The derivation, propagation, storage and gene expression of human embryonic stem cells on human feeders

The tremendous versatility of embryonic stem cells versus the unprecedentedreports describing adult stem cell plasticity have ignited debates as to the choice of onecell type over the ot

GENERAL INTRODUCTION

Several types of stem cells have been discovered from germ cells, the embryo, fetus andadult Each of these has promised to revolutionize the future of regenerative medicinethrough the provision of cell replacement therapies to treat a variety of debilitatingdiseases The tremendous versatility of embryonic stem cells versus the unprecedentedreports describing adult stem cell plasticity have ignited debates as to the choice of onecell type over the other for future applications However, the biology of these mysteriouscells have yet to be understood through a lot more basic research before new therapiesusing stem cell differentiated derivatives can be applied

Everyday, we read and listen to news reports about how stem cells promise torevolutionize medicine and change our lives with panaceas for every imaginable diseaseincluding rhetoric that stem cell therapy will some day delay the process of ageing.Embroiled in the hype and media frenzy are also political agendas, numerous religiousand genuine ethical concerns To further fuel the debate, embryonic stem cell research isoften unjustly associated with reproductive cloning Stem cell research is politicallycharged, receives considerable media coverage, raises many ethical and religious debatesand generates a great deal of public interest

Stem cell research also opens the new field of ‘cell based therapies’ and as suchseveral safety measures have also to be evaluated The hope that someday manydebilitating human diseases may be treated with stem cell therapy is inspired byremarkable examples of whole organ and limb regeneration in animals as well as thehistorical success of bone marrow transplants that have improved the lives of manypatients suffering from leukemia, immunological and other blood disorders Clearly,stem cell research leading to prospective therapies in reparative medicine has thepotential to affect the lives of millions of people around the world for the better and there

is good reason to be optimistic However, the road towards the development of aneffective cell-based therapy for widespread use is long and involves overcomingnumerous technical, legislative, ethical and safety issues

Embryos of most mammals are comprised of a special group of cells that havethe potential to give rise to all the tissues and organs of the fetus and future adult Thisgroup of cells called the inner cell mass (ICM) cells evolves into embryonic stem cells(ESCs) in vitro Unlike other cell types that can only divide a maximum of 50 times or so

in tissue culture dishes (Hayflick & Moorhead 1961), ESCs can divide indefinitelywithout losing their ability to form different cell types Human embryonic stem cells(hESCs) derived from isolation and serial sub-culture of ICMs from 5-day old humanblastocysts hold the promise of revolutionizing the future of medicine by the creation ofearly developmental models for a multitude of human genetic diseases and through thedevelopment of cell and tissue replacement therapies Immense commercial interest aswell as ethical controversy surrounds hESC research

Several improvements in blastocyst culture techniques were a prerequisite forculturing and harvesting good quality blastocysts with large ICMs These breakthroughsnot only led to increased pregnancy rates with blastocyst transfer in patients undergoing

in vitro fertlilization (IVF) cycles but also enabled scientists to derive hESC cell linesfrom human blastocysts hESCs cells were first isolated in 1994 (Bongso et al 1994)while the first continuous immortal hESC lines were established only in 1998 (Thomson

et al 1998)

hESCs are colony forming social cells that are unspecialized This means that ifthey are coaxed properly, ESCs have in theory the ability to turn into any of the celltypes in the human body In contrast, adult stem cells, which are found in adult tissuesand organs, have the ability to transform into only a limited variety of cell types Adult

stem cells are also difficult to isolate and very challenging to grow in culture Thiscoupled with their restricted developmental potential are the main reasons why manyscientists believe that embryonic stem cells are more promising and better alternativesfor developing a wider range of cell based therapies In order for hESCs to retain theirability to form different cell types, they need to be grown on feeder cell supports Thefeeder layer produces growth factors and extracellular matrix components that may help

to keep the hESCs from differentiating into other specialized cells Without the support

of a feeder layer, hESCs spontaneously and uncontrollably differentiate into a milieu ofmixed cell types This is often undesirable for the researcher as specific cell types areoften difficult or near impossible to isolate from this mixed milieu

