CLONAL DERIVATION OF NEURAL STEM CELLS FROM HUMAN EMBRYONIC STEM CELLS

by

Radha Chaddah

A thesis submitted in conformity with the requirements for the degree of Master of Science

IMS University of Toronto

© Copyright by Radha Chaddah 2009

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Clonal derivation of neural stem cells from human embryonic stem cells

Radha Chaddah, M.Sc., 2009 Institute of Medical Science

University of Toronto

Abstract

Clonal culture is crucial for experimental protocols that require growth or

selection of pure populations of cells. Currently, there is no method for deriving neural

stem cells (NSCs) clonally from single human embryonic stem cells (hESCs). Bulk

derivation of neural progenitors from hESCs for cell therapies can lead to a host of

problems including incomplete differentiation leading to proliferation of tumorigenic

clusters in vivo. Clonal derivation allows for the screening and selection of only the most

suitable cells for culture and expansion. We have developed a clonal, serum free method

of generating NSCs and their progenitors directly from hESCs with an efficiency of

1.6%. The NSC colony-forming cell was identified as a TRA-1-60-/SSEA4- cell whose

fate becomes specified in maintenance conditions by inhibition of bone morphogenic

protein (BMP) signalling. This clonal culture method can be scaled up to produce vast

quantities of NSCs for differentiation and use in cell therapies.

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Acknowledgements

I would like to thank the following people for their help: Derek van der Kooy, Peter

Zandstra, Andras Nagy, Sue Runciman, Margot Arntfield, Brenda Coles, Simon Smukler,

Tania Alexson, Phil Karpowicz, Brian deVeale, Laurie Sellings, Jon Draper, Manuel

Alvarez, Raheem Peerani, Mark Ungrin, Ting Yin, and especially Maya Chaddah and

David Marcus. This work was funded by the CIHR, the Canadian Stem Cell Network,

the Regenerative Medicine Development Centre and the Ontario Graduate Scholarship

program.

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Table of Contents Abstract ii Acknowledgements iii List of Abbreviations vi List of Figures viii Chapter 1 Literature Review 1

1.1 Pluripotency 2 1.2 Neural induction and the classical model 8 1.3 Default model of neural induction 9 1.4 Challenges to the neural default model 11 1.5 ES cells and the default model 13 1.6 Neural stem cells 14 1.7 The neurosphere assay 21 1.8 Research aims and hypotheses 22

Chapter 2 Materials and Methods 24 2.1 Propagation and maintenance of hESCs 25 2.2 Neurosphere culture 25 2.3 hESC minimal condition assays 26 2.4 Clonality assays 27 2.5 Immunocytochemistry 28 2.6 RNA isolation, cDNA preparation and Q-PCR 29 2.7 FACS analysis 30 2.8 EB formation 31

2.8 Statistical analysis 31 Chapter 3 Results 32

3.1 Single hESCs differentiate into colony forming 33 neural stem cells without serum or feeder cells

3.2 Neural stem cell colonies can be clonally derived from hESCs 41 3.3 hESC derived neural stem cells are FGF dependent and 45 frequency of neurosphere formation is improved to 1.6% by

cultivation with ROCK inhibitor 3.4 Unlike mESCs, neural specification of hESCs does not occur in 49 minimal culture conditions, but in maintenance culture 3.5 Noggin promotes neural fate specification only in hES 56

maintenance cultures 3.6 Alternate culture conditions enable generation of NSCs with 60 different ground states: culture of hESCs with ROCK inhibitor gives rise to an early lineage neural precursor.

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3.7 Discussion 65 3.8 Conclusion 71

3.9 Future Directions 72 References 76

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List of Abbreviations BMP bone morphogenic protein BSA bovine serum albumin cDNA complementary deoxyribonucleic acid Ctx cortex (cerebral) CC corpus callosum DMEM dulbecco’s modified eagle’s medium dNSC definitive neural stem cell DSHB developmental studies hybridoma bank EB embryoid body EC embryonal carcinoma ES embryonic stem cell EGF epidermal growth factor EpiSC epiblast stem cell FACS fluorescence activated cell sorting FCS fetal calf serum FGF fibroblast growth factor (basic) FITC fluorescein isothiocyanate FOXA2 forkhead box A2 GATA1 globin transcription factor binding protein 1 GATA4 globin transcription factor binding protein 4 GFAP glial fibrillary acidic protein GFP green fluorescent protein HBSS hank’s balanced salt solution hEG human embryonic germ cell hESC human embryonic stem cell hiPS human induced pluripotent stem cell HNF4A hepatocyte nuclear factor 4 alpha ICC immunocytochemistry ICM inner cell mass IGF insulin-like growth factor IgG immunoglobulin G IgM immunoglobulin M iPS induced pluripotent cell LIF leukemia inhibitory factor LIN28 lineage 28 homolog (c. elegans) MAP2 microtubule associated protein 2 MEF mouse embryonic fibroblast feeder cell mEG mouse embryonic germ cells mESC mouse embryonic stem cell mGS multipotent germ line stem cell miPS mouse induced pluripotent stem cell NaHCO3 sodium bicarbonate NES nestin NGS normal goat serum

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NSC neural stem cell O4 o-antigen sulfatide 4 OCT4 octamer binding factor 4 P probability of error PE primitive endoderm PAX6 paired box 6 PBS phosphate buffered saline PGC primordial germ cell pNSC primitive neural stem cell POU5F1 pit-oct-unc class 5 homeobox 1 Q-PCR quantitative polymerase chain reaction RFP red fluorescent protein RNA ribonucleic acid ROCK rho-associated kinase RPE r-phycoerythrin SEM standard error of the mean SFM serum free media SOX1 sex determining region Y box 1 SOX2 sex determining region Y box 2 SSEA4 stage specific embryonic antigen 4 T brachyury TE trophectoderm TGFB transforming growth factor beta TRA-1-60 trafalgar 1-60 TUBB3 beta 3 tubulin 7-AAD 7 aminoactinomycin D

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List of Figures Chapter 1

Figure 1 Properties of pluripotent stem cell lines 6 Figure 2 NSC ontogeny in vivo 16 Figure 3 In vitro model of NSC ontogeny 19 Chapter 3 3.1 Figure 4i-iv Clonal neurospheres grow in defined conditions over 35 4 weeks, express only neural markers, differentiate into neurons and glia and passage clonally long-term. 3.2 Figure 5 Neurospheres grow from single cells rather than 43 multicellular aggregates when cultured at densities of 10c/µl or less. 3.3 Figure 6 Primary neurosphere formation is dependent on FGF, 47 and addition of growth factors and ROCK inhibitor improves survival and frequency of neurosphere formation. 3.4 Figure 7i-ii hESCs default to a mixed ectodermal and endodermal 52 identity in SFM alone. Neurosphere originating cells are TRA-1-60-/SSEA4-, make up 6.14% of cells in confluent colonies, and are characterized by elevated levels of neural and endodermal gene expression. 3.5 Figure 8 Noggin treatment of hESC colonies enhances the 58

generation of NSCs but administration of noggin to low density neurosphere culture has no effect.

3.6 Figure 9 hESC derived NSCs sustain expression of LIN28, 63 and form ES-like colonies that re-initiate OCT4 expression when plated on MEFs.

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Chapter 1

Literature Review

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1.1 Pluripotency

Mammalian development begins with a single cell that divides to form a

mulberry-like (morula) cluster of undifferentiated cells. The first differentiation event

takes place when morula cells segregate into two distinct lineages (Zwaka et al., 2005):

the trophectoderm (TE), which forms the outer membrane of the conceptus; and the inner

cell mass (ICM) which gives rise to the embryo proper. Secretions from trophoblast cells

of the trophectoderm create a fluid filled cavity called the blastocoel (Yamanaka et al.,

2006), while the ICM is compacted and pushed to one end. In a second differentiation

event, ICM cells give rise to primitive endoderm (PE) and epiblast (embryo). The PE

lines the inner surface of the blastocoel including the underside of the epiblast (Zwaka et

al., 2005). At this pre-implantation stage the conceptus is known as a blastocyst, and

contains three distinct cell lineages. With implantation of the blastocyst into the uterine

wall, the TE forms placenta, PE gives rise to the extraembryonic endoderm of the yolk

sacs, and epiblast forms all embryonic tissues (Rossant et al., 2008). The epiblast

differentiates into a pseudostratified epithelium known as primitive ectoderm, from the

proximal part of which a small subpopulation of cells develops into primordial germ cells

(PGCs) (Saitou et al., 2003). Finally, the embryo undergoes gastrulation during which

primitive ectoderm differentiates into endoderm, ectoderm and mesoderm, the three

primary germ layers from which distinct stem cell populations establish all somatic cell

types (Vallier et al., 2005). Germ cells are the only pluripotent cells remaining in the

embryo after gastrulation.

Pluripotent stem cell research began in the 1950s with the study of

teratocarcinomas (Dixon et al., 1952). These are malignant germ cell tumors that contain

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a core of undifferentiated embryonal carcinoma (EC) cells, typically surrounded by a

variety of differentiated cell types that can include representatives of all three germ

layers. Teratocarcinomas can spontaneously arise in the adult testes or ovaries of mice or

humans (Stevens et al., 1954). In fact, the discovery that strain 129 mice had a high

incidence of spontaneous testicular teratocarcinoma formation enabled their use in

experimental analyses (Stevens et al., 1954; Damjanov et al., 1974). Teratocarcinomas

can be serially transplanted between mice because the persistent EC cell component

sustains tumor growth. If the EC component disappears, a benign teratoma may develop.

In 1964 Kleinsmith and Pierce showed that a single EC cell is able to renew itself

limitlessly and differentiate down multiple lineages, thus establishing the existence of

pluripotent stem cells and the criteria by which to define them (Kleinsmith et al., 1964).

Experimentally, teratocarcinomas can be produced by grafting early mouse embryos to

ectopic sites in adult mice. Tumors will then form from embryos ranging from one cell

to the egg cylinder stage (E8), when expression of pluripotency transcription factor

OCT4 is shut off (Solter et al., 1970; Stevens et al., 1970). In 1970, Kahan et al. derived

mouse EC cell lines from ectopic engraftments, initiating an era of the study of

mammalian development using pluripotent cell lines. Subsequently it was shown that EC

cells could contribute to normal chimeric tissue (Brinster, 1974) as well as tumors

(Rossant et al., 1982) when injected into mouse blastocysts. The discovery that antigen

and protein expression of mouse EC cells were similar to that of ICM cells of a pre-

implantation blastocyst-stage embryo (Gachelin et al, 1977; Solter et al., 1978),

suggested that EC cells might represent a malignant counterpart to normal pluripotent

embryonic cells. Thus it was that a direct derivation of pluripotent cell lines was

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attempted from mouse embryos without the ectopic engraftment step that produced

teratocarcinomas. The culture conditions developed for the stable propagation of EC

cells, including the use of feeder cell layers, were used to successfully isolate the first

mouse embryonic stem cells (mESCs) (Evans and Kaufman 1981, Martin 1981) followed

by human embryonic stem cells (hESCs) (Thomson et al., 1998) from blastocyst stage

embryos.

