM cells contained 0.15 ±

0.02% and 0.27 ± 0.05% LSK cells, respectively (Figure 3.9C). Control transduced

cells contained 1.7-fold increase in LSK cells compared to B6 BM cells (Figure

3.9C).

As outlined above, secondary recipients were transplanted with harvested donor BM

and transplanted in limiting dilutions in competition with 4 x 105 freshly isolated B6

BM cells. Analysis of CD45.1+GFP+ chimerism levels 24 weeks after transplant

indicated that WT FGF-2 and NLS/FGF-2 provided little to no contribution in the

peripheral blood (Figure 3.9D). As expected, because of the higher levels of donor

chimerism levels in the primary transplant, slightly higher CD45.1+GFP+ engraftment

levels were also detected in secondary recipients of control transduced cells (~4 ± 8%)

(Figure 3.9D). Comparison of chimerism levels from the control with that of the

previous control group (Figure 3.8A) indicated that chimerism levels from both

groups were similar. This confirms that in this experiment the control cells do not

have an increase in engraftment, but that WT FGF-2 and NLS/FGF-2 transduced

donor cells have poorer stem cell quality and thus poorer engraftment ability.


93

0

10

20

30

40

50

60

0 5 10 15 20 25

Don

or c

him

eris

mG

FP+

0

2

4

6

8

10

12

14

16

Don

or c

him

eris

mC

D45

.1+ G

FP+

Control WT FGF-2 NLS/FGF-2

A.

B. C.

D.

ControlWT FGF-2NLS/FGF-2

0

0.2

0.4

0.6

0.8

1

%LS

K

Control WT FGF-2

NLS/FGF-2

B6 BM


0

1

2

CA

FC/1

05ce

lls

0

1000

2000

3000

4000

5000

Day 7 Day 35

Control

WT FGF-2NLS/FGF-2

CAF

C/1

05ce

lls


0

10

20

30

40

50

60

0 5 10 15 20 25

Don

or c

him

eris

mG

FP+

0

2

4

6

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10

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14

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Don

or c

him

eris

mC

D45

.1+ G

FP+


2

4

6

8

10

12

14

16

Don

or c

him

eris

mC

D45

.1+ G

FP+


A.

B. C.

D.



0

0.2

0.4

0.6

0.8

1

%LS

K

Control WT FGF-2

NLS/FGF-2

B6 BM0

0.2

0.4

0.6

0.8

1

%LS

K

Control WT FGF-2

NLS/FGF-2

B6 BM


0

1

2

CA

FC/1

05ce

lls

0

1000

2000

3000

4000

5000

Day 7 Day 35

Control

WT FGF-2NLS/FGF-2

CAF

C/1

05ce

lls

0

1

2

CA

FC/1

05ce

lls

0

1000

2000

3000

4000

5000

Day 7 Day 35

Control

WT FGF-2NLS/FGF-2

Control

WT FGF-2NLS/FGF-2

CAF

C/1

05ce

lls


Figure 3.9: Transplantation of WT FGF-2 and NLS/FGF-2 cells into primary and secondary recipients.

(A): Day 7 and day 35 CAFC activity of control, WT FGF-2 and NLS/FGF-2 overexpressing cells. (B):

Average CD45.1+GFP+ donor chimerism levels of primary recipients (n=10 per group) transplanted

with control, WT FGF-2 and NLS/FGF-2 overexpressing cells. Chimerism in blood was analyzed on a

monthly basis. (C): LSK analysis of BM harvested from primary recipients 20 weeks after transplant.

(D): Average donor chimerism (CD45.1+GFP+) levels 24 weeks post-transplant of secondary transplant

recipients transplanted (n=5 per group).


94

Discussion In this study we undertook a detailed analysis of the ability of enforced

overexpression of FGF-1 and FGF-2 to maintain and expand hematopoietic cells in

vitro and in vivo. We demonstrate that overexpression of FGF-1 slightly increases the

mitogenic effect of 5-FU BM cells and that both FGF-1 and FGF-2 are almost

exclusively expressed in Lin-Sca-1+c-Kit+ cells, a primitive hematopoietic cell subset.

Finally, we show that secondary recipients transplanted with S130A mutant FGF-1

showed a delayed increase in donor chimerism levels and a 10-fold increase in GFP+

cells, implying that nuclear localized FGF-1 may play a role in maintaining stem cell

quality.

Unfortunately, in in vivo repopulation assays, low donor chimerism levels (~10% to

15%) were detected in primary recipients transplanted with WT FGF-1 and WT FGF-

2 and little to no reconstitution was observed in secondary recipients. This was

unexpected as previous studies had demonstrated that the exogenous addition of FGFs

could maintain whole BM cultures for up to five weeks and generate large numbers of

cells with lymphoid and myeloid long-term repopulating capacity15;16. Additionally,

the use of FGF-1 expanded BM cells as a source of stem cells for retroviral gene

delivery generated 15.5-fold increase in the number of bone marrow-derived

competitive repopulating units per mouse and provided radioprotection and long-term

BM reconstitution with average myeloid and lymphoid chimerisms of 70% and 50%

respectively42.

It is well known that FGFs are exogenous growth factors that activate the cell surface

receptors, thereby inducing activation of intracellular second messengers43. In

addition, the receptor-bound growth factor is endocytosed and translocated across the

vesicular membrane to reach the cytosol44;45. Several lines of evidence exist indicate

that after binding to receptors, FGF-1 and FGF-2 enter the nucleus and that this is

required for mitogenic response in certain cell types46-48. This led us to assume that

both WT FGF-1 and WT FGF-2 may not be entering the nucleus in order to exert its

full mitogenic activity. As a result, two S130 mutants and one NLS/FGF-2 were

created to further examine the effect of nuclear localized FGFs on BM cells.

Immunocytochemistry analysis of S130A and NLS/FGF-2 overexpressing cells

indicated that FGF-1 and FGF-2 were not exclusively localized in the nucleus in all

FGF positive cells. Considering that in only 2% of FGF-1 positive cells the protein


95

was localized in the nucleus, the delayed but marked increase in CD45.1+GFP+

chimerism levels and the significant 10-fold increase in GFP+ cells of S130A

transduced cells in secondary recipients was exceptional. This suggests that the

localization of FGF-1 in the nucleus may play a role in maintaining stem cell quality.

The translocation of FGFs appears to be a dynamic process. For example, FGF-2 has

been shown to enter the nucleus, both when added exogenously46;49 and when

expressed endogenously26;50;51. In the former case, nuclear uptake is cell cycle-

dependent occurring only during G1 to S transition46. In the latter case, only the larger

(22-25kDa) isoforms are found in the nucleus while the 18kDa form remains

cytoplasmic25;26;50;51. Through cell fractionation studies Wiedlocha et al., clearly

showed that WT FGF-1 was found in all fractions (membrane, cytoplasmic and

nucleus) of the cell, S130A was found in the nuclear fraction whereas S130E was

mainly in the cytosolic fraction19. We wish to emphasize that since we have only

analyzed localization of FGF-1 and FGF-2 by immunocytochemistry, it is possible

that we are only able to detect small amounts of FGF-1 and FGF-2 in S130A and

NLS/FGF-2 in the nuclei. Thus, a more sensitive method such as cell fractionation

may be used in future studies.

Based upon previous studies from our group16, we hypothesized that a niche in the

form of stromal cells may increase the mitogenic activity of transduced cells and

maintain stem cell activity. A role is now emerging for the ‘stem-cell niche’ to affect

maintenance, differentiation and regulation of self-renewal of HSCs in vivo52-54. Since

stromal cells include a high proportion of fibroblast cells that respond to FGFs,

especially FGF-2 10;55, we co-cultured WT FGF-1, S130E and S130A transduced cells

with FBMD-1 stromal cells. Unfortunately, co-culturing of transduced cells with a

stromal layer was ineffective as these cells failed to reconstitute the peripheral blood

of recipients.

Heparin or heparin sulfate proteoglycans play a pivotal role in stimulating and

stabilizing the interaction of FGFs to FGFRs8;56;57. One possibility as to why no

dramatic repopulation was observed in FGF-1 and FGF-2 overexpressing cells may be

due to the lack of heparin in cultures. In previous studies involving exogenous FGFs,

heparin was also added exogenously. In our current study, heparin was not added.

Klingenberg et al., has shown that a correlation exists between mitogenic potency and

heparin affinity of FGF-1 phosphorylated mutants21. If the reason for the reduced

repopulation ability of FGF-1, FGF-2, S130 mutants and NLS/FGF-2 was due to the


96

lack of heparin, future overexpression studies with FGFs should involve the addition

of heparin.


97

Acknowledgements We thank Professor C. Jordan from the University of Rochester for providing us with

the MIEV vector. We also thank Geert Mesander and Henk Moes for their assistance

with cell sorting and the staff of the animal facility for taking care of the animals. This

work was supported by the National Institute of Health (R01-HL073710) and the

Ubbo Emmius Foundation.


98

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41. Prudovsky I, Bagala C, Tarantini F et al. The intracellular translocation of the components of the fibroblast growth factor 1 release complex precedes their assembly prior to export. J.Cell Biol. 2002;158:201-208.

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102


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104

105

Chapter 4

Mobilized peripheral blood stem cells provide rapid

reconstitution but impaired long-term engraftment

Joyce S. G. Yeoh, Albertina Ausema, Piet Wierenga,

Gerald de Haan, Ronald van Os



Submitted

Mobilized peripheral blood stem cells have impaired long-term engraftment

106

Abstract In this study, we use competitive repopulation assay to compare the quality and

frequency of stem cells isolated from mobilized blood with stem cells isolated from

bone marrow (BM). Lin-Sca-1+c-Kit+ (LSK) cells were harvested from control BM

and peripheral blood of mice following Granulocyte Colony Stimulating Factor (G-

CSF) administration. LSK cells were used because of their resemblance with human

CD34+ cells. We confirmed that transplantation of phenotypically defined mobilized

peripheral blood (MPB) stem cells results in rapid recovery of blood counts.

However, in vitro results indicated that LSK cells purified from MPB had lower

cobblestone area forming cell (CAFC) day 35 activity compared to BM. Additionally,

evaluation of chimerism after co-transplantation of LSK cells purified from blood and

BM revealed that MPB stem cells contained 25-fold less repopulation potential

compared to BM stem cells. Competitive repopulating unit (CRU) frequency analysis

showed that freshly isolated MPB LSK cells have 8.8-fold fewer cells with long-term

repopulating ability compared to BM LSK cells. Secondary transplantation showed no

further decline in contribution of hematopoiesis relative to BM. We conclude that the

reduced frequency of stem cells within the LSK population of MPB, rather than

poorer quality, causes a reduced repopulation potential of MPB.


107

Introduction The initial source of hematopoietic cells used for transplantations was bone marrow1.

However, due to the faster regeneration of both circulating neutrophils (9-11 days) 2;3

and platelets, peripheral blood stem cells have become the primary source of

hematopoietic stem cells for clinical transplantation over the past 10 years4.

