Therapeutic applications of oligodendrocyte precursors derived from human embryonic stem cells

Therapeutic applications of oligodendrocyte precursors derivedfrom human embryonic stem cellsJason Sharp and Hans S Keirstead
Addresses
Reeve-Irvine Research Center, Department of Anatomy and

Neurobiology, University of California at Irvine, 2111 Gillespie

Neuroscience Research Facility, Irvine, CA 92697-4292, United States

Corresponding author: Sharp, Jason ([email protected]) and Keirstead,

Hans S ([email protected])

Current Opinion in Biotechnology 2007, 18:434–440

This review comes from a themed issue on

Tissue and cell engineering

Edited by Alan Trounson and Andrew Elefanty

0958-1669/$ – see front matter

# 2007 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.copbio.2007.10.006

IntroductionA loss of central nervous system (CNS) myelin is charac-

teristic of several neurological pathologies and trauma

that include multiple sclerosis (MS) [1–3], brain injury

[4], spinal cord injury (SCI) [5], and stroke [6]. Myelin loss

is also linked to psychiatric disorders such as schizo-

phrenia [7], and is correlated with functional deterioration

associated with aging [8,9], and Alzheimer’s disease [10].

Together these disorders affect a patient population of

approximately 10.3 million individuals in the United

States. Although demyelination is concomitant with other

disease processes in each of these disorders, it remains

possible that remyelination can alleviate clinical

deterioration by decreasing axonal degeneration and tran-

section and associated functional deficits [11]. Indeed,

remyelination has been shown to restore saltatory con-

duction in axons [12] and ameliorate locomotor deficits in

animals [13]. Thus, it is relevant to consider the clinical

application of myelinogenic treatments to foster remye-

lination. In experimental models of demyelination,

endogenous [14] or transplanted [15] mature oligoden-

drocytes are incapable of remyelination. By contrast,

current evidence suggests that cell replacement strategies

using oligodendrocyte progenitor cells (OPCs), such as

human embryonic stem cell (hESC)-derived OPCs,

promote remyelination and improve locomotor outcomes

in animals [16]. Although considerable progress has been

made in recent years to demonstrate the effectiveness of

cell-based remyelination treatments, several significant

obstacles must be overcome before this research trans-

lates to the clinic. Particular to hESC-derived OPCs,


these hurdles include determination of preclinical utility

for etiologically disparate demyelinating conditions, miti-

gation of the risks of cell transplantation, large-scale

manufacturing, and regulatory considerations for govern-

ment approval. Here we present the emerging perspect-

ive that stem cell-mediated remyelination of the adult

CNS is a viable therapeutic strategy with potential

beyond remyelination and discuss the challenges to the

development of such therapeutic applications.

Myelin repairEndogenous remyelination

Remyelination has been intensely examined for over

25 years, with convincing demonstrations in both animal

and human disorders. Endogenous repair of axon myelina-

tion is a normal process after demyelinating insults [5].

Newly generated myelin sheaths are readily detectable as

thin and short myelin layers [5,17]. Several lines of evi-

dence point to adult OPCs as the cells that carry out

endogenous remyelination [18]. The adult brain contains

[19] and has the potential to generate [20] myelinogenic

OPCs, though these cells may remain somewhat dormant

until prolonged exposure to growth factors converts them

to proliferating cells similar to those in the developing CNS

[21]. In addition, remyelination is dependent on the re-

expression of developmentally regulated genes [22,23] and

cell division as prerequisites for remyelination [24].