Maintenance of the pluripotent state in mESCs is dependent on the expression of

transcription factors OCT4, SOX2 and nanog, and by signalling through the cytokine

leukemia inhibitory factor (LIF) and BMP4 (Ying et al., 2003). Injection of mESCs into

blastocysts results in the creation of chimeric embryos, illustrating the ability of mESCs

to form all of the tissues of the body. Pluripotent hESCs also express OCT4, SOX2 and

nanog, but self renewal depends on activin/nodal and FGF signalling (Vallier et al.,

2005). As the ethics of the creation of chimeric human embryos are hotly contested,

functional pluripotency must be assessed in vivo by the transplantation of hESCs to

ectopic sites in mouse where they form teratomas (Thomson 1998). Alternately, multi-

lineage differentiation can be assessed in vitro by formation of embryoid bodies (EBs)

(Keller, 1995) or for non-embryonic cells by the selection of ES-like colonies that form

after plating in ES culture conditions using mouse embryonic fibroblast feeders (MEFs),

or MEF conditioned medium (Takahashi and Yamanaka, 2006; Takahashi et al., 2007).

Thus far, a variety of additional pluripotent cell lines have been derived from

embryonic and non-embryonic sources. All of these cell lines depend on the expression

of OCT4 and SOX2 for maintenance of the pluripotent state, however the growth factors

required for self renewal differ, and there is much variability in the propensity of these

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different cell types to give rise to extra-embryonic lineages, and contribute to chimera

formation (Rossant, 2008)(Fig. 1).

Mouse and human embryonic germ (mEG/hEG) cells were the first ES-like cells

with a non-blastocyst origin. These cells were derived from PGCs in the developing

gonad (Matsui et al., 1992; Resnick 1992; Shamblott et al., 1998). Pluripotent ES-like

cells have also been isolated from adult and neonatal testis, deemed multipotent germ line

stem (mGS) cells (Kanatsu-Shinohara et al., 2004; Guan et al., 2006; Seandel et al.,

2007). Perhaps the most striking example of pluripotent cells of non-embryonic origin is

induced pluripotent stem (iPS) cells. These cells were generated by the reprogramming

of adult mouse (Takahashi and Yamanaka, 2006) and human (Yu et al., 2007; Takahashi

et al., 2007) fibroblast cells by overexpression of exogenous transcription factors.

Human and mouse iPS cells greatly resemble mESC and hESCs respectively in their

culture requirements, gene expression and ability to generate multiple tissue types.

Recently, pluripotent cell lines were derived from the epiblast of early post-

implantation mouse embryos (Brons et al., 2007; Tesar et al., 2007), representing the first

variety of pluripotent cells from mouse that bear a striking resemblance to human

pluripotent cells because they require activin and FGF rather than LIF and BMP4 to

sustain growth. In addition, unlike mES cells, mEpiSCs (mouse epiblast stem cells) and

hESCs both exhibit the ability to form extra-embryonic tissues, as judged by spontaneous

TE differentiation, and generation of PE when treated with BMP4 (Brons et al., 2007).

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Figure 1

Fig.1 Properties of pluripotent stem cell lines

Cell Line In Vitro Pluripotency

Chimera Formation

Teratoma Formation

Terato-carcinoma Formation

Spontaneous Trophoblast Differentiation

Growth Factors for Self renewal

mEC + somatic, germline

+ + ? LIF,BMP

mES + somatic, germline

+ ? - LIF,BMP

mEG + somatic, germline

+ ? ? FGF/SCF, then LIF

mGC + somatic + ? ? GDNF, then LIF

mEpiSC + - + ? + FGF, activin

miPS + somatic, germline

+ ? ? LIF

hES + ? + + + FGF, activin

hEG + ? + ? ? FGF hEpiSC + ? + ? ? FGF hiPS + ? + ? ? FGF,

activin

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These observations sparked a debate about the developmental equivalence of hES versus

mES cells, advancing the idea that hESCs were perhaps more closely related to later

stage mEpiSCs (Brons et al., 2007; Tesar et al., 2007), where previously differences in

growth factor dependencies, gene expression, epigenetic status, downstream gene targets

and differentiation potential had been ascribed to differences of species. Paradoxically,

the propensity of hESCs and mEpiSCs to spontaneously generate trophoblast cells in

culture led others to posit that hESCs and mEPiSCs were in fact more closely related to

totipotent pre-blastocyst stage embryos. Complicating matters is that to date mEPiSCs

do not appear to contribute to chimera formation when injected into developing

blastocysts, and that avenue of exploration is not open to human cells.

The confounding properties and vastly differing methods of isolation of all of the

various pluripotent stem cells discussed begs the consideration that in vitro culture has a

potent effect on these cells. In fact, the transforming effects of in vitro culture may call

into question the ability to associate pluripotent cells with in vivo counterparts of a

particular lineage. Indeed the pluripotent state is so transient in vivo, that in vitro

immortalization itself indicates the creation of artifact. Despite such limitations, the

study of the variety of pluripotent cell lines isolated to date continues to inform early

mammalian development, and provides a platform from which an understanding of cell

differentiation may be improved.

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1.2 Neural induction and the classical model

Vertebrate gastrulation describes a period of development that is characterized by

the migration of cells from the surface ectoderm into the interior of the embryo, resulting

in the formation of three distinct germ layers: ectoderm, mesoderm and endoderm. In

vertebrates, cells from the ectoderm ingress through various structures: the blastopore in

amphibians; the embryonic shield in ray-finned fishes (teleosts); or the primitive streak in

reptiles, birds and mammals (amniotes) (Stern, 2005). Gastrulation is followed by

organogenesis, during which ectoderm forms nervous tissue and epidermis (skin),

mesoderm forms bone, blood and muscle, and endoderm creates the lungs, liver and

pancreas.

The process by which ectodermal cells acquire a neural identity during

gastrulation is known as neural induction. Before the molecular mechanisms of this

process were elucidated, a classical model of neural induction was postulated, based on

transplant and explant studies. The generation of neural cell types from ectoderm was

first understood to be reliant on a region of tissue in the underlying dorsal mesoderm.

This region is known as the Spemann organizer in amphibians (Spemann and Mangold,

1924), the shield in teleosts (Oppenheimer, 1936), Henson’s node in chick (Waddington,

1933), and the node in mouse (Beddington, 1994). In 1924 Spemann and Mangold

demonstrated that when organizers (dorsal blastopore lip) of gastrulating newt embryos

were transplanted to the ventral side or prospective belly of host embryos, a second

nervous system developed. Signals from the newt organizer seemingly ‘induced’ the

formation of an ectopic nervous system from ventral ectoderm that would otherwise have

become epidermis. A number of years later, experiments with chick and Xenopus

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seemed to corroborate the inductive power of the organizer. Transplantation of Henson’s

node was able to produce ectopic neural tissue in hosts (Waddington, 1933). In Xenopus,

in vitro culture of pieces of ectoderm with and without organizer tissue showed that

ectoderm became epidermis in the absence of the organizer, but became neural when

combined with the organizer (Holtfretter et al., 1955). Thus a classical model of neural

fate determination emerged that explained neural induction as the result of instructive

signals that emanated from an organizer region common to vertebrates.

1.3 Default model of neural induction

The classical model of neural induction characterized the ground state or ‘default’

fate of ectoderm as epidermis (Holtfretter et al., 1955). In 1989, the experiments of three

different groups challenged this assertion. It was found that when Xenopus ectoderm

from gastrulating embryos was separated from organizer tissue and then dissociated and

cultured as single cells, neural tissue formed upon reaggregation (Godsave and Slack,

1989; Grunz and Tacke, 1989; Sato and Sargent, 1989). It was suggested that the

ectoderm itself produced neural inhibitors whose activity could be suppressed by

isolating cells by dissociation (Godsave and Slack, 1989; Grunz and Tacke, 1989). These

collective findings were the first line of evidence to advance the idea that the default state

of naïve ectoderm might be neural, and that neural development could take place without

the instructive cues of organizer tissue (Dang and Tropepe, 2006).

Meanwhile, the search for a single molecule that might be a neural inducer had been

ongoing for more than 60 years, as the molecular basis for neural specification remained

elusive. The molecular picture began to take shape in the early 1990’s when activin

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signalling was found to influence both mesodermal and neural development (Hemmati-

Brivanlou and Melton, 1992). Activin is a member of the transforming growth factor beta

(TGFB) family of secreted proteins. A mutant activin receptor was used that was able to

knock out the function of the normal activin receptor by forming a heteromeric complex

(also known as a dominant negative, or truncated receptor). In this way, it was found that

inhibition of activin signalling in Xenopus could lead to the neuralization of ectodermal

explants (Hemmati-Brivanlou and Melton, 1994). This was the first molecular piece of

evidence in the puzzle of neural induction, and it supported the hypothesis that neural

specification might arise as a consequence of inhibitory rather than instructive

mechanisms. Concurrently, three neuralizing proteins were isolated and found to be

produced by Xenopus organizers. The first to be identified was noggin (Smith and

Harland, 1992). In addition to the exclusive localization of noggin to the organizer,

neural tissue could be generated from blastula-stage amphibian ectodermal explants

either by treatment with soluble noggin protein or RNA (Lamb et al., 1993). Next

follistatin was shown to be localized to the organizer and able to induce neural

differentiation in Xenopus ectoderm by antagonizing activin signalling (Hemmati-

Brivanlou et al., 1994). Chordin expression also emanated from organizer tissue and

could induce neural differentiation (Sasai et al., 1994). Chordin was subsequently found

to be homologous to the product of an embryonic Drosophila patterning gene called short

gastrulation (sog) (Francois and Bier, 1995). Sog was known to be an antagonist of

decapentaplegic (dpp), the Drosophila homolog of bone morphogenic protein (BMP), and

like activin, also a member of the TGFB superfamily of growth factors (Ferguson and

Anderson, 1992). Further work showed that BMP4 could inhibit neural induction, and

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the truncated activin receptor that led to neuralization of Xenopus ectoderm also inhibited

BMP signalling in addition to activin signalling (Wilson and Hemmati-Brivanlou, 1995).

In turn, noggin, chordin and follistatin were shown to bind to BMP4 ligands in the

extracellular space, thereby blocking BMP signalling and promoting neuralization of

ectoderm (Zimmerman et al., 1996; Piccolo et al., 1996; Fainsod et al., 1997).

Importantly, BMP4 treatment of ectodermal explants promoted the generation of

epidermis rather than neural tissue illustrating that BMP signalling was the key modulator

of fate specification in ectoderm (Wilson and Hemmati-Brivanlou, 1995). From this

series of experiments, starting from the work of Spemann and ending with the elucidation

of the role of BMP4 in neural induction, emerged the neural default model of fate

specification. The underlying assertion of this model is that in the absence of extrinsic

signalling, ectodermal cells become neural, and this fate is achieved during embryonic

development by the inhibition of BMP signalling.