Although multiple hematopoietic growth factors are capable of inducing mobilization

of hematopoietic progenitors, G-CSF is at present one of the most used mobilizing

molecule in clinical protocols5. The efficiency of G-CSF to mobilize bone marrow

precursors and long-term repopulating cells was initially shown in preclinical studies.

Molineux et al., observed a marked increase of the colony-forming unit spleen (CFU-

S) pool in the peripheral blood of mice treated with repeated doses of G-CSF6. In

addition, it was observed that G-CSF alone and in combination with SCF or IL-7

mobilizes hematopoietic precursors capable of both radioprotection and generating

sustained lympho-haematopoiesis in transplanted recipients7. In vitro data from

human patients appear consistent with the concept that the quality of human

mobilized peripheral blood progenitor cells is at least equivalent to that corresponding

to bone marrow grafts8;9.

Surprisingly, despite the prevalent use of hematopoietic stem cell mobilization in

clinical transplantation, few reports exist describing the competitive repopulating

quality of mobilized stem cells compared to bone marrow stem cells following G-CSF

treatment. Most available reports only outlined the differences in the kinetics and

efficiency of engraftment, in homing properties and in cell cycle profiles between

mobilized blood stem cells and those isolated from resting bone marrow5;10-13.

In view of the increased use of peripheral blood stem cells in clinical transplant

settings, it is of relevance to investigate long term functioning of stem cells isolated

from different sources. In this study, we directly assessed the function of mobilized

peripheral blood stem cells compared to control bone marrow stem cells when co-

transplanted in a single recipient mouse in an in vivo competitive repopulation assay.

We show a reduced frequency of repopulating stem cells in purified G-CSF-mobilized

peripheral blood LSK stem cells, which caused a 25-fold reduction in repopulation

potential compared with BM LSK cells.


108

Materials and Methods Mice

Female C57BL/6 (B6), C57BL/6.SJL (CD45.1), (C57BL/6 x C57BL6.SJL) F1

(CD45.1/2) or C57BL/6-Tg(ACTB-EGFP)10sb/J transgenic GFP (GFP) mice were

used as donors, competitors or recipients of blood and marrow stem cells depending

on the experimental model. CD45.1 and transgenic GFP mice were originally

obtained from the Jackson Laboratory (Bar Harbor, Maine) and bred in our local

animal facility. Wild type female B6 mice were purchased from Harlan (Horst, The

Netherlands) and maintained under clean conventional conditions in the animal

facilities of University Medical Centre Groningen (The Netherlands). Mice were fed

ad libitum with food pellets and acidified tap water (pH = 2.8). All animal procedures

were approved by the local animal ethics committee of the University Medical Centre

Groningen.

Mobilization and harvesting of stem cells

Bone marrow cells were harvested by flushing the femoral content with α-MEM

(GibcoBRL, Invitrogen, CA) supplemented with 2% fetal calf serum (FCS;

GibcoBRL, Invitrogen, CA). Pegylated G-CSF (Neulasta) (250µg/kg/mouse)

(Amgen, Thousand Oaks, CA) was used to mobilize stem cells14. Two doses of G-

CSF were subcutaneously administered to donor mice at day -6 and day -3 before

stem cell harvest. Mobilized peripheral blood cells were harvested by cardiac

puncture. Approximately 1 ml of blood was collected and was diluted with 4 ml of

Iscove’s modified DMEM (IMDM; GibcoBRL, Paisley, Scotland) supplemented with

5% FCS (GibcoBRL, Invitrogen, CA) and heparin (25 IU) (Leo Pharma, Breda,

Netherlands). The collected blood cell suspension (5 ml) was centrifuged over an

equal volume of Lympholyte-M (Cedarlane Laboratories Ltd, Hornby, Canada) at 400

x g for 30 minutes at room temperature. After centrifugation, the mononuclear cells

within the opaque interface layer were isolated and washed in IMDM/5% FCS for 5

minutes at 2,000 rpm at 4oC. Alternatively, red blood cells were lysed using

ammonium chloride (NH4Cl) without prior density separation. Nucleated cells were

measured on a Coulter Counter Model Z2 (Coulter Electronics, Hialeah, FL).


109

Isolation of Lin-Sca-1+c-Kit+ cells

Bone marrow and mobilized peripheral blood cells were stained as previously

described15 with biotinylated lineage-specific antibodies Mouse Lineage Panel,

containing anti-CD45R, anti-CD11b, anti-TER119, anti-Gr-1 and anti-CD3e (BD

Pharmingen, San Diego, CA), FITC-anti-Sca-1 and APC-anti-c-kit (BD Pharmingen,

San Diego, CA). Biotinylated antibodies were visualized with streptavidin-PE

(Pharmingen, San Diego, CA). After antibody staining, cells were sorted by a

MoFlow cell sorter (DakoCytomation, Fort Collins, CO). Lin-Sca-1+c-Kit+ (LSK) and

Lin- non-Sca-1+c-Kit+ cells were sorted and used in transplantation assays or in in

vitro CAFC assays.

Long term competitive repopulation ability

Female B6, CD45.1 or transgenic GFP mice were used as donors for competitor cells.

Female B6 mice were used as recipients in all experiments. Recipient mice were

irradiated with 9.5 Gy γ-rays (0.7026 Gy/min) in a CIS Biointernational IBL 637 137Cs-source, 20-24 hours prior to transplantation. For competitive repopulation

determination, unfractionated cells or mobilized peripheral blood LSK cells, were

mixed with competitor cells (unfractionated or LSK bone marrow cells) and

intravenously transplanted into recipient mice. Each transplant group consisted of 8-

10 recipients. Following transplantation, blood samples (60μl) were taken monthly to

determine donor chimerism. Levels of chimerism were determined by detecting the

presence of GFP+ or CD45.1+ and CD45.2+ cells in transplanted mice. To detect

CD45.1+ and CD45.2+ cells, cells were stained with anti-CD45.2 (FITC) and anti-

CD45.1 (PE) antibodies (BD Pharmingen, San Diego, CA) for 30 minutes and

analyzed on a flow cytometer (FACS Calibur; Becton Dickinson Biosciences, San

Jose, CA). In addition, the competitive repopulating index (CRI) was determined. CRI

is a relative measure of the competitive ability of test cells to that of fresh bone

marrow cells. The CRI was calculated by taking the ratio of WBC derived from

mobilized blood cells to bone marrow cells in the circulation and dividing it by the

ratio of mobilized blood cells to bone marrow cells transplanted. A CRI value of one

indicates by definition that mobilized peripheral blood cells and bone marrow cells

have equal competitive ability.


110

Competitive Repopulation Unit assay

B6 recipient mice were transplanted with a series of diluted CD45.1 LSK cells (1,200,

600 and 300) from mobilized blood and control bone marrow and with a fixed number

of B6 competitor cells (5 x 105). Twelve weeks after transplantation, donor cell

contribution in the peripheral blood was determined. Recipients with a contribution of

≥ 5% in both myeloid and lymphoid lineages were considered to be positive.

To evaluate and quantify the repopulating potential of mobilized blood LSK cells and

control BM LSK cells, the frequency of competitive repopulation units (CRU) was

calculated. CRU frequencies per 1,000 LSK were calculated from the resultant

percentage positive recipients by limiting dilution analysis procedures which uses

Poisson statistics16.

Cobblestone area forming cell assays

Cobblestone area forming cell assays (CAFC) were performed as described17-19 to

assess the number of hematopoietic progenitor cells (CAFC day 7) or more primitive

stem cells (CAFC day 35) in mobilized peripheral blood stem cells.

Secondary Transplantations

In one of the competitive repopulation experiments, in which recipients were

transplanted in different ratios with CD45.1 mobilized peripheral blood LSK cells and

CD45.2 bone marrow LSK competitor cells, mice were sacrificed for secondary

transplantations. Bone marrow cells from B6 primary chimeric recipients were

isolated on basis of CD45 isoform. CD45.1 bone marrow cells represent cells derived

from mobilized peripheral blood LSK population (CD45.1 MPB LSK derived bone

marrow) and CD45.2 (B6) bone marrow cells represent cells derived from bone

marrow LSK cells (CD45.2 BM LSK derived bone marrow). Female CD45.1

recipient mice were transplanted with CD45.2 bone marrow LSK derived bone

marrow cells whilst B6 recipient mice were transplanted with CD45.1 mobilized

peripheral blood LSK derived bone marrow cells. Recipient mice were transplanted in

three ratios (1:1, 1:2 and 1:4) with a fixed number (4 x 105) of CD45.1/2 (F1)

competitors. Each transplant group consisted of five recipients and blood samples

(60µl) were taken on a monthly basis to determine donor chimerism and CRI.


111

Statistical Analysis

P-values were calculated using the Mann-Whitney test or the student’s t-test

(assuming unequal variances of the two variables) and were employed to determine

the statistical significance between mobilized blood and normal bone marrow (p <

0.05). Quantification of CAFC at day 7 and 35 was performed by using maximum

likelihood ratio method20. The Poisson-based limiting dilution analysis calculation

was used with a 95% confidence interval to determine significant differences at p <

0.05.


112

Results Unfractionated mobilized peripheral blood cells have reduced repopulation ability

compared to unfractionated bone marrow cells

To compare the repopulation ability of unfractionated bone marrow and

unfractionated mobilized peripheral blood cells, blood cells were extracted from

donor B6 mice following treatment with two doses of G-CSF. Untreated bone marrow

cells were obtained from GFP-Tg or CD45.1 mice. Clonogenic activity of the two cell

sources was measured by seeding in limiting dilutions in a CAFC assay. At day 7,

cells from each source displayed high CAFC activity, with average values of ~1,400

progenitors/106 cells [95% confidence intervals (CI): 908-2294]. No significant

differences were observed between bone marrow and peripheral blood (Figure 4.1A).

Whilst bone marrow cells isolated from either CD45.1 or GFP Tg mice displayed a

high CAFC day 35 activity of 10.5 (95% CI: 6.6-16.8)/106 and 16 (95% CI: 10-

24)/106 respectively, CAFC day 35 frequency in mobilized peripheral blood was only

1.8 (95% CI: 1-3.3)/106 (Figure 4.1A). These results indicate that unfractionated

mobilized peripheral blood cells had significantly lower primitive stem cell activity

compared to bone marrow cells.

To verify the in vitro results, three groups of lethally irradiated mice were

transplanted in a competitive repopulation assay. Donor chimerism levels of

mobilized peripheral blood cells were used to calculate CRI post transplant (Figure

4.1B) and were ~20% (data not shown). As expected, a CRI value of 1 was

maintained throughout the 7 month transplant period for recipient mice receiving

CD45.1 bone marrow mixed with GFP bone marrow, indicating equal competitive

repopulating ability of both sources (Figure 4.1B). However, mice transplanted with

mobilized peripheral blood in competition with either CD45.1 bone marrow or GFP

bone marrow exhibited significantly lower average CRI levels (0.33 ± 0.41 and 0.41 ±

0.29, respectively) at 5 months after transplant (Figure 4.1B).