Examination of failed or diminished remyelination is

further illustrative of the conditions that constitute a

permissive environment for remyelination. Remyelina-

tion is less efficient in old animals than in young animals

[25] and after repeated episodes of demyelination [26],

though this later result is disputed [27]. It is possible that

depletion of myelinogenic progenitors contributes to

remyelination failure. Mature oligodendrocytes are incap-

able of remyelinating axons [14,15] and there is no con-

vincing indication that differentiated oligodendrocytes

are able to revert to a progenitor state. Reactive astro-

gliosis, which manifests in many CNS injuries and dis-

eases, may also contribute to remyelination failure by

enveloping axons and thereby preventing myelinogenic

progenitor access to demyelinated axons [16], or by

expression of molecules that inhibit progenitor matu-

ration or oligodendrocyte myelination. In support of

the latter possibility, astrocytes in demyelinated lesions

expressed Jagged1, an inhibitor of oligodendrocyte differ-

entiation and process outgrowth, whereas expression

in remyelinated areas is negligible [28]. In MS lesions,

the polysialylated form of the neural cell adhesion mol-

ecule (PSA-NCAM) is expressed on axons, and may

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Therapeutic applications of oligodendrocyte precursors derived from human embryonic stem cells Sharp and Keirstead 435

contribute to the decreased remyelination observed in

that disease [29].

Thus, remyelination failure or success is probably a

combination of environmental factors and innate charac-

teristics of endogenous adult oligodendroglial lineage

cells. The success of remyelination during acute stages

of human demyelinating insults, and in experimental

demyelination models, suggests that the therapeutic win-

dow of opportunity is narrow. This period of time pre-

sents an opportunity for transplantation approaches to

repair myelin.

Transplant-mediated remyelination

Transplantation of oligodendroglial lineage cells [16,30],

Schwann cells [31], olfactory ensheathing cells [32], and

various stem cell types or their derivatives [16,33,34] has

been shown to result in remyelination and usually restor-

ation of saltatory conduction. Remyelination may ame-

liorate clinical deterioration by preserving axons, as

chronically demyelinated axons are vulnerable to

degeneration and transaction [11]. Notably, glial trans-

plantation in the mouse hepatitis virus model of demye-

lination has been shown to result in a significant decrease

in axonal loss over time [30]. Although abundant evidence

indicates that transplant-mediated remyelination is

possible and of considerable functional benefit, it is

important to consider the advantages and disadvantages

of each cell type for selection of a candidate population

for clinical application.

Cell replacement therapySelection of a candidate cell type for clinical cell replace-

ment therapy requires the consideration of first, avail-

ability and bankability of the cell type; second,

proliferative ability of the cell type to obtain the

quantities necessary for clinical demands; third, the

genetic stability of the cell type to prevent aberrant

growth or phenotype; and fourth, the purity of the differ-

entiated population. Other considerations, not unique to

cell replacement therapy, include concerns regarding

safety, rejection, ethics, and politics. Several cell types

have the potential to meet the requirements listed above;

these include neural stem cells (NSCs), bone marrow

stem/stromal cells (BMSCs), and embryonic stem cells

(ESCs), among others. Olfactory ensheathing cells meet

some of the requirements listed above, however, it is not

clear if this cell type can be expanded to sufficient

quantities for use in human cell replacement therapy.

Of the cell types discussed above, ESCs currently show

the greatest potential for the widest range of cell replace-

ment strategies.

Use of embryonic stem cells

Stem cells are non-transformed cells that are self-regen-

erative, pluripotent or multipotent for a tissue type, and

highly proliferative. Stem cells provide a nearly limitless


cell supply that can be directed in culture to differentiate

into diverse cell types. Due to the multipotentency of

ESCs, and thereby the ability to direct the differentiation

of the cells into any cell type, ESCs show great promise

for cell replacement therapy. ESCs derive from the inner

cell mass of preimplantation embryos, but can also be

derived by parthenogenetic activation of the egg, somatic

cell nuclear transfer, or in vitro epigenetic re-program-

ming [35–39]. ESCs are novel among other cell types in

that they are totipotent in rodents and multipotent in

humans, they can be almost limitlessly propagated invitro, they can be expanded in defined, rodent-free

media, they have a broad developmental capacity, and

they maintain a normal karyotype and differentiation

potential over years [40,41]. Characterization of ESCs

and the discovery of means to direct their differentiation

are hotly pursued research avenues in many disciplines.