1.4 Challenges to the neural default model

The neural default model provides a simple explanation for neural induction

based on the inhibition of a single signalling pathway, that of BMP. It has been

suggested however, that BMP signalling might not be necessary for neural induction in

chick embryos. This idea took hold as a result of experiments showing that the

production of secreted BMP inhibitors is mistimed with the onset of neuralization (Streit

et al., 1998; Streit et al., 2000). In addition, noggin and chordin were found unable to

induce neural tissue formation in amphibian embryos where FGF signalling was blocked

by a truncated FGF receptor (Launay et al., 1996). A role for FGFs in neural induction

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was further substantiated by chick epiblast explant experiments where FGF signalling

had an early effect on neuralization before the node had even been formed (Wilson et al.,

2000). Parallel to these insights was the growing evidence that a functional organizer

was not necessary for neural induction to occur. Over the course of 10 years, it was

found that the removal of gastrula organizers from zebrafish, chick, mouse and frog did

not prevent the formation of a neural plate (Dang and Tropepe, 2006). For example,

mouse embryos lacking the transcription factor HNF3B (FOXA2), which is crucial for

proper formation of the node, are still able to develop nervous tissue (Klingensmith et al.,

1999). This growing body of evidence suggested that neuralization involving FGFs

seemed to precede the formation of the organizer that was responsible for the production

of extracellular inhibitors of BMPs, calling into question the mechanism of neural

induction as postulated by the neural default model. However, more recent studies

corroborate the importance of the BMP signalling pathway in neural induction and

illustrate that FGFs and even Wnts function to repress BMP signalling in the embryo

before the onset of gastrulation and the development of the organizer (Stern 2007).

Unlike the inhibitory effects of organizer derived molecules that are mediated in the

extracelluar space, FGFs block smad-dependent BMP signal transduction in the

cytoplasm, and in the nucleus FGFs are able to repress the expression of BMP genes

(Dang and Tropepe, 2006). In Xenopus, Wnts are able to downregulate BMP4 gene

expression by activation of transcriptional co-repressors of the iroquois family (Gomez-

Skarmeta et al., 2001). Indeed, transcriptional downregulation of BMP gene expression

defines the onset of neural induction in the ectoderm of all model systems analyzed,

including chick (Munoz-Sanjuan and Hemmati-Brivanlou, 2002). Therefore, although

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there may be variety in the mechanisms by which neural induction is achieved across

species, the central role of BMP inhibition in neural fate specification in vertebrates is

well established.

1.5 ES cells and the default model

In vivo culture environments generally do not allow the recapitulation of complex

signalling environments that include multi-germ layer interactions and their influences on

neural fate specification. However, ES cells do provide a system whereby fate decisions

made by single cells can be studied. As such, mES cells were first used as a system to

investigate the occurrence of default neural fate specification in mammals. These studies

postulated that an ES culture system that allowed for only cell autonomous signalling

could shed light on the innate capability of mammalian ectoderm and its default identity

(Tropepe et al., 2001; Smukler et al., 2006). It was reasoned that single cells plated at

low densities in serum-free and feeder-free environments could create a cell autonomous

signalling environment (Tropepe et al., 2001). After only 4 hours in such an

environment, and largely preceding cell division, the majority of viable cells were found

to express neural markers including SOX1 and nestin (Tropepe et al., 2001; Smukler et

al., 2006). Clumps of mESCs grown in the same conditions did not initiate neural

marker expression, a likely result of increased cell-cell interactions, however these cell

clusters could be neuralized by attenuation of BMP signalling (Tropepe et al., 2001).

These findings suggest that mESCs directly transition to neural cell types in the absence

of extrinsic signalling, and in agreement with amphibian models of neural induction, this

fate specification is dependent on the inhibition of BMP signalling.

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To date, the neural default model has not been successfully investigated using

hESCs, although several studies have explored the effects of the BMP inhibitor noggin on

fate specification in hESC aggregates (Itykson et al., 2005; Sonntag 2007). Other groups

have suggested that inhibition of activin/nodal signalling gives rise to the specification of

hESCs to NSCs through a neural default mechanism (Vallier et al., 2004; Smith et al.,

2007). All of the aforementioned studies rely on hESC culture as multicellular

aggregates or high density monolayers, with primary specification of neural fates

occurring through EB formation or by growth on neural inducing feeder layers. As a

result, these methods examine paracrine mediated cell fate acquisition rather than cell

autonomous mechanisms. In order to accurately assess the default identity of hESCs,

cells need to be dissociated and plated at low densities as single cells, a necessary

prerequisite to achieving a complete blockade of extrinsic signalling.

1.6 Neural stem cells

The existence of a reservoir of somatic cells capable of regenerating tissues was

first postulated by Morgan in 1901. In 1924, Maximow coined the term ‘stem cell’ to

describe wandering resting cells that he postulated could be found in the blood system,

thereby instigating the notion of the presence of a cell capable of regenerating an organ.

The working definition of a stem cell took shape after pioneering experiments with

hematopoietic stem cells (HSCs) (Till and McCullough, 1961). A stem cell can now be

described as a cell that can extensively self renew through symmetric or asymmetric cell

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division, differentiate into the major lineages of the tissue of origin, and display the

capacity to regenerate the tissue or organ of its origin.

For many years it was believed that the CNS did not posses stem cells because

nervous tissue did not appear to regenerate after injury. The isolation of NSCs from the

subependyma of the lateral ventricles was first described in 1992 (Reynolds and Weiss,

1992), offering the first promise of the ability to stimulate endogenous stem cells to

repair neural tissue damaged by injury and disease. In addition, adult NSCs offered a

model system by which to study neural differentiation as they display extensive self

renewal and are to be able to generate all of the primary subtypes of the neural lineage:

neurons, astrocytes, and oligodendrocytes. Obvious technical limitations do not allow for

the regeneration of an entire nervous system in an adult organism as can be done using

HSCs, however transplantation in vivo and differentiation in vitro are used to demonstrate

the ability of NSCs to contribute to all the cell types of the neural lineage.

It is now generally accepted that in mouse there are two types of NSCs (Fig. 2).

Primitive neural stem cells (pNSCs) represent an early and transient population that can

be isolated in vivo from embryonic day (E) 5.5-7.5 epiblast and E7.7-8.5 neuroectoderm

(Tropepe et al, 1999; Hitoshi et al., 2004). Definitive neural stem cells (dNSCs) arise

from pNSCs as the neural lineage becomes restricted. dNSCs are fully committed, FGF

dependent cells that give rise to epidermal growth factor (EGF) responsive secondary

colonies, and first appear in E8.5 neural plate and persist in the subependyma of the

lateral ventricles throughout adulthood (Tropepe et al., 1999; Hitoshi et al., 2004).

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Figure 2

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Fig. 2 NSC ontogeny in vivo

In vivo, NSC development becomes restricted as pNSCs give rise to dNSCs. This

progression is evidenced by the isolation of pNSCs from early embryonic structures

(E5.5-7.5 epiblast and E7.5-8.5 neuroectoderm). dNSCs are derived from later

embryonic tissue (E8.5 neural plate) and in the adult from the subependyma of the lateral

ventricles, a remnant of the germinal zone of the embryo. Unlike embryo derived NSCs

that are constantly dividing to expand the developing nervous system, adult NSCs are a

quiescent population that is responsive to FGF and EGF.

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Using mESCs as a starting point for an in vitro model of neural stem cell

ontogeny (Fig. 3), it was determined that a pNSC-like cell that exhibited characteristics

intermediate to those of mESCs and dNSCs could be isolated by culture in serum free

conditions supplemented with LIF (Tropepe et al., 2001). When pNSCs are passaged

they give rise to dNSCs that are dependent on FGF, but not on LIF (Smukler et al., 2006;

Tropepe et al., 2001).

Cell based therapies using hESCs or hiPS cells offer the promise of regeneration

of damaged nervous tissue. The ability to generate the vast array of neuronal subtypes

and glia that could be utilized to this end may depend on the ability to derive both early

and late neural precursors such as the pNSC and dNSC.

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Figure 3

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Fig. 3 In vitro model of NSC ontogeny

An ES cell based model of NSC development shows that single mES cells grown

in a low density environment free of extrinsic signalling acquire a pNSC identity through

a default mechanism that is negatively regulated by TGFB signalling. In the presence of

LIF, the pNSC maintains an undifferentiated state and proliferates to form a clonal

neurosphere that is made up of dNSCs that have progressed along the neural lineage to a

more mature, FGF dependent state. The dNSC remains in a proliferative state in the

presence of FGF and can be repeatedly passaged, illustrating self renewal. dNSC

containing neurospheres can be differentiated by adherent culture in the presence of

serum, and produce neurons, astrocytes and oligodendrocytes.

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1.7 The neurosphere assay

There are currently no definitive markers that can identify NSCs, or even

distinguish them from their progenitors which may be restricted in lineage potential

and/or be incapable of long term self renewal. Some studies in mouse have identified

molecular markers such as LexA (Capela and Temple, 2006) and Sox2 (Suh et al., 2007)

that are enriched in NSCs and their progenitors. These markers can be used

prospectively to identify NSCs and their progenitors in a mixed population, however, it is

still impossible to obtain pure populations of NSCs using these markers. In addition,

markers like Sox2 are not suitable for prospective isolation of NSCs and progenitors in

ES systems, as ESCs tend to express a vast array of genes including those that mark

NSCs and progenitors in adult populations. As such, the current identification of NSCs

from ESCs must be analyzed retrospectively by the formation of single free-floating

colonies called ‘neurospheres,’ that display multilineage potential and exhibit long term

self renewal. The neurosphere assay was created by Reynolds and Weiss in 1992, and

measures the ability of a single cell to proliferate, self renew, and produce the primary

neural lineages. The number of NSCs present in isolated tissue or generated from ES

cells can be determined by plating single cells at clonal densities in serum free media

with growth factors. Each neurosphere that arises is thought to be the clonal product of a

single NSC or ES cell. The neurosphere assay is limited in that spheres may also arise

from progenitor cells. NSC numbers can be more accurately assessed by scoring larger

spheres (progenitor spheres tend to be small (Louis et al., 2008)), and performing long-

term passage of single colonies as progenitor spheres seem unable to passage repeatedly

(Seaburg et al., 2002). To demonstrate multipotency, single colonies must display an

22

ability to form neurons, astrocytes and oligodendrocytes when differentiated. Implicit in

the formation of the neurosphere itself is the capacity of the NSC to self renew, but long

term self renewal is ideally tested by the consecutive passaging or sub-cloning of single

dissociated colonies at clonal densities for more than 3 or 4 successive generations.

Resulting single neurospheres must also display multilineage potential upon

differentiation. Clonal plating densities need to be established for each starting cell

population to ensure that neurospheres are arising from a single cell rather than by

multicellular aggregation. In order to avoid kinetic aggregation, plates must also remain

undisturbed over the course of sphere formation (Coles-Takabe et al., 2008).

1.8 Research aims and hypotheses

The developmental potential of hESCs has triggered an explosion of research

focused on directing differentiation to specific cell types required for cell replacement

therapies. Many neurodegenerative disorders are the result of the loss or dysfunction of a

single cell type, such as the dopaminergic neuron in Parkinson’s disease. NSCs are the

self renewing, multipotent cells of the central nervous system that can generate all of the

cells in the neural lineage and are thus used as precursors for the production of the vast

quantity of cells that need to be generated for transplantation. Fetal and adult human

brain tissue is an extremely limited source from which to obtain NSCs. However hESCs,

as well as hESC-like hiPS cells are available in a relatively limitless supply for

differentiation into NSCs. In addition, hESCs represent an excellent system whereby the

molecular and cellular aspects of human neural development and differentiation can be

studied.

23

Existing methods for deriving human NSCs and their progenitors from hESCs are

not clonal. In addition, to generate neural precursors using standard protocols, hESCs are

first differentiated through EB or monolayer forming stages that are uncontrolled, and use

serum or feeder layers (Dhara et al., 2008; Elkabetz et al., 2008; Kang et al., 2007; Nistor

et al., 2005; Schuldiner, et al., 2001; Reubinoff et al., 2001; Zhang et al., 2001). Recent

trials with transplanted hESC derived NSCs into Parkinsonian rats highlight the

importance of controlled differentiation and cell selection: while significant functional

improvement was seen, a subset of the heterogeneous mix of transplanted cells

proliferated in undifferentiated tumorigenic clusters (Roy et al., 2006).