Both in vitro CAFC results and in vivo competitive repopulation assays suggest that

unfractionated mobilized peripheral blood cells have lower repopulation ability

compared to unfractionated bone marrow cells. In addition, no apparent differences

were observed between different donor mouse strains. The fact that we used the

mononuclear cell fraction from mobilized blood (which is enriched for stem cells) and


113

unseparated bone marrow cells may underestimate the difference in repopulating

ability.

0

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B.

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Figure 4.1: Unfractionated mobilized peripheral blood cells have reduced repopulation ability in

comparison with bone marrow cells. Unfractionated monunuclear cells from mobilized peripheral

blood (MPB) were obtained from B6 mice following treatment with 2 doses of Peg G-CSF 6 and 3

days before isolation. Control unfractionated bone marrow (BM) cells were collected from CD45.1 and

GFP-transgenic mice and transplanted in equal numbers. (A): Analysis of CAFC day 7 and day 35

activity for CD45.1 BM, GFP BM and MPB. (B): CRI analysis of mice transplanted with B6 MPB +

CD45.1 control BM, B6 MPB + GFP control BM and CD45.1 control BM + GFP control BM. All

mice were transplanted with 2 x 106 donor cells and 2 x 106 competitor cells. Average chimerism levels

at 20 weeks were 42% ± 18 (GFP BM + CD45.1 BM), 20% ±19 (B6 MPB + CD45.1 BM), and 16% ±

21 (B6 MPB + GFP BM). All chimerism levels were converted into CRI values as indicated in

materials and methods.


114

Mobilized peripheral blood cells have a lower frequency of Lin-Sca-1+c-Kit+ cells

We next determined whether the poor repopulation ability of unfractionated mobilized

peripheral blood cells is reflected by the size of the phenotypically defined stem cell

population, LSK. To test this possibility we measured LSK frequencies in bone

marrow cells and mobilized peripheral blood. Typical FACS plots for isolating LSK

cells from bone marrow and mobilized peripheral blood cells are shown in Figure

4.2A and 4.2B. Mobilized peripheral blood differs from bone marrow cells, as they

contain fewer c-Kit+ cells and more Sca-1-c-Kit- cells (Figure 4.2B). This finding is

consistent with that of Levesque et al., who reported that mobilization with G-CSF

results in a down-regulation of c-Kit on mouse hematopoietic progenitor cells in

vivo21. On average, bone marrow cells have a LSK frequency of 0.17% ± 0.1,

significantly different to that of mobilized peripheral blood cells, which have a 3-4

fold lower frequency of 0.05% ± 0.04 (p < 0.05) (Table 4.1). The lower frequency in

mobilized blood may explain the lower repopulation potential of unfractionated

mobilized blood.

To compare mobilized blood and bone marrow LSK on a per cell basis, LSK cells

were isolated from bone marrow and mobilized peripheral blood and placed in

limiting dilution in a CAFC assay to assess clonogenic activity. CAFC day 7

frequency of 250/103 LSK cells (153-402; 95% CI) was recorded for control bone

marrow LSK cells, whilst mobilized peripheral blood LSK cells had a 2.4-fold higher

CAFC activity of (600/103 LSK cells [308-1160; 95% CI]) (Figure 4.2A and 4.2B).

CAFC day 35 frequency for mobilized peripheral blood LSK cells was 3.0/103 LSK

cells (1-8; 95% CI), similar to control bone marrow LSK cells with a CAFC activity

of 6.7/103 (0.9-48; 95% CI) (Figure 4.2A and 4.2B). Thus, these data suggest that,

compared to bone marrow, mobilized peripheral blood LSK cells contain somewhat

more progenitor cells, but slightly fewer stem cells.

It is well known that in normal bone marrow all stem cell activity is contained in the

LSK fraction22;23. To verify that in mobilized blood, stem cell activity is also

restricted to the LSK population, Lin- cells from mobilized peripheral blood were

further fractionated into non-Sca-1+c-Kit+ cells and placed in limiting dilution in a

CAFC assay (Figure 4.2C). As shown in Figure 4.2C, only some day 7 CAFC activity

was detected for Lin- non-Sca-1+c-Kit+ cells, but no CAFC day 35. This demonstrates

that cells outside the LSK gate do not have clonogenic activity. A summary of LSK

frequencies and CAFC day 35 frequencies and a calculation of their total pool size is


115

shown in Table 4.1. Since we did not splenectomize our mice, a considerable number

of LSK cells may have been present in the spleen. However, we aimed at comparing

the phenotypically defined cell population (LSK) from mobilized blood with the same

population in steady state bone marrow.

0.1

1

10

100

1000

10000

7 35

C.

FSC

SSC

Sca-1

c-K

it

BL/6 Mobilized PB Lin- non Sca-1+c-Kit+

nd

CAF

C /1

03ce

lls

Days

87%

A.BL/6 control BM

Lin

5%

Sca-1

c-K

it 0.15%

B.BL/6 Mobilized PB

FSC

SSC

Sca-1

c-K

it

1

10

100

1000

10000

7 35CAF

C /1

03LS

K c

ells

Days

Control BM

MPB

0.04%

3.3%

*

0.1

1

10

100

1000

10000

7 35

C.

FSC

SSC

Sca-1

c-K

it


nd

CAF

C /1

03ce

lls

Days

87%0.1

1

10

100

1000

10000

7 35

C.

FSC

SSC

Sca-1

c-K

it


nd

CAF

C /1

03ce

lls

Days

87%

A.BL/6 control BM

Lin

5%

Sca-1

c-K

it 0.15%

B.BL/6 Mobilized PB

FSC

SSC

Sca-1

c-K

it

1

10

100

1000

10000

7 35CAF

C /1

03LS

K c

ells

Days

Control BM

MPB

0.04%

3.3%

*

A.BL/6 control BM

Lin

5%

Sca-1

c-K

it 0.15%

B.BL/6 Mobilized PB

FSC

SSC

Sca-1

c-K

it

1

10

100

1000

10000

7 35CAF

C /1

03LS

K c

ells

Days

Control BM

MPB

Control BM

MPB

0.04%

3.3%

*

Figure 4.2: Lin-Sca-1+c-Kit+ (LSK) contains the majority of cells with long-term repopulating ability

in both bone marrow and mobilized peripheral blood. Characteristic FACS plot of Lin- and Sca-1+c-

Kit+ sorts for (A): B6 bone marrow (BM) and (B): mobilized peripheral blood (MPB) cells and

comparison in limiting dilution analysis using the CAFC assay. (C): FACS plot of the Lin- non-Sca-

1+c-Kit+ sort for MPB cells placed in CAFC assay.


116

Table 4.1: Average LSK frequencies in bone marrow and G-CSF mobilized blood

Frequency LSK (%) Total LSK cells/2

femurs or per ml blood

CAFC day

35/106

Control BM 0.17 ± 0.1 84837 ± 43361 12537 ± 9695

MPB 0.05 ± 0.04* 8968 ± 6377 2150 ± 1064

Average frequency, total LSK cells and CAFC day 35 frequency in control bone marrow (BM) (n=5)

and mobilized peripheral blood (MPB) (n=6).

* Indicates that LSK frequency is significantly (p < 0.05) lower compared to normal bone marrow

Mobilized peripheral blood stem cells promote accelerated hematological

reconstitution

To directly compare the rates of hematopoietic recovery of peripheral blood cell

values after transplant, 1,250 LSK cells were isolated from bone marrow or mobilized

peripheral blood and transplanted into two groups of lethally irradiated B6 mice.

Circulating blood cells were counted on day 7 and 10 and then once a week to 5

weeks after transplant. WBC counts of recipients transplanted with mobilized

peripheral blood stem cells increased at day 10 and day 14, reaching normal values of

~8 x 106/ml. At days 21 and 35, WBC counts were similar to those of animals

transplanted with bone marrow LSK cells (Figure 4.3). Consistent with the results in

Figure 4.2A and 4.2B (showing the presence of many progenitors/short-term

repopulating cells in blood), these data indicate that transplants with mobilized

peripheral blood stem cells results in faster recovery of WBC number compared to

control LSK bone marrow cells.


117

0

2

4

6

8

10

12

14

0 7 14 21 28 35 42

1250 MPB LSK1250 control BM LSK

WB

C x

106 /m

l

Days after transplant

0

2

4

6

8

10

12

14

0 7 14 21 28 35 42

1250 MPB LSK1250 control BM LSK1250 MPB LSK1250 control BM LSK

WB

C x

106 /m

l

Days after transplant Figure 4.3: Mobilized peripheral blood LSK cells promote faster WBC recovery. Donor mice were

treated with 2 doses of Peg-G-CSF 6 days prior to donor cell isolation. Two groups of lethally

irradiated mice were transplanted with 1,250 LSK cells from control bone marrow (BM) (n=10) or

1,250 LSK cells from mobilized peripheral blood (MPB) (n=6). Following transplant, blood samples

were analyzed for WBC counts (WBC x 106/ml) from 7 days to 12 weeks after transplant.

Repopulation ability of peripheral blood stem cells

To further examine and directly compare the repopulation ability of mobilized

peripheral blood stem cells to bone marrow stem cells, LSK cells from bone marrow

and mobilized peripheral blood were tested in a competitive long-term repopulation

assay in different groups of mice. Recipients were transplanted with various ratios of

LSK cells from blood and LSK cells from bone marrow. A chimerism level of 50%

would be expected if both sources contained the same number of stem cells and

performed equally well. However, the contribution of cells derived from transplanted

mobilized peripheral blood was extremely low, with values of 2.6 ± 3% and 5.6 ± 4%

at 28 weeks when transplanted in a 1:1 ratio (Figure 4.4A). The CRI of peripheral

blood stem cells for each individual mouse is plotted in Figure 4.4B. The average CRI

(0.04 ± 0.04) for all transplanted mice indicate that purified mobilized peripheral

blood stem cells have a severely reduced repopulation potential (25-fold) compared to

bone marrow stem cells (Figure 4.4C). Recipient mice were either female B6 or

female CD45.1/2 mice.


118

0

5

10

15

20

25

30

0 5 10 15 20 25 30

% D

onor

chi

mer

ism

A.

B.

0

0.1

0.2

0.3

0.4

0.5

0.6

0 5 10 15 20 25 30

Com

petit

ive

Rep

opul

atio

n In

dex


C.



1000 MPB LSK + 1000 control BM LSK


1000 MPB LSK + 1000 control BM LSK750 MPB LSK + 750 control BM LSK250 MPB LSK + 500 control BM LSK1600 MPB LSK + 400 control BM LSK

0

0.05

0.1

0.15

0.2

0.25

0 5 10 15 20 25 30

Com

petit

ive

Rep

opul

atio

n In

dex

0

5

10

15

20

25

30

0 5 10 15 20 25 30

% D

onor

chi

mer

ism

A.

B.