Embryonic stem cells are a unique class of cell type for various reasons Mostsignificantly, they can undergo self-renewal for extended periods of in vitro cultivation,have the ability to form teratomas when injected into severely combinedimmunodeficient (SCID) mice and can differentiate into a variety of cell types from all 3primitive germ layers in vitro and in vivo, thus distinguishing them from other adult stemcells

hESCs differ in many ways from mouse embryonic stem cells (mESCs) Severallines of evidence suggest that hESCs and mESCs do not represent equivalent embryonic

cell types In vitro differentiation of hESCs leads to the expression of AFP and HCG,

which are typically produced by trophoblast cells in the developing human embryo,while mESCs are generally believed not to differentiate along this lineage In addition,hESCs express SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 surface antigens prior todifferentiation but only SSEA-1 upon differentiation, while mESCs only express SSEA-

1 prior to differentiation (Thomson et al 1998, Reubinoff et al 2000, Henderson et al2002) The cytokine leukemia inhibitory factor (LIF) has an established facultative role

in keeping mESCs undifferentiated and an exogenous supply of LIF in the culturemedium is sufficient to keep mESCs undifferentiated for prolonged culture periods(Williams et al 1988, Smith et al 1988) hESCs on the other hand do not appear to have aperceivable LIF response (Thomson et al 1998, Reubinoff et al 2000) The molecule orgroup of molecules involved in autocrine or paracrine signaling in keeping hESCsundifferentiated has also not been identified making the culture of undifferentiatedhESCs heavily reliant on feeder layer support

It has been over five years since the first hESC lines were established but ourunderstanding of hESC biology is still limited for it to be exploited for clinicalapplication This hopefully would change once hESC lines become widely and routinelyavailable to all researchers Nevertheless, hESC research needs to be pursuedaggressively if we are to quickly realize the full therapeutic potential of reparative celltherapy and several areas in particular warrant immediate attention

Specifically, advances must be made to improve hESC culture techniques Purerand safer populations of functionally normal undifferentiated hESCs and differentiatedhESC progenitor cell types need to be derived All current 78 NIH listed hESC linesapproved for US government federal research funding have been derived and propagated

on mouse embryonic fibroblast (MEFs) and in the presence of culture mediumcontaining animal based ingredients The use of a feeder layer of animal origin andanimal components in the culture media substantially elevates the risk of the cross-transfer of viruses and other pathogens to the hESCs

Many studies have focused on the differentiation of MEF supported hESCs into arange of clinically useful cell types, while this is important, the development andrefinement of a xeno-free culture system that decreases the risk of hESC contaminationwith adventitious agents while maintaining pure undifferentiated hESC populations

amenable to expansion of cell numbers is critical before any clinical exploitation ofhESC technology can occur New hESC lines need to be derived and bulk-cultured incurrent good manufacture practice (cGMP) conditions according to a xeno-free goldstandard.

The establishment of new hESC lines in cGMP conditions necessitates thedevelopment of an effective cryopreservation protocol that minimizes or restricts thepossibility of early passage hESC seed stock contamination with adventitious agentssuch as viruses and other pathogens during long-term liquid nitrogen storage

Interestingly, hESC lines have heterogeneous genetic backgrounds unlike mESClines that are from inbred mouse strains and appear to behave differently in culture Forexample, not all hESC lines are amenable to bulk and feeder-free culture protocols,doubling times differ considerably between different lines and the degree of spontaneousdifferentiation in vitro also appears to show much variation (Vogel 2002)

Functional genomics data need to be gathered for a better understanding of thegenetic pathways that regulate pluripotency, self-renewal and differentiation Identifying

“master regulators” and as yet undiscovered genes that control hESC self-renewal andimmortality will shed light on cancer genetics as well as have implications in ageingresearch In an effort to better understand the molecular cascades controlling thepluripotent phenotype in hESCs, transcriptional profiling using, microarray, serialanalysis of gene expression (SAGE) and massively parallel signature sequencing(MPSS) technology are being undertaken on undifferentiated and differentiated hESCs(Sato et al 2003, Sperger et al 2003, Brandenberger et al 2004, Ginis et al 2004, Rao2004a) Comparative analysis of the transcriptome profiles using these techniques willreveal several interesting candidate genes that are potentially important to the embryonicstem cell phenotype