Of interest to remyelination, several studies have indi-

cated that rodent ESCs can be directed to differentiate

into neuronal [42,43] or glial [44–46] fates. Most impor-

tantly, from the standpoint of human allogeneic thera-

peutics, human ESCs have been directed to differentiate

into multipotent neural precursors [47,48], low-purity

motor neurons [49], and recently into high-purity OPCs

[16,34]. A growing literature of accordant studies demon-

strates the myelinogenic potential of transplanted neural

cells derived from rodent [33,45] and human [16,34,48]

ESCs in different animal models of demyelination and

dysmyelination.

Therapeutic application of hESC derivates

Any therapeutic to be properly applied in humans is

subject to a risk/benefit analysis. For cell replacement

strategies utilizing a stem cell-derived therapeutic, a

number of adverse effects and advantages must be con-

sidered. On the risk side is the potential for tumorigenesis

resulting from their proliferative nature, central pain

resulting their secretion of growth factors, inflammation

resulting from host rejection, de-differentiation (for

instance, OPC to neural precursor), or trans-differen-

tiation (for instance, OPC to astrocyte). On the benefit

side is the potential to differentiate hESCs into high-

purity cell replacement products for every human tissue

type, the virtually unlimited source of cell replacement

product, their ability to localize to regions of tissue

damage (pathotropism), and their ability to deliver gene

or small molecule-based therapeutics (e.g. biopharmaceu-

ticals or trophic factors) in a manner unlike traditional

gene or small molecule therapy. To enable the regulatory

and widespread acceptance of stem cell-based therapies,

mitigation of their imposed or implied risks is of utter

importance.

Directed differentiation of hESCs

The remarkable proliferative capacity of stem cells and

their ability to adapt their fate within different environ-

ments confers advantages and disadvantages to the use of


436 Tissue and cell engineering

these cells for the treatment of myelin loss. Prediffer-

entiation of stem cells for transplantation is a strategy

designed to harness the power of stem cells while miti-

gating risk.

Tumor formation is a bona fide risk for transplanted

totipotent or multipotent cells, but it is a risk that

decreases with progressive developmental restriction of

the transplant population. Transplanted ESCs can form

teratomas that consist of endodermal, mesodermal, and

ectodermal lineages [48,50] and undifferentiated neural

precursors have been shown to generate nestin-positive,

expanding, and potentially tumorigenic cells following

transplantation [50]. However, more differentiated and

higher purity cells generate neither of these phenotypes

following transplantation.

Differentiation to scarring, or otherwise undesirable cell

types is another risk for transplanted totipotent or multi-

potent cells. Cell–cell interactions, genetic modulation,

and diffusible factors such as bone morphogenic protein,

sonic hedgehog, cytokines, and growth factors, all con-

tribute to the complex regulation of cell differentiation

and pattern formation in vivo. Transplantation of multi-

potent cell populations into cellularly and chemically

complex adult CNS injury sites results in the generation

of astrocytes [51,52]. In addition, these cells migrate

extensive distances from the site of implantation [51–

53]. Because transplanted cells are sensitive to their

implanted environment, predifferentiation of stem cells

to progenitor or mature cells before transplantation is a

technique to restrict their postimplantation fate and

address a defined clinical risk [16,54].