0

0.1

0.2

0.3

0.4

0.5

0.6

0 5 10 15 20 25 30

Com

petit

ive

Rep

opul

atio

n In

dex


C.







1000 MPB LSK + 1000 control BM LSK750 MPB LSK + 750 control BM LSK250 MPB LSK + 500 control BM LSK1600 MPB LSK + 400 control BM LSK

1000 MPB LSK + 1000 control BM LSK1000 MPB LSK + 1000 control BM LSK750 MPB LSK + 750 control BM LSK750 MPB LSK + 750 control BM LSK250 MPB LSK + 500 control BM LSK250 MPB LSK + 500 control BM LSK1600 MPB LSK + 400 control BM LSK1600 MPB LSK + 400 control BM LSK

0

0.05

0.1

0.15

0.2

0.25

0 5 10 15 20 25 30

Com

petit

ive

Rep

opul

atio

n In

dex

Figure 4.4: Mobilized peripheral blood stem cells have reduced repopulation potential compared to

bone marrow stem cells. Mobilized peripheral blood (MPB) stem cells were obtained from donor mice

treated with 2 doses of Peg G-CSF. Both control bone marrow (BM) and MPB were sorted for LSK

cells. (A): Donor chimerism of MPB stem cells transplanted in a 1:1 ratio with competitor BM cells.

(B): Lethally irradiated recipients were transplanted with 1,600 LSK MPB + 400 LSK (n=5), 1,000

LSK MPB + 1,000 LSK control BM (n=6), 750 LSK MPB + 750 LSK control BM (n=10) and 250

LSK MPB + 500 LSK BM (n=4). Chimerism data was converted into a competitive repopulation index

(CRI). CRI values for each individual mouse is shown against weeks post transplant. (C): Average CRI

for all transplanted mice (n=25).


119

Mobilized blood stem cells have a reduced frequency of long-term repopulating stem

cells

To determine whether the inefficiency of mobilized peripheral blood stem cells to

promote long-term repopulation was due to a decline in quality of blood stem cells or

that mobilized blood stem cells contained fewer long-term reconstituting stem cells,

we quantified the frequency of long-term repopulating stem cells in mobilized blood

and bone marrow stem cells. Mice were transplanted with CD45.1 mobilized blood or

control bone marrow LSK cells in limiting dilution (1,200, 600 and 300) with a fixed

number (5 x 105) of B6 competitors bone marrow cells. The repopulation ability of

both mobilized blood and control bone marrow LSK cells was assessed by calculating

the CRU per 1,000 LSK cells. Analysis of the frequency of CRU 12 weeks after

transplant revealed that control bone marrow LSK cells are more competitive in

reconstituting the recipient (17 of 18 = 94% contained ≥ 5% of engraftment) (data not

shown) resulting in a significantly (p < 0.01) higher frequency of CRU (6.6 CRU per

1,000 LSK; 1.6-15; 95% CI versus 0.75 CRU per 1,000 LSK; 0.35-2.8; 95% CI for

mobilized blood LSK) (Figure 4.5). This indicates that mobilized peripheral blood

stem cells have a reduced number of long-term repopulating cells compared to normal

bone marrow. Taken together, these results indicate that reduced repopulation

potential is due to lower frequency of stem cells rather than an impaired function of

mobilized blood LSK cells.

0

5

10

15

20

CR

U/1

000

LSK 6.6

0.75

MPB LSK BM LSK0

5

10

15

20

CR

U/1

000

LSK 6.6

0.75

MPB LSK BM LSK Figure 4.5: Reduced frequency of long-term repopulating stem cells in mobilized blood. Frequency of

competitive repopulation units (CRU) per 1,000 LSK cells with 95% confidence limits of recipient

mice transplanted in limiting dilution (1,200, 600, 300) with CD45.1 mobilized peripheral blood

(MPB) LSK cells and control bone marrow (BM) LSK cells with 5 x 105 B6 bone marrow cells. CRU

frequency of 0.75 CRU per 1,000 LSK (0.35-2.8; 95% confidence limit) was observed for mobilized

peripheral blood LSK group. Control bone marrow LSK group contained a significant (p < 0.01) 8.8-

fold higher CRU frequency (6.6 CRU per 1,000 LSK; 1.6-15; 95% confidence limit).


120

Engrafted mobilized peripheral blood stem cells do not exhaust faster than bone

marrow stem cells

Recipient mice (CD45.1/2) previously transplanted with a mixture of mobilized blood

LSK cells and bone marrow LSK cells were sacrificed 37 weeks after transplant.

Donor CD45.1 mobilized blood LSK-derived bone marrow cells and CD45.2 bone

marrow LSK-derived bone marrow cells were selected, isolated by FACS sorting and

transplanted in limiting dilutions into secondary recipients with 4 x 105 CD45.1/2

competitor cells respectively (Figure 4.6A). Average CRI values of recipients

receiving CD45.1 blood LSK cells or CD45.2 bone marrow LSK cells are shown in

Figure 4.6B. Bone marrow cells originated from LSK blood cells displayed similarly

low CRI levels (~0.14 ± 0.1) as bone marrow derived from bone marrow LSK cells

(~0.1 ± 0.1). This indicates that the quality of stem cells from mobilized blood derived

donor did not decrease after transplantation and that exhaustion of initially engrafted

stem cells is similar for stem cells from both sources.

Reduced repopulation potential of LSK mobilized peripheral blood stem cells in a

non-competitive setting

To model a more clinically relevant protocol, recipient mice were transplanted with

1,250 LSK from pooled bone marrow or 1,250 LSK from pooled mobilized peripheral

blood cells without competitor cells (Figure 4.7). Donor chimerism for mice

transplanted with purified bone marrow stem cells displayed little variation, with

average donor chimerism of 87 ± 8.8% 20 weeks after transplant. In contrast, donor

chimerism of mice transplanted with mobilized peripheral blood LSK cells varied

widely (44 ± 39%), with half of the recipients exhibiting high chimerism levels (80 ±

4.2%) while the other half was below 10%. This suggests that the frequency of cells

that contribute to long-term repopulation is at limiting dilution; 1,250 MPB LSK cells

contain very few (0, 1, or 2) stem cells. This is consistent with our CRU data (0.75

CRU/100 LSK). Reducing the dose of bone marrow LSK to 625 led to more variation

in chimerism but overall levels were still higher (52 ± 34%) than those observed in

some recipients transplanted with 1,250 mobilized peripheral blood LSK.


121

A.

B.

0

0.1

0.2

0.3

0.4

0 5 10 15 20

CR

I


CD45.1 MPB LSK derived-BM cells CD45.2 BM LSK derived-BM cells

9Gy

CD45.1 MPB LSK derived-BM + 4x105 CD45.1/2

CD45.2 BM LSK derived-BM + 4x105 CD45.1/2

and

CD45.1 or B6 recipient

9Gy

1000 CD45.1 MPB LSK + 1000 CD45.2 control BM LSK

37 weeks, sacrifice Sort BM for CD45.1

and CD45.2

CD45.1 MPB LSK derived BM

CD45.2 BM LSK derived BM

FITC

PE 86%

3%

B6 recipient

A.

B.

0

0.1

0.2

0.3

0.4

0 5 10 15 20

CR

I


CD45.1 MPB LSK derived-BM cells CD45.2 BM LSK derived-BM cells CD45.1 MPB LSK derived-BM cells CD45.2 BM LSK derived-BM cells

9Gy



and9Gy9Gy



and

CD45.1 or B6 recipient

9Gy


9Gy9Gy


37 weeks, sacrifice Sort BM for CD45.1

and CD45.2

CD45.1 MPB LSK derived BM


FITC

PE 86%

3% CD45.1 MPB LSK derived BM


FITC

PE 86%

3%

B6 recipient

Figure 4.6: Similar long-term functioning of engrafted mobilized blood and bone marrow stem cells.

(A): Primary recipients were transplanted with CD45.1 mobilized peripheral blood (MPB) LSK and

CD45.2 bone marrow (BM) LSK cells. Thirty-seven weeks after transplant, recipient mice were

sacrificed and their bone marrow was isolated and the CD45.1 and CD45.2 fractions were separated.

CD45.1 MPB LSK-derived BM and CD45.2 BM LSK-derived BM cells were isolated separated by

FACS sorting and transplanted in different ratios (1:1, 1:2 and 1:4) with 4 x 105 CD45.1/2 F1

competitor cells into lethally irradiated CD45.1 or B6 secondary recipients. (B): Average CRI values of

secondary recipients receiving CD45.1 mobilized blood LSK-derived bone marrow cells or CD45.2

bone marrow LSK-derived bone marrow cells.


122

0

20

40

60

80

100

0 5 10 15 20 25

0

20

40

60

80

100

0 5 10 15 20 25

A.

B.

1250 LSK Control BM

1250 LSK MPB

% D

onor

chi

mer

ism


% D

onor

chi

mer

ism


0

20

40

60

80

100

0 5 10 15 20 25

0

20

40

60

80

100

0 5 10 15 20 25

A.

B.

1250 LSK Control BM

1250 LSK MPB

% D

onor

chi

mer

ism


% D

onor

chi

mer

ism


Figure 4.7: Mobilized peripheral blood stem cells have reduced long-term repopulation ability in a

non-competitive setting. (A): Lethally irradiated B6 recipients (n=7) were transplanted with 1,250 LSK

from CD45.1 bone marrow (BM). Donor chimerism levels are shown for each recipient. (B): Lethally

irradiated B6 recipients (n=6) transplanted with 1,250 LSK purified from CD45.1 mobilized peripheral

blood (MPB) cells. Donor chimerism levels are shown for individual recipients.


123

Discussion In the present study, we exploited a murine model to evaluate the quality of mobilized

peripheral blood stem cells in in vitro and in vivo assays. Our data demonstrated that

unfractionated mobilized peripheral blood stem cells have a 5-fold reduction in CAFC

day 35 activity and a 5-fold reduction in stem cell repopulation potential compared to

unfractionated control bone marrow cells (Figure 4.1). Mobilized blood stem cells,

selected on basis of LSK expression, displayed an even greater fold reduction (25-

fold) in long-term repopulation potential when transplanted in competition with

normal bone marrow stem cells (Figure 4.4). Limiting dilution analysis showed that

this was a result of fewer stem cells rather than less potent stem cells as mobilized

blood stem cells have a 8.8-fold decrease in CRU compared to bone marrow stem

cells (Figure 4.5).

To compensate for the possible differences in stem cell frequency our experiments

were performed using a phenotypically defined stem cell population; Lin-Sca-1+c-Kit+

(LSK). Although LSK cells are not a pure stem cell population, in steady-state bone

marrow they contain all cells with long-term repopulation ability, very much like

CD34+ cells in humans22;23. The overall LSK cell number in mobilized peripheral

blood was markedly lower than in the bone marrow pool (Table 4.1). In mobilized

blood, fewer c-Kit+ cells were observed. These data are consistent with a down-

regulation of c-Kit on mouse hematopoietic progenitor cells in vivo resulting from

proteolytic cleavage as previously reported21. G-CSF administration also causes a

change in cell surface marker expression of CD3424. Importantly, we found no in vitro

stem cell activity in cells negative for either c-Kit or Sca-1, documenting that also in

mobilized blood all stem cells are contained in the LSK population.