To address the problems created by the bulk propagation of mixed populations of

cells in undefined conditions, we endeavored to create a defined one step clonal method

of deriving NSCs from single hESCs. Based on studies done with mESCs (Tropepe et

al., 2001; Smukler et al., 2006), we hypothesized that single pluripotent hESCs cells

would directly transition to a neural identity in minimal culture conditions and proliferate

to form NSC colonies. We further hypothesized that this transition may occur along a

default pathway of neural fate specification.

24

Chapter 2

Materials and Methods This chapter has been submitted for publication: Radha Chaddah, Margot Arntfield, Susan Runciman, and Derek van der Kooy (2009). Clonal derivation of neural stem cells from human embryonic stem cells.

25

2.1 Propagation and maintenance of hESCs

H9 (Thomson et al 1998) and CA1 hESCs were used in all experiments. The CA1

hESCs are fully characterized and approved by the Stem Cell Oversight Committee at the

Canadian Institute of Health Research. (Correspondence concerning the isolation and

characterization of the CA1 hESCs should be directed to Andras Nagy, Samuel

Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada M5G

1X5; nagy@mshri.on.ca.). hESCs were maintained on mitotically inactivated MEFs in

media containing 80% knock-out DMEM and 20% knock-out serum replacement

(Invitrogen), 1% non-essential amino acids, 1mM L-glutamine, 0.1mM 2-

mercaptoethanol, and 4ng/ml basic FGF (bFGF; represented throughout as ‘FGF’).

Confluent colonies were disaggregated into ~100 cell clumps using 0.1% type IV

collagenase (Invitrogen) and passaged 1:3 or 1:4 every 4-7 days. For noggin pre-

treatment, 100ng/ml of mouse recombinant Noggin/Fc chimera (R&D systems) was

added to fresh media each day until colonies became confluent (4-7 days).

2.2 Neurosphere culture

hESCs were incubated at 37 degrees for 15-20 minutes in tripleE Express for

dissociation, and then quenched with MEF conditioned media before being centrifuged.

Cells were resuspended and triturated with a small borehole pasteur pipette before being

filtered through a 40µm cell strainer. The resulting single-cell suspension was plated at

10 cells/µl in chemically defined SFM with growth factors. SFM (Reynolds and Weiss,

1996) contained a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) and F-

12 nutrient (Gibco) with 0.6% D-glucose, 5mM Hepes, 3mM NaHCO3, 2mM glutamine,

26

25 µg/ml insulin, 100 g/ml transferrin, 20nM progesterone, 60 µM putrescine, and 30 nM

sodium selenite (all Sigma). Growth and survival factors added singly or in combination

included 1400U/ml human LIF (Chemicon), 10ng/ml bFGF, 2µg/ml heparin, and

20ng/ml EGF (all Sigma), 1x B27 (Invitrogen), and 100ng/ml recombinant Noggin/Fc

chimera (R&D systems), and rho-kinase inhibitor Y-27632 (Calbiochem) used at a final

concentration of 10uM. hESCs were plated in the centre 24 wells of 48 well coated plates

(Nunclon) and at 2 weeks were re-fed with an equal volume of plating media (300µl).

Floating sphere colonies >75µm were scored at 4 weeks, except neurospheres cultured

with rho-kinase inhibitor that were collected every 11 days. To assess self renewal of

neurospheres, 4 week old colonies were collected in bulk and floated in a 10cm Petri dish

where colonies >75µm were picked for passage. Spheres were dissociated into single

cells by incubation in trypsin/EDTA(Sigma) for 10 minutes, followed by manual

trituration with a small borehole pasteur pipette. Single neurosphere cells were then

plated at 10 cells/µl. Secondary sphere colonies were quantified after 4 weeks. For

differentiation, a single neurosphere per well was plated on matrigel (BD Bioscience)

coated plates in SFM supplemented with 1% fetal calf serum (FCS; Hyclone). Spheres

were re-fed at 7 days, and cultured for 14 days. Undifferentiated and differentiated

neurospheres were collected for Q-PCR analysis, or fixed for ICC.

2.3 hESC minimal condition assays

hESCs were dissociated into a single-cell suspension using tripleE Express

(Invitrogen), and plated at 1 cell/µl in defined serum-free media (described above)

without growth factors. Cells were collected for Q-PCR analysis or fixed for ICC analysis

27

at 4h, 24h, and 3d time-points. For analysis by ICC, cells were plated at on

laminin/poly-L-ornithine coated culture plates (Nunclon). To assess the survival of

individual hESCs they were plated at 50 cells/well in 48 well plates. At 4h, 24h, and 3d

viable cells were scored by trypan blue exclusion.

2.4 Clonality assays

For mixing experiments, H9-RFP and CA1-GFP cells (provided by Jon Draper)

were prepared for the neurosphere assay as previously described. Equal proportions of

two types of cells were plated at final densities of 5, 10, 20 and 50 cells/µl. The following

combinations of cells were used: H9 and H9-RFP; CA1 and CA1-GFP; H9-RFP and

CA1-GFP. A control for frequency of sphere formation was performed for each cell type

in each experiment. At 2 and 4 weeks, floating spheres were scored for chimerism. For

single cell per well experiments, H9-RFP cells were prepared for the neurosphere assay

as previously described. A single cell per well was sorted into a minimum of 2,016 wells

(per experiment) of 96 well plates using the FACS Aria 3-laser system. After three

hours, all wells were scored for the presence of single cells. Wells containing single cells

(80.4% single wells contained one cell, 19.6% of wells contained no cell) were scored at

2 weeks for the presence of a sphere colony >50µm in diameter.

2.5 Immunocytochemistry

Adherent cells and colonies were fixed with 4% paraformaldehyde in PBS (pH

7.3) for 20 minutes at room temperature and then washed with 3x with PBS. Cells were

permeabilized with 0.3% Triton X-100 for 5 minutes and then washed twice with PBS

28

before being blocked with PBS containing 1% bovine serum albumin (BSA) and 10%

normal goat serum (NGS) for one hour at room temperature. Blocking solution was

washed off with PBS before primary antibodies were applied. Antibodies were diluted in

PBS containing 0.3% triton and 1% NGS and applied to cultures overnight at 4ºC.

Primary antibodies used were: mouse monoclonal human specific anti-NES (IgG)

(Chemicon) at 1:200, mouse monoclonal anti-TUBB3(IgG)(Sigma) at 1:500, rabbit

polyclonal anti-GFAP (IgG)(Dako) at 1:1000, mouse monoclonal anti-O4

(IgM)(Chemicon), at 1:200, rabbit polyclonal anti-SOX1 (IgG)(Chemicon) at 1:100,

mouse monoclonal anti-MAP2 (IgG)(Chemicon) at 1:250, mouse monoclonal anti-OCT4

(IgG)(BD Biosciences) 1:200, mouse monoclonal anti-FOXA2 (IgG) (DSHB) at 1:10 and

goat monoclonal anti-FOXA2 (IgG)(Santa Cruz) at 1: 200. After overnight incubation,

cultures were washed 3x with PBS before secondary antibodies were applied.

AlexaFluor secondary antibodies were used at a dilution of 1:400 and included: 488 goat

anti-rabbit, 488 goat anti-mouse, 568 goat anti-rabbit, 568 goat anti-mouse, 568 donkey

anti-goat, and 647 goat anti-mouse (Molecular Probes). Secondary antibodies were

suspended in PBS with 1% NGS and applied for 2 hours at 37ºC. Cultures were then

washed 3x before applying Hoechts dye 33258 (0.015mg/ml stock solution diluted to

0.001 mg/ml in PBS; Sigma) to stain the nuclei of all cells. Hoechts was applied for 10

minutes at room temperature after which cells were washed 3x with PBS. Positive,

negative and secondary only controls were used for each experiment. All controls were

stained simultaneously using the same method, except for the omission of primary

antibodies from secondary only wells. Fluorescence was quantified only when negative

and secondary only controls were negative for staining. Cells in the differentiation and

29

neural default assays were quantified by assessing the number of positively stained cells

as a percentage of dapi positive nuclei in at least 4 photographed fields for a minimum 4

colonies in a minimum of 3 individual experiments. Cell fluorescence was visualized

using a motorized inverted microscope (IX81; Olympus). Fluorescent images were

captured using Olympus MicroSuite version 3.2 image analysis software and MicroSuite

Five Biological Suite (Soft Imaging System Corp.)

2.6 RNA Isolation, cDNA preparation and Quantitative RT-PCR

Total RNA was isolated using an RNeasy extraction kit (Qiagen) according to

instructions with optional DNase treatment. The amount of total RNA was quantified

using a NanoDrop ND-1000 spectrophotometer. cDNA was prepared from 150–500 ng

RNA using 200 U SuperScript III RNase H–Reverse Transcriptase (Invitrogen) and

0.2 ng random hexamer primer (6-mer, Fermentas). QPCR was performed using pre-

designed TaqMan gene expression assays (Table 1, Applied Biosystems) with TaqMan

universal PCR master mix (Applied Biosystems) under universal cycling conditions

(95°C for 10 minutes; 95°C for 15 seconds, 60°C for 1 minute for 40 cycles) on a

7900HT Fast Real-Time PCR System (Applied Biosystems). Reactions were done in

triplicate and always included a negative control (no template). Relative quantification

(ΔΔCt) was done using RQ Manager Software (Applied Biosystems). β-Actin and 18S

were used as endogenous controls as they had consistent expression across all samples.

A dilution curve of hESC RNA was used to determine that efficiencies of targets were

similar to those of endogenous controls.

30

TaqMan Gene Expression Assays

Gene Gene Expression Assay ID# 18S Hs99999901_s1 β-Actin Hs99999903_m1 OCT4 Hs01895601_u1 LIN28 Hs00702808_s1 SOX2 Hs00602736_s1 NES Hs00707120_s1 SOX1 Hs00534426_s1 PAX6 Hs00240871_m1 TUBB3 Hs00801390_s1 FOXA2 Hs00232764_m1 HNF4A Hs00766846_s1 GATA4 Hs00171403_m1 GATA1 Hs00231112_m1 T Hs00610080_m1 2.7 FACS Analysis

Fluorescence activated cell sorting was performed on CA1 and H9 cells. hES

colonies were dissociated into single cell suspensions (as above). Cells were suspended

in blocking solution containing Hank’s Balanced Salt Solution (HBS)(Gibco) with 2%

FCS (Hyclone) before adding primary antibodies TRA-1-60 (IgM)(Chemicon; 1:100) and

SSEA4 (IgG)(DSHB; 1:5) at 4°C for 30 minutes. Secondary antibodies used were: goat

anti-mouse R-phycoerythrin (RPE ) conjugate and goat anti-mouse fluorescein (FITC)

conjugate both at 1:100 (Southern Biotech). Secondaries were applied for 30 minutes at

4°C. Cells were then washed twice and strained through a 40µm cell strainer. 7-Amino-

actinomycin D (7-AAD) was added to mark dead and dying cells. Live cells were sorted

at low pressure (20PSI) by a FACS Aria 3-laser system (Beckton-Dickinson) using FACS

DiVa software version 5.02. Cells were sorted into 4 separate fractions (+/+, -/-, +/- and

31

-/+) based on RPE and FITC fluorescence. All fractions were kept on ice until plated in

the neurosphere assay, or collected for Q-PCR. Unstained hESCs were used as negative

controls, and RPE-only, FITC-only and RPE plus FITC labeled cells were used as

positive controls to set gates for sorting, and to assess survival of sorted cells.