With regards to remyelination, it is crucial that the

implanted cells acquire a myelinogenic phenotype, both

for therapeutic effectiveness and to avert the risk of

tumorigenesis and scarring. Predifferentiation of ESCs

before transplantation has been demonstrated to produce

benefits in animal models without the formation of

tumors or enhancement of scarring [16,33,55]. In a recent

series of studies from our laboratory, hESCs were pre-

differentiated into high-purity OPCs; the first demon-

stration that hESCs can be directed to differentiate into a

high-purity neural population [34]. This protocol has

since been independently repeated and improved upon

[56]. Transplantation of these cells into spinal cord

injured animals demonstrated pathotropism, cell survival

and differentiation, enhanced remyelination, and signifi-

cantly improved locomotor outcomes [16]. Follow-up

studies indicated that the procedure was safe, in that

the transplant was not associated with tumor formation,

scarring, tissue pathogenesis, or behavioral decline [57]. It

is possible that the proclivity of multipotent cells to

spontaneously differentiate into oligodendrocytes within

the injured CNS milieu [58] might act to further support

the directed differentiation to OPCs, and thereby act as a


safeguard against de-differentiation or trans-differen-

tiation of these cells. If so, such a prospect would be

expected to contribute to the successful and safe clinical

application of these cells in CNS injuries [58]. Impor-

tantly, histological regeneration and behavioral recovery

following transplantation of myelinogenic cells has now

been demonstrated by multiple independent laboratories

[59–62].

Clinical challengesThe success of differentiated, myelinogenic stem cell

populations to augment remyelination in experimental

models does not necessarily predict clinical success. The

complex, reactive, and oftentimes multifocal nature of

human demyelinating disorders presents several chal-

lenges that must be overcome before cell replacement

therapies can become clinical realities. The therapeutic

potential of hESC-derived OPCs will depend upon con-

ditions specific to the particular disease or injury. To

illustrate this point, we consider the pros and cons of

therapeutic application of hESC-derived OPCs in two

conditions with demyelination: acute spinal cord injury

(SCI) and multiple sclerosis (MS).

SCI is typically a focal injury of the spinal cord that results

in inflammation, progressive hemorrhagic necrosis,

edema, demyelination, and cellular destruction. Demye-

lination is a persistent feature of SCI [5]. In the case of

spinal cord contusion injury in humans, the lesion is

usually singular and spacially defined and can be grossly

assessed through neurological testing and imaging. Fea-

tures detrimental to the survival and integration of trans-

planted cells, such as inflammation, excitotoxicity, and

free radical-mediated lipid oxidation, peak within the first

days after injury, then decline [63]. Reactive gliosis

begins within two to four weeks after SCI [63,64], and

eventually contributes to the formation of a physical

barrier to axonal regeneration and remyelination [16].

Work from our laboratory strongly suggests that the gliotic

environment of scarred, chronic lesions contributes to the

failure of remyelination by prohibiting transplanted

OPCs from remyelinating [16]. Thus, effective treatment

of SCI using hESC-derived OPCs seems possible and

probably necessitates transplantation of the cells during a

brief therapeutic window: following acute inflammation

and before scar formation [16,65].

The conditions governing therapeutic applications of

OPCs to MS are different than acute SCI. MS is a poorly

understood, autoimmune and chronic disease of the CNS

characterized by multifocal loci of inflammation and

myelin destruction [66]. This myelin loss compromises

the exposed axons and leads to their gradual loss. Although

the mechanism of axonopathy is not fully understood, it

probably involves the deregulation of calcium and associ-

ated energy deficits [67]. As a result, the loss of axons

contributes to the irreversible clinical deficits. As in SCI,



MS is prone to glial scarring that may contribute to the

failure of endogenous OPCs to remyelinate, despite their

presence within demyelinated MS plaques [68]. Although

intracerebroventricular administration of myelinogenic

populations does produce widespread dissemination and

myelination in animal models [69], glial progenitors do not

migrate through normal tissue [70]. The clinical usefulness

of diffuse administration is further hindered by risks of

cellular emboli, such as cerebrospinal fluid occlusion or

occlusive stroke. Furthermore, cell transplanted into MS

disease foci would be subject to the same demyelinating

process that caused the initial pathology. Thus, the disease

process and underlying inflammation may compromise the

transplant, the transplant could exacerbate autoimmune

attack, or both. While inflammation may exacerbate rejec-

tion of the transplant and thereby decrease remyelination,

it has also recently been shown to enhance migration and

remyelination by transplanted cells [69,71]. However, MS

patients are not likely to benefit from this potential effect

of inflammation, as they are invariably on immunosuppres-

sive regimens. Therefore, unlike SCI, the pathology of MS

is not currently considered amenable to transplant-

mediated remyelination strategies.