Purified stem cells with the same phenotype do not always have the same functional

properties. One in five Thy-1lowSca-1+Lin- c-Kit+ cells isolated from young bone

marrow provides long-term reconstitution following transplantation into irradiated

mice25;26. Poorer repopulating efficiencies were reported for stem cells isolated from

other sources. Only 1 in 10 bone marrow stem cells from old mice engraft and 1 in 75

cyclophosphamide/G-CSF mobilized spleen Thy-1lowSca-1+Lin-c-Kit+ cells provided

long-term multilineage engraftment 27;28. Purification of mobilized spleen stem cells

using signalling lymphocytic activation molecule (SLAM) family markers,

CD150+CD48- resulted in a ~2-fold reduced long-term engrafting capacity when


124

compared to the same population in young bone marrow stem cells29. This suggests

that fewer “true” stem cells are present in the Thy-1lowSca-1+Lin-c-Kit+ stem cell

population in mobilized blood. Our CRU data also indicate that LSK cells from blood

are inherently worse than LSK from bone marrow.

In our study, transplantation of purified stem cells from peripheral blood displayed a

rapid increase in recovery of WBC counts demonstrating an increase in short-term

engraftment, consistent with clinical and experimental data2;3;30;31. The observed

consistency between clinical and experimental data validates the use of LSK cells in

the murine system as a model to study the quality of mobilized peripheral blood stem

cells. Murine LSK, much like human CD34+ cells, are predominantly progenitor cells

but contain the vast majority of long-term repopulating stem cells.

The observed reduction in the frequency of stem cells rather than a faster deterioration

of blood stem cells suggest that recipients should receive higher cell dose of

mobilized blood stem cells to compensate for the decrease in repopulation potential.

In clinical studies, patients receiving mobilized peripheral blood typically receive a

much higher CD34+ cells dose than the bone marrow group30;32. Currently, 15-20 x

104 CFU-GM/kg or 2-2.5 x 106 CD34+ cells/kg is generally the agreed minimum

threshold below which rapid hematopoietic reconstitution may not occur4;33.

However, our data cautions the use of the frequency of a phenotypically defined

population such as CD34+ in human or LSK in mice, as the only parameter for

decisions about a minimum cell dose. The presence of fewer long-term repopulating

cells within this population, will increase the risk of re-growth of host (malignant)

haematopoiesis as shown in our non competitive transplants (Figure 4.7), increases.

Interestingly, a ~7-fold difference in CRI was observed between transplants with

unfractionated mobilized peripheral blood cells (CRI of 0.31 ± 0.1) (Figure 4.1) and

transplants with mobilized purified blood stem cells (CRI of 0.04 ± 0.04) (Figure 4.4).

This could be caused by the difference in cells used (mononuclear cells for MPB and

unseparated (>80% granuloid) cells from bone marrow, or it may suggest a potential

role of accessory cells to facilitate engraftment of mobilized peripheral blood cells. It

was reported that a CD8+/TCR- cell population from the donor bone marrow

facilitates engraftment of purified allogeneic bone marrow stem cells34. Mobilized

peripheral blood LSK cells may be more dependent on accessory cells than bone

marrow stem cells. Thus, it is possible that during the purification of LSK cells from


125

mobilized peripheral blood, accessory cells facilitating engraftment were lost, leading

to a further decrease in repopulation of purified mobilized peripheral blood stem cells.

In summary, although mobilized peripheral blood stem cells promote faster

haematological recovery, our competitive and non-competitive transplant model

suggest that their lower stem cell frequency affects the long-term repopulation

potential of blood LSK stem cells.


126

Acknowledgements We wish to thank Geert Mesander and Henk Moes for their assistance with cell

sorting and the staff of our animal facility for taking care of the mice. No conflicts of

interest exists between authors. This work was supported by grants from the European

Union (EU-LSHC-CT-2004-503436) and Ubbo Emmius Foundation.


127

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Proc.Natl.Acad.Sci.U.S.A 1995;92:10302-10306.


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27. Morrison SJ, Wandycz AM, Akashi K, Globerson A, Weissman IL. The aging

of hematopoietic stem cells. Nat.Med. 1996;2:1011-1016.

28. Morrison SJ, Wright DE, Weissman IL. Cyclophosphamide/granulocyte colony-

stimulating factor induces hematopoietic stem cells to proliferate prior to

mobilization. Proc.Natl.Acad.Sci.U.S.A 1997;94:1908-1913.

29. Yilmaz OH, Kiel MJ, Morrison SJ. SLAM family markers are conserved among

hematopoietic stem cells from old and reconstituted mice and markedly increase

their purity. Blood 2006;107:924-930.

30. To LB, Roberts MM, Haylock DN et al. Comparison of haematological recovery

times and supportive care requirements of autologous recovery phase peripheral

blood stem cell transplants, autologous bone marrow transplants and allogeneic

bone marrow transplants. Bone Marrow Transplant. 1992;9:277-284.

31. Wierenga PK, Setroikromo R, Kamps G, Kampinga HH, Vellenga E. Peripheral

blood stem cells differ from bone marrow stem cells in cell cycle status,

repopulating potential, and sensitivity toward hyperthermic purging in mice

mobilized with cyclophosphamide and granulocyte colony-stimulating factor. J

Hematother Stem Cell Res. 2002;11:523-532.

32. Korbling M, Anderlini P. Peripheral blood stem cell versus bone marrow

allotransplantation: Does the source of hematopoietic stem cells matter? Blood

2001;98:2900-2908.

33. Bender JG, To LB, Williams S, Schwartzberg LS. Defining a therapeutic dose of

peripheral blood stem cells. J.Hematother. 1992;1:329-341.

34. Kaufman CL, Colson YL, Wren SM et al. Phenotypic characterization of a novel bone marrow-derived cell that facilitates engraftment of allogeneic bone marrow stem cells. Blood 1994;84:2436-2446.

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CHAPTER 5

Summarizing Discussion and Future Perspectives


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Summarizing Discussion Expansion and maintenance of self renewing primitive hematopoietic stem cells

(HSCs) would have major implications in the areas of stem cell transplantation and

gene therapy. The ultimate goal of many scientists is to successfully expand and

maintain stem cells in their primary functional characteristic, namely their ability to

engraft and sustain long-term hematopoiesis. Establishing the ideal in vitro growth

conditions to expand and maintain HSCs has proven to be difficult. Recently, strong

evidence is emerging indicating that the large family of fibroblast growth factors

(FGFs) and FGF receptors (FGFRs) may play a key role in stem cell maintenance.

Chapter 1 provides a conceptual overview on FGFs and their receptors in

maintaining tissue homeostasis in HSCs, neural stem cells (NSCs) and embryonic

stem (ES) cells, in order to retain the self renewal capacity of stem cells.

Commonalities do exist between these three distinct stem cell systems. We summarize

evidence that FGFs are growth factors crucial for the regulation and culturing of all

three of these stem cell systems.

In Chapter 2 we studied the role of FGFs to maintain HSC function by culturing

unfractionated bone marrow (BM) cells in serum-free media supplemented only with

FGF-1, FGF-2 or the combination of both (FGF-1 + 2). Bone marrow cells were

cultured for a total of five weeks and then competitively transplanted into lethally

irradiated hosts. Cells cultured in FGF-1 + 2 showed a 5-fold and 1.5-fold increase in

repopulating units after culturing for one and five weeks respectively. In addition, we

co-cultured Lin-Sca-1+c-Kit+ (LSK) with unfractionated BM cells for a total of five

weeks in FGF-1 + 2 and transplanted without competitors into lethally irradiated

recipients. Overall, the data demonstrated that FGF cultured BM cells can be

maintained for up to five weeks while retaining long-term repopulating ability

(LTRA) and that all FGF-induced stem cell activity was derived from the LSK

population. Furthermore, the data signifies the importance of the stem cell niche, a

specialized microenvironment that houses, regulates and protects stem cells1. We

could not culture purified LSK cells in serum-free medium supplemented only with

FGF-1 and/or FGF-2. However in the presence of unfractionated BM cells, LSK cells

proliferated, suggesting that FGFs may be acting on other cell types to induce stem

cell activity in vitro. Receptors for FGF-1 have been shown to be present on primitive

hematopoietic cell subsets2. Therefore, it is also highly probable that FGFs maintain


135

stem cells by acting on FGFRs expressed on the stem cells. Additionally, non-LSK

cells present in the stem cell niche may carry FGFRs and therefore be responsive to

FGFs, playing an important role in the maintenance of stem cells. We speculate that

BM elements in the co-culture act as a pseudo niche facilitating the proliferation of

stem cells in vitro. These results demonstrate that we can maintain stem cell in culture

for up to five weeks with LTRA.

Quantitative PCR (QPCR) analysis in Chapter 3 demonstrated that FGF-1 and FGF-2

were predominantly expressed in LSK cells. The presence of both FGFRs2 and high

expression levels of FGFs on LSK cells strongly implies that FGFs may regulate

HSCs by autocrine signaling. To further examine the role of FGFs on HSCs, in

Chapter 3 we retrovirally overexpressed FGF-1 and FGF-2 in 5-Fluorouracil (5-FU)

treated BM cells. In addition, to assess the role of nucleocytoplasmic trafficking of

FGFs in hematopoietic cells, two mutant isoforms which altered the phosphorylation

status of FGF-1 were created and overexpressed. In the first mutant, the serine residue

at phosphorylation site 130 was exchanged for glutamic acid (S130E). This mutant

was expected to mimic the phosphorylated state of FGF-1, constitutively exporting

FGF-1 to the cytoplasm3. For the second mutant, the serine was exchanged for alanine

(S130A) which was hypothesized to prevent FGF-1 phosphorylation and remain

localized in the nucleus3. Higher molecular weight isoforms of human FGF-2 (21-22,

22.5, 24 and 34kDa) contain and upstream nuclear localization signal (NLS) which

translocates FGF-2 into the nucleus4;5. The smaller 18kDa isoform is confined to the

cytoplasm6-8. To create a nuclear localized isoform of mouse FGF-2, an artificial NLS

was inserted upstream of FGF-2 (NLS/FGF-2).

Fluorescent images of hematopoietic cells overexpressing wild-type (WT) FGF-1,

S130E or WT FGF-2 retroviral vectors showed that both FGF-1 and FGF-2 were

expressed predominantly in the cytoplasm. In contrast, overlay images of

hematopoietic cells overexpressing S130A and NLS/FGF-2 revealed that 2% of FGF-

1 positive cells and 11% of FGF-2 positive cells were localized in the nucleus

respectively. Although FGF-1 and FGF-2 mutant proteins were not predominantly

localized in the nucleus, compared to WT FGF-1, S130E and WT FGF-2 nuclear

localization did increase. More sensitive methods such as cell fractionation studies

should be performed to clarify these results. Recently, using cell fractionation studies,

Wiedlocha et al. showed that WT FGF-1 was found in all fractions (membrane,

cytoplasmic and nucleus) of the cell, S130A was found in the nuclear fraction


136

whereas S130E was mainly in the cytosolic fraction9. These data indicated that the

translocation of FGFs appears to be a dynamic process and each FGF can be regulated

differently.