2.8 EB Formation

In order to confirm the pluripotent ability of CA1 and H9 hESCs, colonies were

disaggregated into ∼100 cell clumps (as described) and cultured in hanging drops (Dang

et al., 2002) in media containing 58% dH20, 15% FCS (Hyclone), 100µM ß-

mercaptoethanol (Gibco), 100U/ml penicillin, 100µg/ml streptomycin, 20% high glucose

DMEM, 1mM L-glutamine, 10mM hepes, 0.6% glucose, 0.1% NaCO3 (all Sigma). 30µl

drops containing approximately 3000 cells were cultured for 2 days before being rinsed

onto uncoated plates (Phoenix Biomedical) and cultured for an additional 3 to 12 days.

Alternately, suspensions of single cells such as sorted cells and NSCs were aggregated by

force using a ‘spin EB’ method (Ungrin et al., 2008). EBs were collected for analysis by

Q-PCR.

2.9 Statistical analysis

Statistics were calculated using 2-way ANOVA, one-way ANOVA and

Bonferroni posttests. Error bars represent the standard error of the mean (sem) of three or

more replicates using at least two different cell lines. Single asterisks (*) indicate

statistical significance, with p values reported in figure legends.

32

Chapter 3

Results

This chapter has been submitted for publication: Radha Chaddah, Margot Arntfield, Susan Runciman, and Derek van der Kooy (2009). Clonal derivation of neural stem cells from human embryonic stem cells.

33

3.1 Single hESCs differentiate into colony forming neural stem cells

without serum or feeder cells

In order to investigate the ability of single hESCs to proliferate in vitro to form

neural colonies in the absence of serum, feeder layers or EB formation, we employed

conditions originally used to isolate brain-derived NSCs that have since been adapted for

the growth of clonal neurospheres from single mESCs (Tropepe et al., 2001; Reynolds

and Weiss, 1996). hES colonies were dissociated to single cell suspensions and plated at

low density in defined serum-free conditions. By 28d in vitro, floating neurospheres had

formed that ranged in diameter from 50µm to 300µm. An average sized colony of

150µm contained approximately 10,000 cells (Fig. 4a). As spheres derived from

restricted progenitors tend to be smaller in size (Louis et al., 2008), only spheres ≥75µm

were included for analysis. Q-PCR showed that neurospheres expressed the neural

markers SOX1, NES, PAX6 and TUBB3, but not the endodermal markers FOXA2,

HNF4A, GATA4, nor mesodermal marker T (also known as brachyury), indicating that

these colonies are neural in character (Fig. 4b). In addition, neurospheres do not express

pluripotency genes OCT4 (also known as POU5F1) or LIN28, indicating the absence of

cells that could be teratocarcinoma initiating in vivo, however, this does not rule out the

possibility that tumors consisting of differentiated cells may arise.

To determine the potential of neurospheres to form neurons and glia, single

colonies were placed on matrigel-coated plates and differentiated in the presence of

serum for 14 days. By ICC, differentiating cells express the neural precursor marker

NES, early and mature neuronal markers TUBB3 and MAP2 respectively, neural

precursor and astrocyte marker GFAP and oligodendrocyte marker O4 (Fig. 4c-g). All

34

single colonies differentiated to contain proportions of neurons, astrocytes and

oligodendrocytes. We did not find any unipotent or bi-potent spheres. Differentiated

neurospheres from H9 and CA1 cells respectively, produced similar proportions of

neurons and glia (Fig. 4h). Q-PCR analysis of differentiated spheres shows expression of

neural markers only, with no induction of extra-neural differentiation resulting from

adherent culture in FCS (Fig. 4j)

In order to assess long-term self renewal, primary neurospheres were passaged at

clonal density each month. Neurospheres passaged clonally for 11 months (Fig. 4k,l)

indicating the continued generation of NSCs in addition to progenitors that do not self

renew beyond 3 or 4 passages (Seaburg et al., 2002). Moreover, there was a 20-fold

expansion of the NSC population at each subsequent passage, suggesting a large increase

of NSCs with continued culture. In addition, Q-PCR of passaged neurospheres revealed

that neural gene expression remained stable over time (Fig. 4m), suggesting the sustained

propagation of pure populations of NSCs and their progenitors. To further investigate the

commitment of hESC derived NSCs to the neural lineage, we plated late passage

neurospheres on MEFs to see if they could generate ES-like colonies, or differentiate to

endodermal or mesodermal cell types. This is a colony selection method that is used in

the derivation of iPS cells to identify clones that have been successfully reprogrammed

(Takahashi and Yamanaka, 2006; Takahashi et al., 2007). After 7 days of cultivation in

hESC maintenance conditions, cells were collected and analyzed by Q-PCR (Fig. 4n).

This analysis revealed that extra-neural gene expression was not initiated, further

suggesting that hESC derived NSCs are intractable to differentiation to other cell types.

35

Figure 4i

36

Figure 4ii

37

Figure 4iii

38

Figure 4iv

39

Figure 4i-iv Clonal neurospheres grow in defined conditions over 4 weeks, express

only neural markers, differentiate into neurons and glia and passage clonally long-

term. (a) A floating clonal neurosphere at 28 d, grown from a single hESC. (b) A

comparison of hESC versus primary neurosphere gene expression by QPCR revealed that

clonal NSC colonies have an entirely neural phenotype: they express neural markers

(NES, SOX1, PAX6, TUBB3) but not pluripotency genes (OCT4, LIN28) endodermal

genes (FOXA2, HNF4A, GATA4) or mesodermal genes (GATA1, Brachyury).

(c,d,e,f,g) Differentiation potential was assessed by plating single neurospheres on

matrigel-coated plates in SFM with 1% fetal calf serum (FCS) for 14 days.

Differentiated neurospheres produced (c) TUBB3+ and (g) MAP2+ neurons (shown here

growing out of a whole sphere), (e) GFAP+ astrocytes and (f) O4+ oligodendrocytes,

while many cells still expressed the neural precursor marker (d) NES. All nuclei were

counterstained with dapi (blue). (h) Quantification of protein expression by ICC showed

that differentiated CA1 and H9 neurospheres produced similar proportions of

differentiated cells, so data was pooled (2-way ANOVA showed no significant main

effect of cell type (p=0.71)). Differentiated cell types consisted of neural precursors

(45.9% ± 6.9% NES+), neurons (37.9% ± 8.3% TUBB3+, and 15.2% ± 4.2% MAP2 +),

astrocytes (34.7% ± 4.2% GFAP+), and oligodendrocytes (5.8% ± 1.9% O4+).

(i) Overlapping expression of GFAP and NES accounted for marker quantification being

>100%. Cells were double-labeled with GFAP (red) and NES (green) to reveal that a

population of the differentiated cells were producing both proteins (yellow), and that this

overlapping expression accounts for combined marker expression in (h) being >100%.

40

(j) Q-PCR of gene expression of differentiated late passage neurospheres confirms that

differentiation only produces neural cell types. (k,l,m) Neurospheres passaged clonally

long-term and stably expressed neural markers. (k) Primary CA1 neurospheres (passage

0) were dissociated and plated as single cells at clonal densities in SFM + FGF each

month for 11 months. (l) Floating 11th passage neurospheres formed from single

dissociated 10th passage neurosphere cells. (m) Comparison of gene expression in

passaged CA1 neurospheres versus hESCs. Passaged neurospheres maintain stable

expression of neural markers. (n) Late passage (p5-7) CA1 spheres plated on MEFs do

not re-initiate expression of endodermal, mesodermal or pluripotency genes. Scale bars

a,k=100µm, c,d,e,f,i=50µm. g= 200µm.

41

3.2 Neural stem cell colonies can be clonally derived from hESCs

To be sure that neurospheres were forming clonally from a single cell instead of

by multicellular aggregation, mixing experiments were performed with GFP+ and GFP-

CA1 cells. Equal numbers of GFP+ and GFP- cells were plated at final densities of 5,

10, 20, 50 and 100 cells/µl, and the resulting neurospheres were scored for chimerism.

The same experiment was also performed with RFP+ and RFP- H9 cells. Clonal density

was established as that which yielded only spheres of one color. Only all clear spheres

were quantified, as there was a difficulty in ascertaining the presence of clear cells in red

or green colonies. For this reason, we also performed these mixing experiments using

RFP+ H9, and GFP+CA1 cells, as chimerism was more easily discernable in all

neurospheres. Regardless of the cell combinations used, at 5 and 10 cells/µl, only

unmixed spheres were found, indicating a clonal origin (Fig. 5a,b,d), while at densities of

20-100 cells/µl, some spheres of mixed colors formed, indicating that these colonies had

arisen by aggregation (Fig. 5c,d).

When single hESCs were plated at 10cells/µl, 0.14% of the starting single cell

population formed neurospheres. To further establish that neurospheres can arise from a

single cell, and to investigate the frequency at which this occurs, we plated single H9-

RFP cells in microwells and checked for sphere formation at 2 and 4 weeks. After

plating, all wells were assessed for the presence of a single cell, and only wells

containing single cells were tracked for colony growth. In total, 5 colonies formed from

3,881 single cells in three separate experiments, representing 0.11% of the starting

population. This frequency is similar to that of neurosphere formation when hESCs are

plated at 10cells/µl (0.14%), reflecting the clonal density established by our mixing

42

experiment (Fig. 5e). Microwell derived colonies did not survive beyond 2 weeks and

indeed were smaller in size than spheres grown at densities of 10cells/µl, indicating that

factors supporting the long-term propagation of neurospheres are excreted by

neurospheres themselves.

43

Figure 5

44

Fig. 5 Neurospheres grow from single cells rather than multicellular aggregates

when cultured at densities of 10c/µl or less.

(a,b,c,d) When equal numbers of RFP and GFP hESCs were plated together, spheres of

one color (a,b) that indicate a clonal origin, only arose at 10 cells/µl and 5 cells/µl (d).

Some chimeric spheres (c) arose at densities of 20 cells/µl and 50 cells/µl (d), indicating

that these colonies had formed by the aggregation of 2 or more cells. (e) Single cell per

well experiments confirmed that roughly 1 in 900 cells was capable of forming a clonal

neural colony in serum free conditions. A comparison of sphere formation from single H9

cells/well versus 10 cells/µl (3000 cells/well) showed that the frequency of sphere

formation of isolated single cells was equivalent to when cells were plated at 10c/µl

(t(4)=1.22, p=0.32). Scale bars a,b,c=100µm

45

3.3 hESC derived neural stem cells are FGF dependent, and

frequency of neurosphere formation is improved to 1.6% by cultivation

with ROCK inhibitor.

NSCs derived from embryonic and adult cells are dependent on different growth

factors for their propagation in vitro. As previously described, mESC derived primary

neurospheres originate from pNSCs that rely on LIF to mediate self renewal, where

passaging of these neurospheres yields dNSCs dependent on FGF (Smukler et al., 2006;

Tropepe et al., 2001; Hitoshi et al., 2004). Late embryonic and adult mouse forebrain

ventricular tissue contains NSCs that require FGF or EGF to form primary and secondary

colonies (Tropepe et al., 1999). To ascertain whether there is a specific growth factor

dependency of the hESCs that survive to proliferate as NSCs, hESCs were plated at

clonal densities in wells containing LIF, FGF, EGF, and the culture supplement B27

either singly or in combination. At low cell densities, hESCs were dependent on FGF,

but not LIF, EGF or B27 (Fig. 6a).