In principle, hESCs are capable of differentiating into all cell types in the adulthuman, therefore they have the potential to provide a source of tissues for replacement indiseases in which native cell types are inactivated or destroyed However, the wide-ranging diverse nature of human diseases and technical shortcomings will in most caseslimit the promise of hESC replacement therapy to a few common human diseases Incontrast, the value of hESCs as a developmental model in helping us gain insight tovirtually all human diseases with a genetic basis appears limitless hESCs also holdpromise in screening and toxicity testing assays in the pharmaceutical industry

Established hESC lines provide a convenient tool for investigating celldifferentiation in a way that is pertinent to human embryonic development, providinginsights into the causes of birth defects and diseases such as cancer that involve aberrantcell proliferation and differentiation Perhaps even more powerful than generatinghealthy tissues from existing hESC lines for cell replacement therapy, would be theability to generate diseased hESC lines with genetic defects through somatic cell nucleartransfer (SCNT) These diseased and genetically abnormal hESC lines could produce anunlimited quantity of diseased cell types that will be an invaluable resource and model tostudy the disease phenotype and its genetic basis Several technical hurdles need to bebypassed before the enormous implications of ES cell technology for understanding andcuring human diseases can be realized

hESC research opens up new vistas in the fields of medicine For instance,treating hESCs with the right combination of growth factors may induce the formation ofdopaminergic neurons or perhaps insulin secreting beta cells of the pancreas Thespecialized neurons and beta cells derived from precursor hESCs can then be returned topatients suffering from Parkinson’s disease or diabetes to correct the defects of themalfunctioning organ or tissue However, cellular therapy using hESC derived

specialized cell types will very likely be useful for treating only certain human diseases.Diseases that involve and affect multiple cell types and organs may not be treatable using

a cell based therapeutic approach Thus, Parkinson’s disease and Type I diabetes areoften singled-out as the two most promising targets for a cell based therapeutic approach

Such treatments from stem cell research will be “cell-based therapies”.Presently, doctors administer fluids (injections), solids (pills) or surgical intervention.For the first time, treatment may be by the administration of cells directly into the body.Thus, several added precautions have to be taken before cell-based therapeutic productscan be released into the market Firstly, stringent tests have to be conducted to ensurethat the specialized cell types, which are returned to the patient, are totally pure Nocontaminating undifferentiated hESCs should be present because they have the potential

to divide and replicate and produce a tumor if an undetected renegade hESC isaccidentally injected into the patient Secondly, specialized cell types derived from

hESCs must be rigorously tested in vitro and in animal models in vivo to show that they

can restore normal physiological function in disease models Thirdly, several hurdles inthe manipulation and differentiation of hESCs must be overcome before the technologycan be successfully transferred to the bedside Cell replacement therapies require thegrowth of large numbers of hESCs Thus, large-scale hESC culture strategies usingbioreactors need to be developed to generate sufficient numbers of cells High efficiencydirected differentiation strategies via spontaneous, co-culture or genomics approaches,safer and purer populations of hESCs and their differentiated progeny, clinicallycompliant xeno-free hESC lines and xeno-free storage systems are very urgent areas thatneed investigation

The studies in this thesis address some of these urgent issues, more specificallythe derivation and propagation of xeno-free hESC lines, the xeno-free storage of hESCs

8and the understanding of the molecular genetics of hESCs that can help identify genesinvolved in the maintenance of pluripotency and commitment to differentiation events.

LITERATURE REVIEW

Regeneration in invertebrates and vertebrates

Man has long been fascinated by the regenerative abilities of certain animals.Regeneration is a remarkable physiological process in which remaining tissues organize

to reform a missing body part All species possess the ability to regenerate damagedtissues, the degree of regeneration, however, varies considerably among species Suchdifferences in regenerative capacity are perhaps indicative of specific mechanisms thatcontrol the different types of regeneration

Several invertebrates like the Planarian flatworm and the Hydra regeneratetissues with speed and precision Planarians are spectacular examples of whole bodyregeneration by an invertebrate; a planarian sliced into 50 pieces will regenerate 50 newplanarians from each piece

The majority of higher vertebrates are incapable of any form of whole organregeneration, even though they had all the necessary instructions and machinery togenerate the tissue during embryonic development (Wolpert et al 1971, Brockes 1997)