Unfortunately, transplantation studies with WT FGF-1, WT FGF-2, S130E and

NLS/FGF-2 overexpressing cells resulted in low donor chimerism levels in primary

recipients and little to no reconstitution in secondary recipients. This was unexpected

as previous studies from our group (as described in Chapter 2) had shown that the

unfractionated BM cells treated with exogenously added FGF-1 and/or FGF-2 were

capable of long-term repopulation2;10. These results suggest that the constitutive

overexpression of FGF-1 and FGF-2 does not increase the repopulating potential of

hematopoietic cells. Interestingly, secondary recipients transplanted with S130A

overexpressing cells showed a delayed but marked increase in donor chimerism levels

and a significant increase in GFP+ cells. These results strongly suggest that nuclear

localized FGF-1 may play an important role in maintaining stem cell quality.

In vivo competitive transplantation assay is the ‘gold standard’ to test whether BM

derived cells are indeed HSCs with the potential for reconstituting all hematopoietic

lineages. The competitive repopulation assay11 has two key features. The first is that it

enables the detection of a very primitive class of hematopoietic stem cells and the

survival of lethally irradiated mice transplanted with very low numbers of such cells.

The second is the use of a limiting dilution experimental design to allow stem cell

quantitation.

To highlight the effectiveness of this assay, in Chapter 4 we use the competitive

transplantation assay to compare the functional qualities of mobilized peripheral

blood (MPB) stem cells to normal BM stem cells. Mobilized peripheral blood has

been used for the past 10 years in place of BM as a source of stem cells for

transplants. Their ease of collection and ability to promote faster regeneration of

neutrophils12;13 and platelets makes them a primary source for HSC transplantation.

Transplantation of MPB stem cells in competition with BM stem cells demonstrated

that MPB stem cells have a reduced long term repopulation potential. This impairment

in repopulation potential was due to the presence of fewer stem cells rather than a

decrease in stem cell quality. In actual fact, secondary transplantation of MPB stem

cells indicated that the quality of stem cells from MPB did not decrease after

transplantation and that exhaustion of initially engrafted stem cells is similar for both


137

BM and MPB stem cells. Clearly, in a clinical setting, more blood stem cells must be

transplanted to compensate for the decrease frequency of stem cells.

Future Perspectives In this thesis the exogenous and endogenous effects of FGF-1 and FGF-2 were

examined. We have shown that FGFs, in particular FGF-1 and FGF-2 play an

important role in regulating and maintaining stem cells.

In total, 22 FGFs exists (not including spliced forms) and we have shown that two out

of 22 FGFs are able to maintain HSCs. Most stem cell studies carried out are

restricted to FGF-1 or FGF-2. From mouse knock-out studies it has appeared that

other FGFs are more potent. For example, deletion of FGF-414, FGF-815-18, FGF-919

and FGF-1020 result in embryonic lethality whereas FGF-1 and FGF-2 knockout

mice21 are viable and fertile. Given their pleiotropic effects, similar receptor binding

properties, overlapping patterns of expression and sequence similarities, functionally

redundancy is likely to occur. It will be interesting to assess the effects of other FGFs,

in particular FGF-4, FGF-8, FGF-9 and FGF-10 on stem cells. Such studies would

increase our understanding on the biological role of FGFs on HSC maintenance and

regulation.

The mechanistic action of FGFs on stem cells remains unknown. Our results suggest

that the intracellular function of nuclear localized FGF-1 is biologically significant.

This indicates that the nuclear import/export trafficking pathway of FGFs may be key

to understanding the mechanistic action of FGFs. Future studies should be aimed at

assessing the trafficking pathway of FGFs in stem cells. Firstly, it will be interesting

to determine which FGFs bind to which FGFR and to what affinity. It should be noted

that a large number of splice variants of FGFR genes exist and must be taken into

account. Secondly, it would be appealing to determine the stage of the cell cycle

which enables the precise cueing of the nuclear localization of FGFs. This may

provide valuable information as to how nuclear localized FGF-1 (S130A) maintained

stem cell quality. Thirdly, it would be interesting to determine whether all FGFs,

which are highly homologous, have the same trafficking pathway.

The competitive transplantation assay serves as the only tool to detect long-term

repopulating stem cells with the potential for reconstituting all hematopoietic lineages.

We highlighted the effectiveness of this assay to study the qualities of MPB stem cells


138

compared to normal BM stem cells. Blood cells mobilized with Granulocyte-Colony

Stimulating Factor (G-CSF) have reduced repopulation ability due to a lower

frequency of stem cells. Many growth factors capable of migrating stem cells from the

BM to the blood exist. Each mobilizing agent, alone or in combination with

chemotherapeutic agents affects the stem cell differently. It will therefore be

interesting to examine the effects of different mobilization regimes and whether this

will improve the stem cell frequency and repopulating ability of blood stem cells. This

knowledge may be relevant and change techniques used in established clinical

application.


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References 1. Schofield R. The relationship between the spleen colony-forming cell and the

haemopoietic stem cell. Blood Cells 1978;4:7-25.


3. Klingenberg O, Widlocha A, Rapak A et al. Inability of the acidic fibroblast growth factor mutant K132E to stimulate DNA synthesis after translocation into cells. J.Biol.Chem. 1998;273:11164-11172.

4. Arnaud E, Touriol C, Boutonnet C et al. A new 34-kilodalton isoform of human fibroblast growth factor 2 is cap dependently synthesized by using a non-AUG start codon and behaves as a survival factor. Mol.Cell Biol. 1999;19:505-514.

5. Dono R, James D, Zeller R. A GR-motif functions in nuclear accumulation of the large FGF-2 isoforms and interferes with mitogenic signalling. Oncogene 1998;16:2151-2158.

6. Bugler B, Amalric F, Prats H. Alternative initiation of translation determines cytoplasmic or nuclear localization of basic fibroblast growth factor. Mol.Cell Biol. 1991;11:573-577.

7. Florkiewicz RZ, Baird A, Gonzalez AM. Multiple forms of bFGF: differential nuclear and cell surface localization. Growth Factors 1991;4:265-275.

8. Quarto N, Finger FP, Rifkin DB. The NH2-terminal extension of high molecular weight bFGF is a nuclear targeting signal. J.Cell Physiol 1991;147:311-318.

9. Wiedlocha A, Nilsen T, Wesche J et al. Phosphorylation-regulated nucleocytoplasmic trafficking of internalized fibroblast growth factor-1. Mol.Biol.Cell 2005;16:794-810.


11. Harrison DE. Competitive repopulation: a new assay for long-term stem cell functional capacity. Blood 1980;55:77-81.

12. Goldman J. Peripheral blood stem cells for allografting. Blood 1995;85:1413-1415.

13. Korbling M, Champlin R. Peripheral blood progenitor cell transplantation: a replacement for marrow auto- or allografts. Stem Cells 1996;14:185-195.



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15. Meyers EN, Lewandoski M, Martin GR. An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination. Nat.Genet. 1998;18:136-141.

16. Reifers F, Bohli H, Walsh EC et al. Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis. Development 1998;125:2381-2395.

17. Shanmugalingam S, Houart C, Picker A et al. Ace/Fgf8 is required for forebrain commissure formation and patterning of the telencephalon. Development 2000;127:2549-2561.


19. Colvin JS, Green RP, Schmahl J, Capel B, Ornitz DM. Male-to-female sex reversal in mice lacking fibroblast growth factor 9. Cell 2001;104:875-889.


21. Miller DL, Ortega S, Bashayan O, Basch R, Basilico C. Compensation by fibroblast growth factor 1 (FGF1) does not account for the mild phenotypic defects observed in FGF2 null mice. Mol.Cell Biol. 2000;20:2260-2268.

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143

Nederlandse Samenvatting


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Nederlandse samenvatting Stamcellen zijn primitieve cellen met het vermogen zichzelf te vernieuwen en te

differentiëren in andere celtypen. Ze hebben het unieke vermogen schade in weefsels

te herstellen en/of andere cellen te activeren voor dit herstelproces. Stamcellen

worden onderverdeeld in embryonale of volwassen stamcellen. De studie beschreven

in dit proefschrift is gefocust op stamcellen uit het beenmerg (BM), die bloedcellen

produceren. Deze cellen worden hematopoietische stamcellen genoemd (HSCs).

HSC’s zijn in staat zich te vernieuwen en te differentiëren in een verscheidenheid van

gespecialiseerde bloedcellen zoals rode- (erytrocyten) en witte bloedcellen

(leukocyten). HSCs zijn verantwoordelijk voor het dagelijks aanmaken van miljarden

bloedcellen. Het is vanwege deze eigenschappen dat HSCs in de kliniek gebruikt

worden bij de behandeling van kanker, voornamelijk leukemie en lymfoma, kankers

onstaan uit bloedcellen. Ze worden na zware chemotherapie en/of radiotherapie

getransplanteerd om vernieuwe bloedcelaanmaak te garanderen.

HSCs kunnen geïsoleerd worden uit het BM en de bloedcirculatie. Het aantal

stamcellen in beenmerg en de bloedcirculatie is echter erg klein, daar er in BM slechts

minder dan 1 stamcel per 105 BM cellen aanwezig is. Het gevolg hiervan is dat

onderzoekers zijn gaan zoeken naar kweekmethodes met als doel meer primitieve

HSCs te genereren voor transplantatie. Om dit te verwezenlijken hebben onderzoekers

muizen-HSCs blootgesteld aan groeifactoren in een in vitro milieu.

Groeifactoren zijn kleine eiwitten die op cellulair niveau verschillende effecten

teweeg kunnen brengen. Er bestaan vele soorten groeifactoren. In dit proefschrift zijn

voornamelijk proeven beschreven die zich richten op de effecten van fibroblast

groeifactoren (FGFs) op de HSCs. FGFs zijn groeifactoren die specifiek reageren op

fibroblasten door het hele lichaam. Fibroblasten produceren de bouwstoffen van

fibreus weefsel, dat overal in het lichaam te vinden is. Tot op heden zijn 22 FGFs van

mens en muis bekend. FGFs zijn vooral bekend van wege hun functie tijdens

embryogenese wanneer de FGFs de ontwikkeling van organen zoals de longen en

ledematen besturen. Bij volwassenen spelen FGFs een belangrijke rol bij herstel van

weefsels, regeneratie, metabolisme en angiogenesis. FGFs verzorgen een biologische

respons door zich te binden aan gespecialiseerde eiwitten die receptoren genoemd

worden. Tot op heden zijn er in gewervelde dieren vier FGF-receptoren (FGFR)

geïdentificeerd, namelijk Fgfr1- Fgfr4.