Cell survival and proliferation is extremely limited in the minimal culture

conditions provided by SFM. We assessed cell survival by plating 50 cells per well and

then scoring live cells by trypan blue exclusion at various time points. When 50 cells/well

were plated in SFM alone, 69.7±7.9, 38.6±4.9, and 15.5±3.3 % of cells survived to 4h,

24h, and 3d, respectively (doublets and aggregates were not scored). The addition of FGF

and B27 improved survival to 87.92±2.8, 65.8±5.8, and 33.5±8.3% at 4h, 24h, and 3d

(Fig. 6b). It has been shown that addition of rho-associated kinase (ROCK) inhibitor Y-

27632 diminishes the dissociation-induced apoptosis that challenges hESC survival after

dissociation to single cells (Watanabe et al., 2007). Survival of hESCs in SFM with FGF

46

and B27 can be further improved by the addition of ROCK inhibitor (Fig. 6c) such that

the frequency of sphere generation is increased by 11-fold to 47.1±9.7 spheres per 3000

cells (1.6% of the starting hESC population).

47

Figure 6

48

Fig. 6 Primary neurosphere formation is dependent on FGF, and addition of

growth factors and ROCK inhibitor improves survival and frequency of

neurosphere formation.

(a) A comparison of culture conditions with addition of different growth factors reveals

that the ability of single hESCs to form neurospheres was reliant on the addition of FGF.

One-way ANOVA showed a significant main effect of growth factors (F(7,31)=56.46,

p<0.0001), while Bonferroni’s multiple comparison tests showing a significant difference

between FGF and LIF (p<0.05) and FGF+B27 compared to LIF+B27 (p<0.05)

illustrating that neurospheres are FGF-dependent, and not LIF-dependent. (b) Single cell

survival in SFM vs. SFM with growth factors represents the survivors as a percentage of

starting cell populations. 2-way ANOVA showed that the survival of single cells in both

conditions is diminished over time (F(2,30)=42.40, p<0.0001) and that the addition of

growth factors to single cells in SFM improves survival (F(1,30)=19.23, p=0.0001). (c)

Addition of ROCK inhibitor boosts the frequency of neurosphere generation 12x from

0.14% to 1.6%.

49

3.4 Unlike mESCs, neural specification of hESCs does not occur in

minimal culture conditions, but in maintenance culture

As previously described, the first neurally specified cells in vertebrates arise

during gastrulation from the ectoderm (Spemann and Mangold, 1924) through a process

of release from the inhibition of local BMPs (Hemmati-Brivanlou and Melton, 1994).

BMPs are members of the TGFB superfamily of molecules that are strong inhibitors of

neural differentiation (Wilson and Hemmati-Brivanlou, 1995). Noggin, chordin and

follistatin secreted from a specialized group of dorsal mesodermal cells (organizer/node)

bind BMPs extracellularly, preventing them from binding to their receptors on ectoderm

cells and promoting neuralization (Zimmerman et al., 1996; Piccolo et al., 1996; Fainsod

et al., 1997). The underlying assertion of this model is that in the absence of extrinsic

signalling, ectodermal cells (and even earlier embryonic cells) become neural. This

‘default’ mechanism of neural fate specification was elucidated by experiments in which

single cells from early cleavage and gastrulating embryos were cultured at low density in

serum free conditions, mimicking an environment free of extrinsic signalling. In these

conditions cells autonomously adopted a neural identity (Godsave and Slack, 1989;

Grunz and Tacke, 1989; Sato and Sargent, 1989). Low density culture in minimal,

serum-free conditions with mESCs yielded similar findings, as these cells adopted a

neural phenotype within 4 to 24 hours (Smukler et al, 2006; Tropepe et al., 2001). The

direct default to a neural identity was ascertained to be the first step in an mESC based

model describing the in vitro ontogeny of NSCs (Fig. 3) (Smukler et al., 2006).

Based on these studies, we hypothesized that single pluripotent hESCs would

directly transition to a neural identity in minimal conditions by a default mechanism. In

50

order to test this, we cultured dissociated single hESCs at 1 cell/µl in SFM with no

growth factors (neural default conditions), and collected the cells at various timepoints

for analysis by Q-PCR. By day 3, hESCs had begun to downregulate expression of

pluripotency and mesodermal genes, but expression levels of both endodermal and neural

genes were either upregulated or not significantly down regulated from hES levels

(Fig. 7a). Taking into account the longer cell cycle time of hESCs (analysis at day 3),

this was still a different outcome than the experiments with mESCs that showed

exclusively neural gene expression by 24 hours. This result made us question the

assumption of our original hypothesis that NSCs would transition directly from

pluripotent hESCs in culture— ie, is it really the pluripotent cells in hES colonies that

give rise to NSCs? To address this assumption, as well as the confounding effect of

using a mixed population of starting cells, we sorted cells from confluent hESC colonies

labeled with cell surface antigens TRA-1-60 and SSEA4. These markers are expressed

on pluripotent hESCs and become downregulated during differentiation (Henderson et

al., 2002). We then cultured all four fractions of TRA-1-60 and SSEA4 sorted cells in

the clonal neurosphere assay to determine which fraction(s) could give rise to NSC-

containing neurospheres. Surprisingly, the pluripotent (double positive) cells did not

form neurospheres. In fact it was the TRA-1-60–/SSEA4– fraction that contained the

sphere-originating cells (Fig. 7b). TRA-1-60–/SSEA4– cells produced clonal

neurospheres at a frequency of 0.7% (adjusted to control for loss of cells in FACS), and

in confluent colonies TRA-1-60–/SSEA4– cells represent only 6.1% of the mixed

population (Fig. 7c). This means that in 3000 cells plated, roughly 180 cells will be

TRA-1-60–/SSEA4–, and with a sphere formation frequency of 0.7%, should produce

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about 12 neurospheres. In fact, when unsorted cells were placed in the neurosphere

assay, the frequency of sphere production is 0.14% , or 4.1±0.5 spheres per 3000 cells,

indicating that even in mixed cultures, it may only be the single -/- cells that give rise to

spheres. These data suggest that neural fate specification is taking place in the hES

maintenance cultures, and cells primed for neural differentiation are those that survive to

proliferate into spheres.

To establish an expression profile of the neurosphere forming cell, gene

expression of TRA-1-60–/SSEA4– was compared to TRA-1-60+/SSEA4+ cells by Q-PCR.

This analysis revealed a down-regulation of pluripotency gene OCT4 along with an

upregulation in the neural genes SOX1 and PAX6 and sustained expression of

endodermal genes FOXA2, HNF4A, and GATA4 in the double negative fraction (Fig.

7d). We predict that the TRA-1-60–/SSEA4– starting population contains a mix of cells

of separate neural and endodermal identity. This is supported by studies on colony

morphology demonstrating that extraembryonic endoderm-like cells tend to ring the

outsides of hES colonies, while small clusters of neuroepithelial-like cells are contained

within (Peerani et al., 2007).

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Figure 7i

53

Figure 7ii

54

Fig. 7i-ii hESCs default to a mixed ectodermal and endodermal identity in SFM

alone. Neurosphere originating cells are TRA-1-60-/SSEA4-, make up 6.14% of cells

in confluent colonies, and are characterized by elevated levels of neural and

endodermal gene expression.

(a) A comparison of gene expression by Q-PCR of confluent hESCs in maintenance

conditions versus single hESCs at low density in SFM for 3 days shows that when hESCs

are free of extrinsic signals their phenotype shifts from pluripotent to mixed endodermal

and ectodermal. 2-way ANOVA revealed a significant interaction between gene

expression and time in SFM alone (F(10,54) =5.46, p<0.0001). Bonferroni posttests

showed a significant decline in the pluripotency gene LIN28(p<0.01), as well as the

mesoderm gene Brachyury(p<0.001), along with a significant increase in expression of

the neural gene SOX1 (p<0.001). (b) When single hESCs were sorted on TRA-1-60 and

SSEA4 expression and the resulting 4 fractions plated at clonal density in SFM + growth

factors, only cells from the double negative fraction could form neurospheres, indicating

that the neurosphere-originating cell in an unsorted population is a TRA-1-60–/SSEA4–

cell. One-way ANOVA showed a significant main effect of starting cell population

(F(6,34)=10.06, p<0.0001), while Bonferroni’s posttest revealed a significant difference in

the sphere forming capacity of TRA-1-60+/SSEA4+ cells vs. TRA-1-60–/SSEA4– cells

(p<0.05). (c) FACS shows that a minority of cells in confluent hES colonies are TRA-1-

60–/SSEA4– (6.14±1.35%), supporting the idea that neurospheres are arising solely from

this fraction. The majority of hESCs in confluent colonies are TRA-1-60+/SSEA4+

(65.78±3.23%), with the remaining 7.12±2.31% being TRA-1-60+/SSEA4–, and

21.24±3.84% being TRA-1-60–/SSEA4+. The FACS plot shown represents a single sort,

55

while data cited is based on an n of 7. (d) A comparison of mRNA expression by Q-PCR

in TRA-1-60–/SSEA4– versus TRA-1-60+/SSEA4+ cells showed significant differences in

pluripotency, neural and endodermal gene expression (significant interaction between

sorted fraction and gene expression by 2-way ANOVA: (F(10,58) =14.67, p<0.0001)).

TRA-1-60–/SSEA4– fraction, which contains the neurosphere forming cell, is

characterized by the significant upregulation of neural genes SOX1(p<0.001),

PAX6(p<0.001), and endodermal genes FOXA2(p<0.001), HNF4A(p<0.001), and

GATA4(p<0.001), and downregulation of OCT4(p<0.001) (all by Bonferroni posttests).

56

3.5 Noggin promotes neural fate specification only in hES

maintenance cultures.

Neural fate specification in the mammalian embryo relies on the inhibition of

BMPs by antagonists such as noggin (Munoz-Sanjuan and Brivanlou, 2002; Zimmerman

et al., 1996). Neuroectodermal differentiation of hESCs is improved by culture with

noggin, however this has been achieved by concomitant growth on transgenic MS5-Wnt1

stromal feeders that facilitated neural differentiation (Sonntag et al., 2007). In another

study, noggin treatment was administered to multicellular hESC aggregates that

proliferated in minimal conditions and were highly but not exclusively enriched for

neural progenitors (Itykson et al., 2005). As previously mentioned, neural default studies

suggest that noggin promotes neural fate specification by inhibiting paracrine signalling

in vivo (Zimmerman et al., 1996), while single cells grown at low cell densities will

acquire a neural fate autonomously (Godsave and Slack, 1989; Grunz and Tacke, 1989;

Sato and Sargent, 1989). This suggested that in order to use noggin to direct

differentiation in our clonal assay, it might be more effective to administer it to hESC

colonies in maintenance culture conditions rather than to single cells in neurosphere

forming conditions. We added noggin to hESC maintenance cultures each day for 4-7

days before colonies reached confluency. We then plated these noggin treated hESCs in

the clonal sphere-forming assay. Noggin treated hESCs produced significantly more

neurospheres than controls (Fig. 8), indicating that inhibition of BMPs facilitates the

process of neuralization of hESCs. To test the effects of noggin on single hESCs in low

density minimal conditions (SFM + growth factors), we grew hESC colonies to

confluency in the absence of noggin, and then added noggin to our neurosphere culture.