Of the higher vertebrates, mammals appear to have limited regenerative ability, a off perhaps for more proficient wound healing ability The most striking examples ofwhole organ regeneration in mammals are that of antler regeneration in Elks, and inhumans, liver regeneration after partial hepatectomy (Kiessling & Anderson 2003)

trade-Most tissue repair events in mammals are dedifferentiation independent eventsresulting from the activation of pre-existing stem cells or progenitor cells In contrast,some vertebrates like the salamanders regenerate lost body parts through thededifferentiation of specialized cells into new precursor cells These dedifferentiatedcells then proliferate and later form new specialized cells of the regenerated organ Stemcells or progenitor cells are the common denominator for nearly all types of

regeneration They are either already pre-existing, as in the case for mammals or created

by the process of dedifferentiation

The process of retina and limb regeneration in urodele amphibians involvescomplex dedifferentiation and redifferentiation events Following limb amputation, thewound is quickly covered by an epithelium that provides the necessary signals for theunderlying tissues to dedifferentiate, proliferate, and form the blastema also known asthe amphibian regeneration bud Blastema tissues then undergo redifferentiation to formmuscle, bone and other mesodermal tissues to enable the reconstruction of the amputatedlimb Major cell signaling pathways activated in the blastema during this process are thefibroblast growth factor (FGF) and transforming growth factor (TGF) pathways (Tsonis1996) Additionally, the blastema appears to express the phosphorylated version of thetumor suppressor gene, retinoblastoma (Rb) that is found highly expressed in manydiverse tumors Cancer cells share similarities with blastema cells in that they are bothdedifferentiated and pluripotent An animal with powerful regenerative capabilities isoften refractory to spontaneous or experimentally induced cancer; this is true for theamphibia Spontaneous tumors are difficult to find in this class of vertebrates (Tsonis andDel Rio-Tsonis, 1988) Studies in the Hydra have identified a family of Wnt proteins,produced during Hydra budding and at the tip of a decapitated Hydra when its head starts

to regrow (Hobmayer et al 2000) Thus, the FGF, TGF and Wingless-Type MmtvIntegration Site Family (Wnt) signaling pathways appear to play important andoverlapping roles in developmental, cancer, regeneration and stem cell biology

Plant meristems

Plants but not most animals have the remarkable capacity to regenerate from vegetativeparts Many terminally differentiated plant organs, tissues and cells retain their capacity

to regenerate For example, a stem segment broken off from Opuntia will regenerate a

new plant Stem cells derive their name from their similarity to the stem of a plant.Indeed, the etymological origins of the term “stem cell” can be traced back to earlybotanical monographs documenting the regenerative competence of plant meristems(Kiessling & Anderson 2003) Stem cells that are totipotent are also found in plants inthe shoot apical meristems (SAM)

Whole plants can be regenerated by either organogenesis or somaticembryogenesis by tissue culture In somatic embryogenesis, regenerating tissuerecapitulates embryonic development while in organogenesis, organs form directlywithout embryogenesis Studies have shown that whole carrot plants can be regeneratedfrom single vegetative cells (Stewart 1958) This means that differentiated plantvegetative cells retain the ability to revert into a totipotent state

Definition of a stem cell

Three basic categories of cells make up the human body viz., germ cells, somatic cellsand stem cells Somatic cells include the bulk of the cells that make up the human adultand each of these cells in their differentiated state has its own copy or copies of thegenome with the only exception being cells without nuclei viz., red blood cells Germcells are cells that give rise to gametes viz., eggs and sperm The canonical definition of

a stem cell is a cell with the ability to divide indefinitely in culture and in the livingorganism whilst retaining the potential to give rise to mature specialized cell types(Alison et al 2002) When a stem cell divides, the daughter cells can either enter a pathleading to the formation of a differentiated specialized cell or self-renew to remain astem cell, thereby ensuring that a pool of stem cells is constantly replenished in the adultorgan This mode of cell division characteristic of stem cells is asymmetric and is a

necessary physiological mechanism for the maintenance of the cellular composition oftissues and organs in the body Other attributes of stem cells include the ability todifferentiate into cell types beyond the tissues in which they normally reside This isoften referred to as stem cell plasticity Stem cells are also believed to be slow cyclingbut highly clonogenic and generally represent a small percentage of the total cellularmake up of a particular organ (Gardner 2002)