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Dit proefschrift richt zich op handhaving en groei van jonge HSCs onder invloed van

FGFs in zowel in vitro als in vivo studies. Hoofdstuk 1 geeft een algemeen overzicht

van de regulerende rol van FGFs bij de handhaving van zich zelf vernieuwende

stamcellen om veroudering van HSCs, neurale stamcellen (NSCs) en embryonale

stamcellen (ES) tegen te gaan. Veroudering gaat gepaard met een langzaam

achteruitgaan van weefselfunctie, inclusief het afnemen van de groei van nieuwe

cellen en een afname van het herstelvermogen. Veroudering wordt ook geassocieerd

met de toename van het ontstaan van kanker in alle weefsels waarin stamcellen

aanwezig zijn. Dit geeft aan dat de balans tussen proliferatie, overleving en

differentiatie van stamcellen streng gereguleerd moet zijn. Deze waarnemingen

suggereren dat er een link bestaat tussen verouderingsprocessen en de rol van

stamcellen in het vernieuwingproces van stamcellen. We veronderstellen dat FGFs

met hun receptoren weefsel-homeostase tijdens veroudering ondersteunen door

vernieuwing, handhaving en proliferatie van HSCs, NSCs en ES te reguleren.

In hoofdstuk 2 bestudeerden we de effecten van de groeifactoren FGF1, FGF2, alleen

en in combinatie, op normale muizen BM cellen in kweken met serum-vrij medium.

De BM cellen die op deze manier, gedurende 1,3 en 5 weken met FGFs gekweekt zijn

werden onderzocht op hun vermogen gedurende lange tijd nieuwe bloedcellen te

vormen na transplantatie. Daartoe werden ze gemengd met vers geïsoleerde BM

cellen. Dit celmengsel werd geïnjecteerd in muizen die vooraf een hoge

bestralingsdosis toegediend kregen om er voor te zorgen dat de endogene cellen van

de muis niet meer functioneel zouden zijn. De gekweekte cellen en de vers

geisoleerde BM cellen verschillen op het Ly5 (CD45) locus, wat ervoor zorgt dat de

cellen aan de hand van hun fenotype nog te onderscheiden zijn na transplantatie. Deze

manier van transplantatie staat bekend als de competitieve transplantatie assay, en

stelt ons in staat te bewijzen dat met FGF gekweekte BM cellen inderdaad HSCs zijn.

Na transplantatie werd het bloed van de getransplanteerde muis geanalyseerd.

Wanneer getransplanteerde cellen, die behandeld waren met FGF-groeifactoren, na 24

weken terug gevonden worden in het bloed kan aangenomen worden dat er stamcellen

aanwezig waren. Er wordt in dat geval aangenomen dat deze stamcellen zorgen voor

kunnen lange termijn repopulatie, dus permanente bloedeelvorming. In hoofdstuk 2

laten we zien dat de stamcelactiviteit van BM cellen gekweekt met FGFs tot 5 weken

in stand gehouden kan worden, doordat deze cellen in de ontvanger muizen een lange

termijn repopulatie teweeg brengen. Vervolgens is gekeken of deze stamcelactiviteit


146

afkomstig was van al bestaande HSCs of dat ze ontstaan zijn uit een andere populatie

cellen. Stamcellen uit BM kunnen gezuiverd worden door selectie op basis van hun

primitieve HSC-fenotypering. Stamcellen hebben geen lineage marker, maar wel Sca-

1 and c-Kit stamcel-markers (LSK). Deze cellen worden LSK-cellen genoemd. De

LSK cellen werden gezuiverd en gedurende 5 weken verder gekweekt en in

combinatie met normale BM cellen met alleen FGF-1+2 in serum vrij medium. Na 5

weken werden alle cellen getransplanteerd in muizen die een dodelijke

bestralingsdosis toegediend hadden gekregen. Analyse van het bloed liet zien dat de

geïsoleerde en gezuiverde stamcellen bijdragen aan de bloedcelvorming na

transplantatie. Dus de stamcelactiviteit in FGF gestimuleerde BM kweken is

afkomstig van LSK stamcellen.

In hoofdstuk 3 onderzoeken we de rol van FGFs bij de intrinsieke regulatie en

handhaving van HSCs. De hypothese was dat FGFs van belang zijn voor het

handhaven van de stamcelactiviteit van HSC. Als het niveau van FGFs in stamcellen

verhoogd zou worden door retrovirale overexpressie, zou de stamcelactiviteit

verhoogd worden of langer gewaarborgd kunnen zijn. Om dit te onderzoeken zijn BM

cellen van met 5-fluorouracil (5-FU) behandelde muizen getransduceerd met FGF-1

of FGF-2. 5-Fluorouracil is een middel dat dient om beenmergcellen te verrijken voor

primitieve cellen en deze te activeren. Dit verbetert de transductie van stamcellen.

Het doel van de transductie was om in stamcellen een extra FGF-1 of FGF-2 gen in te

brengen. FGFs komen dan tot overexpressie hetgeen een hogere niveau van deze

eiwitten in de cel oplevert. De met het FGF-1 of FGF-2 gen getransduceerde cellen

werden getest in het in vivo competitieve transplantatiemodel. De cellen die extra

FGF-1 of FGF-2 produceren, hadden echter geen voordeel boven cellen die geen extra

FGFs produceerden. Ook op de lange termijn, na doortransplantatie van

getransduceerde cellen in secundaire ontvanger muizen, leverde een extra FGF-1 of

FGF-2 geen voordeel op voor hematopoietische stamcellen.

Er wordt gesuggereerd dat de locatie van FGFs in de cel belangrijk is voor zijn

functie. FGFs aanwezig binnen in de cellen zouden verantwoordelijk zijn voor de

mitogene effecten. Om dit te onderzoeken hebben we verscheidene mutanten van FGF

gecreëerd om de invloed van localisatie van FGF op zijn functie in hematopoietische

stamcellen te onderzoeken. Twee mutaties werden aangebracht in het FGF-1 gen.

Voor de eerste mutant werd het eiwit S130E ontwikkeld, dat de gefosforyleerde fase

van FGF-1 zou moeten nabootsen, waardoor deze naar het cytoplasma van de nucleus


147

getransporteerd kon worden. De tweede mutant, S130A, bootst de niet

gefosforyleerde fase van FGF-1 na, en veronderstelt dat FGF-1 in de kern gehouden

wordt. Om de FGF-2 van het cytoplasma over te brengen naar de kern werd een

kunstmatig nucleair localisatiesignaal (NLS) ingebracht vóór het FGF-2 gen

(NLS/FGF-2). Kwantitatieve analyse middels polymerase chain reaction (PCR) liet

zien dat zowel FGF-1 als FGF-2 duidelijk tot expressie komt in de LSK cellen. Dit

suggereert dat de FGFs een cruciale rol spelen bij de HSC regulatie en handhaving.

Fluorescentie beelden van hematopoietische cellen met overexpressie van FGF-1,

S130E en FGF-2 laten zien dat zowel FGF-1 als FGF-2 duidelijk voornamelijk

aanwezig zijn in het cytoplasma en nauwelijks in de celkern. Daarentegen laten

beelden van hematopoietische cellen met overexpressie van S130A en NLS/FGF-2

zien dat in respectievelijk 2% van de FGF-1 positieve cellen en 11% van de FGF-2

positieve het eiwit vooral in celkern aanwezig is. Deze bevindingen suggereren dat er

wel meer cellen zijn waar FGF zich in de kern bevindt maar dat de translocatie van

FGFs een dynamisch proces zou kunnen zijn waardoor dit toch in relatief weinig

cellen te zien was. Een schematische voorstelling van het transportmechanisme van

celkern naar cytoplasma (Figuur 1) laat zien dat FGF de cel binnenkomt door zich aan

FGFR te binden. Eenmaal binnen het cytoplasma gaan de FGFs de kern binnen met

behulp van hun NLS. Om de kern te verlaten moet het FGF-eiwit in gefosforyleerde

bv. (S130E) staat zijn. In een niet gefosforyleerde staat (S130A) blijft ermeer FGF-

eiwit in de kern.

De in vivo competitieve transplantatiestudies met S130E en NLS/FGF-2 dat de

endogene expressie van deze mutanten net als de normaal voorkomde FGF-1 en FGF-

2 eiwitten geen toename van het repopulatiepotentieel van hematopoietische cellen

bewerkstelligt. Stamcellen waar de niet gefosforyleerde vorm van FGF-1 (S130A) tot

overexpressie was gebracht, lieten na transplantatie ook geen verbeterd repopulatie

vermogen zien. Maar als deze cellen werden gezuiverd en doorgetransplanteerd in

secundaire ontvanger muizen werd een interessante bevinding gedaan. In eerste

instantie leken deze cellen het minder goed te doen, maar na verloop van tijd bleken

ze op de lange duur een verhoogd repopulerend vermogen te hebben. De niet

gefosforyleerde vorm van FGF-1 (localisatie in celkern) zou dus een belangrijke rol

kunnen spelen bij het handhaven van de kwaliteit van hematopoietische stamcellen.


148

FGF

FGFR

FGF

Cytoplasma

Celkern

NLSP

FGF

FGF

Cellulairestress

S130E

FGF

FGFR

FGF

Cytoplasma

Celkern

NLSNLSPP

FGF

FGF

Cellulairestress

S130E

Figuur 1: Transportmechanisme van fibroblast groeifactoren (FGFs). FGF komt de cel binnen door

zich aan FGF-receptoren (FGFR) te binden. Eens binnen het cytoplasma, gaan de FGFs de kern binnen

met behulp van hun nucleair localisatiesignaal (NLS). Om de kern te verlaten moet het FGF-eiwit in

gefosforyleerde (bv. S130E) staat zijn. Het FGF-eiwit zal de cel verlaten tijdens cellulaire stress.

Hematopoietische stamcellen kunnen geïsoleerd worden uit bloed of BM. De

voornaamste bron van HSCs voor klinische transplantaties was BM. Door een

stamceldonor of een patient gedurende enkele dagen een hematopoietische groeifactor

toe te dienen (meestal Granulocyte Colony Stimulating Factor; G-CSF) migreren

stamcellen vanuit het beenmerg naar het bloed. Het is veel makkelijker en

aangenamer voor de stamceldonor of patient om stamcellen uit het bloed te halen dan

uit het beenmerg. Het proces van migratie van stamcellen van beenmerg naar bloed

wordt stamcelmobilisatie genoemd. Daarom zijn, mede als resultaat van de

toegenomen opbrengst, stamcellen uit het bloed de voornaamste bron van stamcellen

voor transplantaties geworden. In de meeste gevallen levert dit een versneld herstel

van het aantal rode en witte bloedcellen op. Er zijn echter weinig studies uitgevoerd

die de kwaliteit uit het bloed en BM op de lange termijn hebben vergeleken. In

hoofdstuk 4 passen we de competitieve transplantatie assay toe om de bloed

stamcellen met die van de normale BM stamcellen te vergelijken. Competitieve

transplantatie assay is de “gouden standaard” om te bewijzen dat de geteste cel

daadwerkelijk een HSC is. De test donor cel wordt gemengd met BM cellen en

geïnjecteerd in een muis, die eerder een hoge dosis bestraling heeft gekregen om zijn


149

eigen bloed producerende cellen te doden. Wanneer de muis herstelt en alle typen

bloedcellen weer aanwezig zijn (die een genetische marker van de test donor dragen)

kan worden beoordeeld of de geïnjecteerde cellen stamcellen bevatten (Figuur 2).