Although hESC-derived OPCs may be well suited for

transplant-mediated remyelination, the environment of

human demyelinating pathologies is not. Thus, the more

immediately approachable demyelinating pathologies

will be those that present relatively fewer environmental

challenges, such as acute SCI.

Future directions for hESC derivatesRegardless of the pathology being addressed, allogeneic

transplants into immune privileged CNS sites necessi-

tates long-term immunosuppression [72]. Induction of

tolerance in transplant recipients to circumvent the use

of immunosuppression, via transplantation of hESC-

derived hematopoietic derivates, or somatic cell nuclear

transfer (SCNT)-mediated generation of stable trans-

plant populations with the genetic make-up of the donor,

may one day prove efficacious, safe, and commercially

viable [37,73,74]. It may also be possible genetically

target MHC antigens on hESC derivates to overcome

rejection for widespread use [75].

Due to their inherent pathotropism, hESC derivates will

probably prove useful for delivery of biopharmaceuticals

to sites of injury or disease. Unlike traditional gene

therapy, transgene expression can be induced in the

transplant population immediately before or following

implantation, providing a level of efficacy and safety

not obtainable using current gene therapy regimens.

Genetic modifications of hESC-derived OPCs may also

prove useful in overcoming molecular obstacles to remye-

lination, such as knock out or knock down of Notch1 to

prevent Jagged1-mediated inhibition, or expression of

genes to address gliotic scarring. Using OPCs to deliver


biopharmaceuticals obviates the need for protracted

intraspinal biopharmaceutical administration and miti-

gates the associated complications [76]. As transplanted

OPCs can remain in the CNS for prolonged periods [19–

21,27,68], the cells can serve as pumps capable of secret-

ing trophic factors, such as glial-derived neurotrophic

factor (GDNF). GDNF-secreting OPCs may be useful

in promoting neuronal survival and plasticity in con-

ditions such as Parkinson’s disease [77], and other degen-

erative disorders [78]. Another potential application of

hESC-derived OPCs is the treatment of brain cancers. A

growing literature indicates that pathotropic neural pro-

genitor cells migrate toward tumors and halt progression

tumor [79–81]; it is possible that hESC-derived OPCs will

do the same. Again, modification of the OPCs to express

anti-cancer agents might further reduce tumor burden

[82–84]. To titrate administration of biopharmaceuticals,

transgene expression may be controllable using an on or

off operator to control transcription.

High-purity populations of hESC derivates will probably

prove useful in screens for conventional drug discovery, as

well as pharmacokinetic/pharmacodynamic studies, and

toxicology. Crucial to this research direction are contin-

ued advances in developmental biology, to elucidate the

conditions that will enable stem cell scientists to direct

the differentiation of hESCs to specific human cell popu-

lations.

As the generation of different human cell populations is

realized, the field will begin to combine these prep-

arations in complex systems to model physiologically

relevant circuits or tissues. For example, generation of

a complex layered retinal-like structure may prove useful

in transplant approaches to treat age-related macular

degeneration [85], and the differentiation and combi-

nation of human cortical or brainstem neurons, human

spinal motor neurons, and human muscle may prove

useful in the study of motor systems, motor neuron

regeneration, and preferential motor reinnervation.

Concluding remarksThe preclinical use of hESC-derived OPCs to treat SCI is

the culmination of over two decades of research in SCI

injury pathogenesis, myelin regeneration, stem cell

biology, and cell replacement therapy. The relatively

advanced preclinical development of this approach to

CNS repair over other stem cell-based approaches is

the result of a defined therapeutic target, the develop-

ment of a high-purity human cell population to address

that target, a receptive host environment, and the clinical

and commercial scalability of hESCs. For clinical appli-

cation to be contemplated, it is imperative that putative

hESC-based therapies pass vigorous safety testing. In

addition, hESC-based culture methods and derivates will

need to overcome multiple regulatory and manufacturing

hurdles. These challenges are currently being addressed,



and their resolution will facilitate preclinical and clinical

initiatives for all hESC-based treatments.

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Therapeutic applications of oligodendrocyte precursors derived from human embryonic stem cells