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In this case, there was no increase in the number of spheres produced (Fig. 8), indicating

that inhibition of BMPs by noggin mediates the hESC to NSC transition by inhibiting

paracrine signalling in high density ES maintenance cultures, rather than inhibiting

autocrine signalling in low-density, minimal culture conditions.

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Figure 8

59

Fig. 8 Noggin treatment of hESC colonies enhances the generation of NSCs but

administration of noggin to low density neurosphere culture has no effect.

When noggin was administered as a “pre-treatment” to hESCs grown to confluency in

maintenance conditions, these cells went on to produce significantly more neurospheres

than when noggin was added directly to single hESCs in neurosphere forming minimal

conditions (“Noggin in SFM”). Data is represented as a fold of control (neurosphere

formation from single cells grown in SFM + growth factors in the absence of noggin).

2-way ANOVA shows a main effect of treatment (F(1,8)=7.17, p=0.03). This result

illustrates that noggin-mediated neuralization takes place when cells are grown at high

densities, rather than at low (clonal) densities where paracrine signalling is minimized.

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3.6 Alternate culture conditions enable generation of NSCs with

different ground states: culture of hESCs with ROCK inhibitor gives

rise to an early lineage neural precursor.

There is evidence that cells possessing an ability to revert back to a pluripotent

phenotype can be found in early mESC derived NSC colonies. The neural precursors

(pNSCs) contained in primary neurospheres derived from mESCs retain OCT4

expression, can form multi-germ layer EBs and contribute to embryonic tissues when

aggregated with morula cells (Tropepe et al., 2001; Akamatsu et al., 2009). As

previously described, these pNSCs are LIF-dependent and FGF-independent. When

these colonies are dissociated and re-plated, they give rise to dNSCs that are FGF-

dependent and LIF independent. dNSCs do not express OCT4, and can not form other

embryonic tissues when grown in serum, or aggregated with ICM cells. As hESC-

derived primary neurospheres did not sustain expression of OCT4, we predicted that the

NSCs contained within were fully committed to the neural lineage, resembling dNSCs.

We chose to explore the extent of lineage commitment of primary hESC derived

neurospheres by testing their ability to form multi-germ layer EBs, and ES-like colonies

when plated on MEFs. We also chose to investigate this issue with primary neurospheres

derived with ROCK inhibitor (to be designated hereafter as “ROCK spheres”) as these

colonies differed from ‘standard culture’ (SFM + FGF) neurospheres by their sustained

expression of LIN28 (Fig 9a, Fig 4b). As previously mentioned, the frequency of ROCK

sphere generation also differed significantly from standard culture spheres (Fig. 6c). For

EB formation, neurospheres were disaggregated, then re-aggregated either in hanging

drops, or by centrifugation, and then cultured in serum. Regardless of the method used,

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cells from ROCK and standard neurospheres would not aggregate to form EBs. This

could be a consequence of changes in the expression of cell adhesion molecules such as

E-cadherin that are downregulated during differentiation (Karpowicz et al., 2009),

possibly making neurosphere cells intractable to aggregation as EBs. Next we plated

standard and ROCK neurospheres onto MEFs and cultured them in hESC maintenance

conditions for 7 days to see if the neural precursors could form ES-like colonies.

Morphological analysis showed that standard primary neurospheres were not able to form

ES-like colonies when plated on MEFs. However, ES-like colonies did form from

ROCK spheres. Q-PCR analysis of these ES-like colonies showed that multi-germ layer

expression was re-initiated (Fig 9a). Most interestingly OCT4 expression is dramatically

upregulated in ES-like colonies grown from ROCK neurospheres. These data indicate

that neurosphere culture with ROCK inhibitor allows for the isolation or generation of an

early neural precursor akin to a pNSC. This result also indicates that culture of the same

starting cell population of hESCs can produce NSCs with differing ground states.

We also tested the effects of BMP inhibitor noggin to facilitate neuralization of

hESCs in the presence of ROCK inhibitor. As we previously found that noggin mediated

BMP inhibition is most effective in high density cultures with active paracrine signalling,

we pre-treated hESCs with noggin for 6-7 days before plating cells at clonal density in

SFM + FGF + ROCK inhibitor. In contrast to standard neurosphere cultivation, there

was no significant increase in the number of NSCs compared to controls (Fig. 9b). It is

possible that increased cell survival caused by culture with ROCK inhibitor sufficiently

altered the minimal serum-free neurosphere culture environment so as to create a

paracrine signalling environment. In light of our finding (discussed earlier) that noggin

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acts by blocking BMP signalling in a paracrine fashion, we could perhaps have improved

the output of NSCs had we added noggin directly to neurosphere culture in addition to

pre-treatment of the starting hESC population.

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Figure 9

64

Fig. 9 hESC derived NSCs sustain expression of LIN28, and form ES-like colonies

that re-initiate OCT4 expression when plated on MEFs. (a) A comparison of gene

expression by Q-PCR of hESCs, primary ROCK spheres and ROCK spheres plated on

MEFs. It is interesting to note the similarity of gene expression in ROCK primaries and

standard neurospheres, with the exception of sustained LIN28 expression in ROCK

spheres (compare with Fig. 4b). When ROCK primaries are plated on MEFs, their

transcriptional profile becomes ES-like. A significant interaction between culture

environment and gene expression was revealed by 2-way ANOVA (F(20,66)=25.19,

p<0.0001). Bonferroni posttest comparison of ROCK primaries vs. ROCK spheres on

MEFs shows significant OCT4 upregulation (p<0.001), as well as significant

upregulation of endodermal and mesodermal genes whose expression had been shut off in

primary ROCK spheres (FOXA2, HNF4A, GATA4, T, p<0.001). These data suggest

that an early neural precursor with pluripotent ability can be obtained from hESCs by

culture with ROCK inhibitor. (b) Comparison of ROCK sphere formation using noggin

pre-treated and untreated hESCs. As opposed to standard neurosphere culture, noggin

pre-treatment of hESCs does not increase neural progenitor numbers in neurosphere

culture with ROCK inhibitor.

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3.7 Discussion

This body of work reports the development of an assay that allows for the

generation of clonal NSCs from single hESCs in completely defined conditions. In the

absence of serum, feeder layers, or EB formation, single hESCs differentiate into colony

forming NSCs that can be propagated and expanded long-term, and produce progenitor

cells that differentiate into neurons and glia. The importance of this work lies in the

clonality of the method. The ability to select and expand single hESC-derived NSCs is

crucial for a variety of reasons. hESC colonies are heterogeneous, containing cells with

differing developmental potential. Non-specific and bulk selection of neuroepithelial

rosettes from hESC cultures, and or uncontrolled differentiation to neural subtypes

through an EB stage has produced mixed populations of cells, some of which retain

unsuitable capabilities such as teratoma formation in graft recipients (Brederlau et al.,

2006). Commitment to the neural lineage must be stable and complete before cells are

expanded for the purpose of transplantation, and this process relies on the ability to select

and expand single cells. Single cell selection and propagation will also enable exclusion

of cells with various chromosomal abnormalities including trisomies of chromosome 17q

and 12 that have been reported in several hESC lines (Draper et al., 2004). Also, a

variety of hESC lines have one or more genomic alterations commonly observed in

human cancers including aberrations in gene promoter methylation (Maitra et al., 2005).

The propensity of hESCs maintained in vitro to undergo genetic and epigenetic

alterations, as well as the standard heterogeneity of colonies makes clonal selection of

untransformed and specified cells a necessary step in the differentiation and expansion of

hESCs to transplantable cell types. Clonal propagation as enabled by our method also

66

addresses safety concerns regarding fetal NSC transplants, currently being challenged as

a viable cell therapy. A recent report cited donor-derived multiple tumor formation

following fetal NSC transplant in a recipient with ataxia telangiectasia (Amariglio et al.,

2009). When combined with screening techniques such as live cell sorting using surface

markers of transformation like CD30 (Herszfeld et al., 2005), our method could mitigate

the dangers of this therapeutic approach.

The current study defines the clonal neurosphere-forming cell as a TRA-1-60-

/SSEA4- cell that grows within hESC colonies, and reveals that BMP inhibition with

noggin mediates the acquisition of this identity. It is possible that pluripotent hESCs

could form neurospheres directly, but that the cell death associated with dissociation of

pluripotent cells to single cells prevents this. Cloning of single pluripotent hESCs is

notoriously difficult, but perhaps this has less to do with the sensitivity of single cells

than the culture requirements of colony-forming cells. Initial studies on the dependence

of undifferentiated culture of hESCs on bFGF examined heterogeneous populations of

cells for the presence of FGF receptors, but assumed it was the undifferentiated fraction

that possessed them (Xu, et al., 2005; Xu, C., et al., 2005). It has since been shown that

all known FGF receptors are expressed on the sub-fraction of hESCs that lack OCT4

expression and are incapable of forming pluripotent colonies. A signalling loop is

established between differentiated cells that receive FGF and in turn secrete IGF for

which pluripotent, clonogenic hESCs have receptors (Bendall et al., 2007). We propose

that in the minimal conditions we use to generate clonal neurospheres, cell densities are

so low that paracrine signalling ceases between differentiated cells and pluripotent cells,

such that there is not enough IGF for the pluripotent cells to survive. Being FGF-

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dependent, the TRA-1-60-/SSEA4- fraction is sustained by growth factor

supplementation, and proliferates to form clonal NSC colonies. Interestingly, it has been

shown that a population of SSEA4+/CD133+ cells derived from human embryonic

forebrain is enriched for neurosphere initiating cells (Barraud et al., 2006). However,

neurospheres derived from these fetal cells do not generate oligodendrocytes upon

differentiation, suggesting that this population of cells contains a more restricted neural

progenitor than SSEA4- hESC derived NSCs. This may seem counterintuitive but it has

been recently shown that among mouse ES cells heterogeneous for pluripotency and

germ cell marker Stella, the Stella negative cells can give rise to Stella positive cells

(Hayashi et al., 2008).