Daarnaast kan de competitieve assay gebruikt worden om de frequentie van de

stamcellen te meten en om de kwaliteit van twee stamcelpopulaties te vergelijken. De

transplantatie van bloed stamcellen gemengd met normale BM stamcellen laat zien

dat bloed stamcellen minder vermogen tot repopulatie van de bestraalde muis

bezitten. Dit kan liggen aan een mindere kwaliteit of minder “echte” stamcellen in het

bloed; “echte” stamcellen bezitten de eigenschap om onbeperkt gedurende lange tijd

rijpe bloedcellen te produceren. Wij hebben gevonden dat het lagere repopulerend

vermogen van bloedstamcellen is te wijten aan een afname van frequentie van

stamcellen in het bloed.

Muis type BMuis type A

Bestraling

Muis type B

Bloed analyseerd

Type A

Type B

+Muis type BMuis type A

Bestraling

Muis type B

Bloed analyseerd

Type A

Type B

Type A

Type B

+

Figuur 2: Competitieve transplantatie assay. De testcellen van muis A worden gemengd met verse

beenmerg (BM) cellen van muis B. De celsuspensie wordt geïnjecteerd in lethaal bestraalde muizen.

Na transplantatie wordt het bloed van de getransplanteerde muizen geanalyseerd om de repopulatie

capaciteit van de testcellen van muis A te bepalen.


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In hoofdstuk 5 tenslotte, vatten we onze bevindingen samen en bediscussiëren we de

toekomstperspectieven. De FGF eiwit familie is groot en de eiwitten zijn zeer

homoloog, wat betekent dat ze erg op elkaar lijken. Het verwijderen van FGF-1 bij de

muis heeft bijvoorbeeld geen effect, waardoor we mogen concluderen dat de

functionele taak overbodig is. We hebben in onze studies slechts 2 van de 22 FGFs

getest. Het zal daarom erg interessant zijn de effecten van de overige 20 FGFs op

HSCs te onderzoeken zowel in vitro als in vivo. Verkregen resultaten hiervan zullen

waardevolle informatie verschaffen betreffende de biologische rol van FGFs in HSCs.

De mechanistische aspecten van FGFs zijn nog onbekend. De nucleaire localisatie van

FGF-1 blijkt waarschijnlijk een rol te spelen in de handhaving van de hoedanigheid

van de stamcellen. Toekomstige studies zouden gericht moeten zijn op het vraag

waarom er een vertraging optrad als gevolg van de effecten van de S130A mutant. Het

zal interessant zijn vast te stellen hoe de nucleaire gelocaliseerde FGF-1 de stamcel

hoedanigheid handhaaft omdat deze waardevolle informatie zou kunnen verschaffen

over de mechanistische aspecten van FGFs.

De bloedstamcellen die gemobiliseerd zijn met G-CSF hebben een beperkt

repopulatievermogen ten gevolge van een lage frequentie van stamcellen. Er bestaan

vele groeifactoren die in staat zijn stamcellen te doen migreren van BM naar bloed.

Elke gemobiliseerde groeifactor beïnvloedt de stamcel verschillend. Het zal daarom

interessant zijn de effecten van verschillende factoren van mobilisatie te onderzoeken

en of dit de stamcel-frequentie en het repopulatie-vermogen van de bloed stamcellen

zou kunnen verbeteren. Deze kennis zou van relevantie kunnen zijn en zou technieken

kunnen modificeren zodat die toegepast zouden kunnen worden in de kliniek om het

succes van stamceltransplantatie te verhogen.

151

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Acknowledgements

As I sit back and reflect on the past four years and four months I can’t help but think

how quickly time has passed by. Naturally, these same thoughts and feelings never

went through my mind whilst in the crux of my PhD. I guess what they say is true –

sometimes it’s hard to see the bigger picture when you are standing in the middle of it.

The knowledge I have gained, the places I have seen and the many people involved

that have now become friends have all made this an incredible experience I will

forever remember. To these friends who have made it all possible, thank you.

Firstly, I would like to thank my supervisor Professor Gerald de Haan for accepting

me into the Department of Stem Cell Biology. Your steer and support was

instrumental towards the completion of this thesis. This opportunity has most

certainly helped further my scientific career. Special thanks to Ronald, my co-

promoter for your advice and assistance on experiments, proof reading my

manuscripts and for the constant repartee.

A big thank you goes to the members of the reading committee, Edo Vellenga, Paul

Coffer and Albrecht Mueller for reading and providing critical comments on my

thesis.

To my invaluable paranymphs, Bertien and Ellen you have provided the local support

and knowledge required to sort out the difficult intricacies towards my graduation.

Without both your help I would have never been able to complete all the experiments

that have formed the basis of this thesis. Bert, I really appreciated the many hours you

spent with me in the CDL. I will miss our chats and your great sense of humour. To

my roommates, Leonie, Alice and Lenya thank you for advice, guidance and, most

importantly, laughter. Leonie, I’m so pleased that you went through the graduation

procedures and formal documents with me. Lenya, I will miss your politically

controversial outlook on life and science. Your honesty is refreshing and sadly I will

never meet another Lenya. To my friends Sandra, Isabelle and Kyrjon, thanks for

being there when I needed to share a joke with you, when I had to let my frustrations

out or when I had some great gossip I badly needed to share. Many thanks to my

friends and colleagues in Radiation and Stress Cell Biology and Hematology for your

critical input. To my students Eelo and Alexandra, thank you for helping out with the

work. Your efforts were much appreciated.

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Gerry, I can not thank you enough for everything you have done for me. Any

bureaucratic problems I had, whether it be my taxes, the IND or the university, you

solved it with a smile. If it wasn’t for your dream, I would not have been able to

graduate. Your kind-heartedness shines through in the many ways you selflessly help

others and I am delighted to have had the pleasure of meeting you.

My Mondays to Fridays would not have been complete without bumping into my

fellow international friends, Daniella, Larissa, Heni, Jola and Jarir. The overcooked

vegetables and fried food at the hospital was easier to take with the good company.

Special thanks to Babs, my fellow Aussie. Our lunches in the garden, frantically

chatting away, always put me in good spirits for the remainder of the day. Many

thanks for helping me with the Dutch summary. I don’t know what I would have done

without your help.

Groningen would have never been the same without my friends, Joe, Hermione,

Mensur, Jackie, Alex, Jim, Brad, Tim, Meagan, John, Susan, Tom, Anna, Leah, Ina,

Paul Hoban, Paul Hogarth, Greg, Karolien, Richard, Glen, Lucy, Jeanette, Josh, Tina,

Marcella, Thomas, Claire, and Karen. May our crazy skiing stunts, holidays and wild

parties continue wherever we are in this world. To the girls, the nights out at Da

Vinci’s will always form some of my fondest memories. There is nothing better than

pizza, pasta and lots of Rosé and whilst some of you had the luxury of Red Bull’s

wings to carry you through the Friday, I only had our canteen’s milk and cheese at my

disposal. The lack of culinary diversity never dampened my hungover spirit as I just

had to think back to the previous night’s fun and shenanigans to realise it was all

worth it. Alex and Jim, thank you for the chocolates, spring rolls, coffee sessions and,

most importantly, your perfectly timed phone calls. Josh and Tina, you deserve more

for your artistic talents than just food and booze but I’m glad you lowered your rates

for my cover. To my friends Michelle, Nathan, Rebecca, Zheng and Eu-Jin thank you

for all your continuous support from a distance. Always remember that “wherever you

are it is your friends who make your world” (William James).

Most importantly, a big thank you to my loving family. I am grateful for all the love

and support my family in Malaysia and Australia have given me all these years. To

my parents-in-law, Albert and Pauline, thank you for your care and continuous

prayers for me. We have been blessed in so many ways and will always try to remain

thankful. To my husband Chevy, thank you for your love and constant encouragement

especially when it came to solving mathematical problems. The laughter and fun we

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shared in Groningen will always resonate in my memories as you made living so far

away from home seem not so far at times. You always made sure that I had everything

I needed to be happy and to achieve my goals, especially when it came to techno

wizardry. Without you I most definitely would not have travelled so much.

To my dearest mum and brother, I thank you from the very bottom of my heart for all

that you have done for me, not only during these past four years but throughout my

life. You were both always there for me whenever I needed to hear your voices or

have my own voice heard by a compassionate ear. I could always depend on either of

you to provide the motivation I needed, in the form of a supporting email or phone

call, wrapped in a bit of homely cosiness. I hope that I have made you both proud of

me and continue to do so in the next phase of my life.

With love

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Curriculum vitae

Personal Details

Full Name: Joyce Siew Gaik Yeoh

Date of Birth: 26 November 1979

Country of Birth: Penang, Malaysia

Nationality: Australian

Education

PhD in Department of Cell Biology, Section Stem Cell Biology, University Medical

Centre of Groningen, May 2002 – August 2006

“Regulatory Role of Fibroblast Growth Factors on Hematopoietic Stem Cells”

Anticipated Graduation February 2007

Hons., BSc. in Pathology, University of Western Australia, Graduation, 2001

Work Experience

Research fellow in Department of Ophthalmology, University of Aberdeen, Scotland,

2006 – present

Production scientist in Verigen Transplantation Services International and Verigen

Australia Pty Ltd, 2001 to 2002

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Papers published

Fibroblast growth factor-1 and 2 preserve long-term repopulating ability of

hematopoietic stem cells in serum-free cultures

Stem Cells 2006, 24 (6) 1564 – 1572

Fibroblast growth factors as regulators of stem cell self renewal and aging

Mechanism of Ageing and Development, in press

Mobilized peripheral blood stem cells provide rapid reconstitution but impaired long-

term engraftment

Bone Marrow Transplantation, submitted

Abstracts

In Vitro Generation of Hematopoietic Stem Cells

Joyce Yeoh, Ellen Weersing, Bert Dontje, Edo Vellenga, Ronald van Os, Gerald de

Haan

International Society of Experimental Hematology, Paris, France 2003

Fibroblast growth factors regulate stem cell functioning in vitro and in vivo.

Joyce Yeoh, Ronald van Os, Ellen Weersing, Bert Dontje, Edo Vellenga, Leonid

Bystrykh, Gerald de Haan

American Society of Hematology, San Diego, United States of America 2004




Dutch Program Tissue Engineering, Ede, The Netherlands 2005




International Society of Experimental Hematology, Glasgow, Scotland 2005

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