While there is still much to discover about the molecular mechanisms that governstem cell fate decisions and self-renewal, transcriptome profiling studies havehighlighted several properties believed to be common to all stem cells at the molecularlevel These essential attributes of “stemness” are proposed to include (i) active Januskinase signal transducers and activators of transcription, TGFβ and Notch signaling, ii)the capacity to sense growth hormones and interaction with the extracellular matrix viaintegrins, iii) engagement in the cell cycle, either arrested in G1 or cycling, iv) a highresistance to stress with upregulated DNA repair, protein folding, ubiquitination, anddetoxifier systems, v) a remodeled chromatin, acted upon by DNA helicases, DNAmethylases, and histone deacetylases and vi) translation regulated by RNA helicases ofthe Vasa type (Ramalho-Santos et al 2002, Ivanova et al 2002)

Totipotency, pluripotency and multipotency

Stem cells can also be classified as totipotent, pluripotent and multipotent Totipotency isthe ability to form all cell types of the conceptus, including the entire fetus and placenta.Such cells have unlimited capability They basically can form the whole organism Earlymammalian embryos are clusters of totipotent cells

In mammals, the fertilized egg, zygote and the first 2, 4, 8 and 16 blastomeresresulting from cleavage of the early embryo are examples of totipotent cells Proof that

these cells are indeed totipotent arises from the observation that identical twins areproduced from splitting of the early embryo However, the expression “totipotent stemcell” is perhaps a misnomer because the fertilized egg and the ensuing blastomeres fromearly cleavage events cannot divide to make more of them Although these cells have thepotential to give rise to the entire organism, they do not have the capability to self-renewand by strict definition therefore, the totipotent cells of the early embryo should not becalled stem cells.

Multipotency is the ability of giving rise to a limited range of cells and tissuesappropriate to their location for example, blood stem cells give rise to red blood cells,white blood cells and platelets, while skin stem cells give rise to the various types of skincells Some recent reports suggest that adult stem cells such as haemopoietic stem cells,neuronal stem cells and mesenchymal stem cells could cross boundaries and differentiateinto cells of a different tissue (Bjornson et al 1999, Jackson et al 1999, Clarke et al 2000,Karuse et al 2001) This phenomenon of unprecedented adult stem cell plasticity hasbeen termed “transdifferentiation” and appears to defy canonical embryological rules ofstrict lineage commitment during embryonic development

Pluripotency is the ability to form several cell types of all three germ layers(ectoderm, mesoderm and endoderm) but not the whole organism Pluripotent stem cellshave in theory the ability to form all the 200 or so cell types in the body There are fourclasses of pluripotent stem cells in humans, other primates and mice These areembryonic stem cells, embryonic germ cells, embryonic carcinoma cells and recently thediscovery of a fourth class of pluripotent stem cell, the multipotent adult progenitor cellfrom bone marrow (Smith 2001)

It is generally assumed that the range of potential fates for human embryonicgerm cells (hEGCs) will be limited compared to human embryonic stem cells (hESCs)

because hEGCs are much further along in the schema of embryonic development Thenumber of groups working with hESCs continues to expand rapidly and this coupledwith the deluge of exciting experimental reports on hESCs appears to haveovershadowed much of the interest in hEGCs

Human embryonal carcinoma (hEC) cell lines are derived from tumors of germcell origin and have long served as the human counterpart of murine EC cells forstudying human development and differentiation in vitro (Andrews 2002) hEC cell linesare capable of multi-lineage differentiation in vitro but being of tumor origin areunfortunately mostly aneuploid making them unsuitable for cell replacement therapies.Both hESC and hEC cell lines express similar stage specific embryonic antigens andtumor rejection antigens on the surfaces of their cells hEC lines also express thepluripotency controlling transcription factor Oct-4, grow in colonies and aremorphologically similar to hESC with individual cells displaying a high nucleus tocytoplasmic ratio Several hEC cell lines also require the support of a feeder layer toretain pluripotent characteristics Not all hEC cell lines are pluripotent and some feeder-independent hEC lines have been reported to be nullipotent