Neural fate specification in mESCs takes place in the absence of extrinsic signals,

paralleling a model of BMP inhibition in vivo (Smukler et al., 2006; Tropepe et al.,

2001). Although studies using hESCs have supported the concept of neural

differentiation by default, the analyses were not clonal (Vallier et al., 2004). In our

clonal neural default assay, overwhelming cell death and the substantial heterogeneity of

starting cell populations confound this experimental approach. However, our finding that

pluripotent hESCs do not become entirely neural when grown in serum-free conditions in

the absence of growth factors does not rule out a neural default program. hESCs could

still be adopting a neural phenotype via a default mechanism, but this happens in high

density hESC maintenance conditions, because of the heterogeneity of cells in these

colonies. This heterogeneity creates local signalling microenvironments (akin to the

regional inhibitory environment that leads to neural specification in the embryo) that can

influence cell fate along extra-embryonic endodermal or neuroectodermal lineages

68

(Peerani et al., 2007). Indeed, analysis of the behaviour of sorted cells in neural default

conditions combined with morphological analyses of hES colonies suggests that the

TRA-1-60-/SSEA4- fraction of cells is also heterogeneous, containing both endodermal

and neural specified cells. However, it is only neurally specified cells that survive and

proliferate to form colonies in serum-free media, suggesting that the neural cells of the -/-

fraction have a survival advantage. These minimal conditions may also select for dNSCs

rather than the pNSCs that are selected from mESCs in neural default conditions

(Smukler et at., 2006) because first, the human cells have been specified previously in

maintenance hES culture conditions and second, only differentiated FGF-dependent

hNSCs cells survive in minimal neural culture conditions. The lack of dependence of

hESCs and their early progeny on exogenous LIF means that it is impossible to use LIF

in minimal culture conditions to isolate a pNSC from hESCs as is done in mouse ES

cultures. However, the addition of ROCK inhibitor promotes the survival or generation

of a pNSC-like neural progenitor that displays residual plasticity. The plasticity of ROCK

spheres could be a direct or indirect result of cell survival. The pNSC may be sensitive to

dissociation-induced caspase-mediated apoptosis in which case ROCK inhibitor promotes

its survival. Otherwise increased cell survival could indirectly create an alternate

signalling microenvironment in which paracrine signalling is no longer inhibited at

10cells/ul. This scenario could either support the survival of the pNSC, or cause the

generation of the pNSC itself.

The question of the developmental equivalence of hESCs could be addressed by

assessing the type of NSC that one is able to derive from them. If hESCs are truly more

like later stage EpiSCs than blastocyst stage cells one may predict that the neural

69

precursor isolated from them may be of a later stage, like a dNSC. Our results from

culture with ROCK inhibitor illustrate the shortcomings of this approach. The fact that

we are able to isolate pNSC-like AND dNSC-like neural progenitors from the same

starting cells using different culture conditions calls into question the reliability of

assigning labels of in vivo equivalence to a variety of pluripotent cells cultured on

different platforms. In attempting to define the culture requirements of certain

populations of cells, one may simply be selecting for growth responsiveness to some

conditions while favoring the survival of a subpopulation of heterogeneous starting cells

with distinct capabilities. While understanding the initial developmental status of

different pluripotent cell lines is important for defining starting conditions when

differentiating cells for therapeutic uses, culture conditions themselves can be a greater

determinant of cell fate and plasticity than stage of isolation. A recent study (Chou et al.,

2008) found that cell lines from mouse blastocysts could be isolated using mEpiSC/hESC

conditions (FGF and activin). These ‘FAB’ stem cells stably express OCT4, SOX2,

nanog, and SSEA1 for over 30 passages, however they are unable to make teratomas or

chimeras. Interestingly, treatment with LIF and BMP for one week induces FAB stem

cell developmental potential, and the ability to make teratomas and chimeras is sustained

even when cells are returned to their original culture environment. Another recent study

illustrates that the pathways used to sustain undifferentiated ES cells depend on the

culture milieu rather than the innate requirements of ES cells themselves, so that self-

renewal requirements vary depending on culture conditions (Ying et al., 2008). The

finding that hESCs autologously derive their own feeder cells in maintenance conditions

70

(Bendall et al., 2007) is further evidence that in vitro culture can create unique

microenvironments that may bear little relation to in vivo developmental states.

Certainly it is possible that embryo-derived pluripotent cell lines become

transformed in culture, and are tissue culture artifacts. It is also possible that these early

lineage cells are more easily reprogrammed. It has been suggested that in vitro culture of

mGSCs reprograms a minority of these cells into pluripotent stem cells (Thomson et al.,

1998). A recent report showed that NSCs from adult mouse are more easily

reprogrammed to ES cells, requiring only one (OCT4) of the Yamanaka factors (Kim et

al., 2009). Perhaps pNSCs are also more amenable to reprogramming, in this case by

alternate culture milieu. When OCT4 is shut off in vivo in the mouse (E8), this signifies

an end of an ability to derive pluripotent cell lines (Vallier et al., 2005). It could

therefore be suggested that the re-initiation of OCT4 expression when ROCK spheres are

plated back down on MEFs signifies a reprogramming event. For clinical utility, it will

be important to establish at what stage during long-term passage (if at all) the ability to

reprogram, or the residual plasticity of ROCK spheres is lost.

Understanding the clonal derivation of stable and non-plastic neural progenitors

has applications beyond hESC therapies. The creation of hiPS cells from adult human

fibroblasts has prompted a rush of efforts to direct differentiation of these potentially

patient specific cells to functional, transplantable types (Wernig et al., 2008; Abeliovich

and Doege, 2009). So far, hESC biology provides the best allowable approximation to

human development. Lessons from ES studies will continue to inform the mode and

manner in which iPS cells adopt a pluripotent phenotype and how they can be made to

differentiate away from it. It has been proposed that differentiating hESCs into later cell

71

types will require sequentially mimicking early axis formation and then germ layer

development before specialized cells can be obtained (Rossant, 2008). However, the

cloning of Dolly by somatic cell nuclear transfer, and the generation of iPS cells

demonstrates the proof of principle that the identity of a cell can be directly changed

without sequentially following or reverting the process of development. Perhaps most

exciting is the notion that cellular reprogramming to specific cell types may be achieved

directly through culture in appropriate conditions, without genomic insertion and/or

forced overexpression of pluripotency associated genes. Just like knowledge of gene

expression of pluripotent cells was exploited by Yamanaka to discover candidates whose

expression was capable of reprogramming somatic cells, understanding of growth factor

dependencies and critical signalling pathways as they relate to various pluripotent states

could be similarly utilized to discover the means to achieve cellular reprogramming by

induction with signalling molecules rather than exogenous gene expression.

3.8 Conclusion

This thesis reports the development of a one-step clonal method where single H9

and CA1 hESCs proliferate in defined, serum-free conditions to form floating

neurosphere colonies that are comprised of NSCs and their progenitors. Q-PCR analysis

of these neurospheres shows expression of neural precursor markers, but not expression

of early endodermal and mesodermal markers, indicating that these colonies are entirely

neural in character. Neurosphere colonies generated from single NSCs could be

differentiated to form TUBB3 and MAP2 positive neurons, GFAP-positive astrocytes and

O4-positive oligodendrocytes by immunocytochemical staining and Q-PCR, illustrating

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multipotentiality. Neurospheres also passaged long term (11 months) to form clonal

descendent spheres, exhibiting self renewal and thereby confirming the presence of NSCs

in addition to progenitors that may also display multipotentiality but do not passage

repeatedly. Expansion of NSCs by passage was robust and therefore easily scaleable for

clinical needs. Interestingly, hESCs become primed for the acquisition of a neural

identity in maintenance conditions, unlike mESCs that transition directly to a neural

identity by default when placed in minimal culture conditions that minimize TGFB

signalling (Smukler et al., 2006; Tropepe et al., 2001). Nevertheless, the transition from

hESC to NSC is negatively regulated by BMP signalling which, when inhibited by

addition of noggin to maintenance cultures, leads to an increase in NSC numbers.

Finally, with the addition of ROCK inhibitor to clonal cultures, we show that

growth factor environment can alter the ground state of our neurosphere cells such that

we are able to isolate an early lineage neural precursor that possesses the ability to revert

back to a pluripotent phenotype.

3.9 Future directions

Neural default

The heterogeneity of hESC colonies represents a challenge to single cell assay

systems using unsorted cell populations. To be able to definitively test the default

identity of hESCs, homogeneous fractions of hESCs need to first be sorted from hESC

cultures. Then these cells can be placed in short term conversion assays where extrinsic

signalling is blocked by serum free and growth factor free culture combined with

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extremely low cell densities. Ideally, single cell Q-PCR would be utilized to assess the

changing fate of single cells over a 3 day period, during which cell death in minimal

conditions could be mitigated by the use of ROCK inhibitor (provided it be found to be

solely survival enhancing rather than inductive). One challenge to this approach is the

tendency of hESCs to autologously give rise to feeder cells. In my current study, the

heterogeneity of starting cell cultures made the elucidation of the default identity of

hESCs impossible. Perhaps fate mapping of single pluripotent hESCs could provide

further insight onto the possibility that these cells are able to autologously generate feeder

cells in neural default conditions, in addition to the clarification of the default identity of

hESCs. Once a reliable system has been established that included the use of bona-fide

pluripotent hESCs, the mechanisms of neural fate specification in this mammalian system

could be reliably investigated.

Clinical use of hESC-derived NSCs and mechanisms governing

lineage progression.

In the interest of more definitively ascertaining the safety of our hESC-derived

NSCs for cell based therapies, it would be useful to characterize the in vivo

differentiation potential of ROCK and standard neurospheres by injecting these cells

subcutaneously into mice to determine if they will form teratocarcinomas. The prediction

would be that standard neurospheres would not form teratocarcinomas, where primary

ROCK spheres would. The robust generation of NSCs achieved with ROCK inhibitor

would be preferential for clinical applications, thus, it would be useful to passage ROCK

spheres and assess gene expression at each subsequent passage to determine if and when

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gene expression of these colonies matched standard neurosphere expression. Only when

ROCK spheres display the same gene expression profile and inability to revert to a

pluripotent phenotype will they be safe for cell based therapies. Here the prediction

would be that when LIN28 expression is shut off, ROCK spheres would no longer

possess the ability to revert to a pluripotent phenotype. The caveat here is that while

teratocarcinoma formation may be avoided by use of fully committed NSCs, solid tumors

containing neural cells (but not EC-like cells) may still form.

LIN28 blocking experiments could also be performed to investigate the possibility

that sustained LIN28 expression is the means by which ROCK spheres maintain their

plasticity, perhaps by preventing lineage progression from pNSCs to dNSCs. A recent

study reported that LIN28 functions to block micro-RNA mediated differentiation in stem

cells (Viswanathan et al., 2008). Using short hairpin RNAs and siRNAs to knockdown

LIN28 in neurosphere culture with ROCK inhibitor, we could investigate whether the

maturation of primary to mature microRNAs that is inhibited by LIN28 is the mechanism

by which pNSCs progress to a dNSC identity. Here the prediction would be that hESCs

plated in neurosphere forming conditions with ROCK inhibitor and LIN28 knockdown

would yield fully committed dNSCs lacking plasticity.

Finally, it would be interesting to further investigate the identity of the

neurosphere forming cell in relation to culture with ROCK inhibitor. To this end, hESCs

could be sorted based on TRA-1-60 and SSEA4 expression, and then the resulting four

fractions plated in the neurosphere assay with ROCK inhibitor. This could shed light on

the issue of the neurosphere initiating cell in these conditions, as it may be that ROCK

75

inhibitor is promoting the survival of an earlier lineage cell (akin to a pNSC) that is not

found within the TRA-1-60-/SSEA4- fraction.

hIPS cells

It will be beneficial to apply the culture techniques and insights from the present

study to the generation of hiPS cell derived NSCs. Using our clonal method and

characterization data as a benchmark for successful differentiation of NSCs from

pluripotent cells it will be possible to gauge the capability of hiPS cells to form clonal

neurospheres that can be assessed for multilineage potential by differentiation. If it were

possible to optimize the neural default assay in hESCs, it would be particularly

interesting to assess whether iPS cells obtain a neural identity through a default

mechanism, or if directed differentiation would need to be achieved by other neural

inducing factors or culture conditions. If bona fide NSCs could be obtained from hiPS

cells using the method outlined in this study, it would be a fruitful step toward obtaining

autologously derived neurons and glia for treatment of diseased or damaged neural tissue.

76

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