A new class of pluripotent adult stem cells from the bone marrow has beenrecently discovered In a series of experiments, Jiang et al (2002) isolated mousemultipotent adult progenitor cells (MAPCs) from murine bone marrow and demonstratedthat these cells express telomerase and that a single MAPC could be expanded clonallyinto a large number of daughter cells Additionally, under appropriate conditions,MAPCs differentiate into ectoderm, endoderm and mesoderm and are capable ofgenerating chimaeric mice when injected into mouse blastocysts Also, reporter genemarked MAPCs contribute to adult tissues when injected into the veins of adult mice(Jiang et al 2002) Although extremely promising, MAPCs are rare cells in the bone

marrow and difficult to isolate It is also still unclear if these cells are truly biologicallyequivalent to hESCs and if they can be expanded indefinitely whilst retaining their long-term differentiation potential More data needs to be collected from human MAPCs asmost of the current experimental data are derived from studies in the murine model.

Classification of stem cells

Germ cells of the gonads

Mammalian stem cells are usually classified according to their tissue of origin Theovary and testis contain oogonia and spermatogonia which have been referred to as thestem cells of the gonads In adult mammals, only the germ cells undergo meiosis toproduce male and female gametes which fuse to form the zygote that retains the ability

to make a new organism thereby ensuring the continuation of the germ line In fact thezygote is at the top of the hierarchical stem cell tree being the most primitive andproducing the first two cells by cleavage This unique characteristic of germ cells istermed as developmental totipotency Intriguingly, Oct-4 an embryonic transcriptionfactor critical for the maintenance of pluripotency continues to be expressed in the germcells but is absent in other peripheral tissues (Yoshimizu et al 1999, Pesce et al 1998)

Adult stem cells

Adult stem cells also known as somatic stem cells can be found in diverse tissues andorgans The most common adult stem cells are the hematopoietic stem cells, mesechymalstem cells and neuronal stem cells Adult stem cells have also been isolated from severalother organs such as the brain (neuronal stem cells), skin (epidermal stem cells), eye(retinal stem cells) and gut (intestinal crypt stem cells) (Spradling et al 2001) However,

not all organs and tissues may contain stem cells The molecular marking and lineagetracing of pancreatic cells have revealed that some organs like the islet component of thepancreas do not contain any stem cells (Dor et al 2004)

Although many somatic stem cells have been very well characterized and isolated

in rodents, the human equivalents of these adult stem cells have been difficult to identifyand difficult to expand in vitro This could reflect innate differences between human androdent cell physiology Human somatic stem cells appear to display telomere dependentreplicative senescence while rodent stem cells do not (Wright & Shay 2000)

Hematopoietic stem cells

The best-studied adult stem cell is the hematopoietic stem cell (HSC) HSCs have beenused widely in clinical settings for over 40 years and form the basis of bone marrowtransplantation success Unfortunately, HSCs like many other adult stem cells are rareand difficult to isolate in large numbers from their in vivo niche For example, onlyapproximately 1 out of 10,000 bone marrow cells is a HSC (Spradling et al 2001)

Mesenchymal stem cells

Mesenchymal stem cells (MSCs) are another well-characterized population of adult stemcells MSCs are prevalent in bone marrow at low quantities (1 out of 10,000 - 100,000mononuclear cells) It is thought that they respond to local injury by dividing to producedaughter cells that differentiate into multiple mesodermal tissue types, including bone,cartilage, muscle, marrow stroma, tendon, ligament, fat and a variety of other connectivetissues (Short et al 2003) The ease of culture has greatly facilitated the characterization

of MSCs In addition, recent studies have shown that the MSCs can also differentiateinto neuron-like cells expressing markers typical for mature neurons, suggesting that

adult MSCs may be capable of overcoming germ layer commitment Several reports hintthat MSCs can form a variety of cell types and tissues including fat cells, cartilage, bone,tendons and ligaments, muscle cells, skin cells and even nerve cells (Short et al 2003).

Neuronal stem cells

The discovery of neuronal stem cells has indicated that cell replenishment is possiblewithin the brain Neuronal stem cells have been isolated from various regions of thebrain including the olfactory bulb (Pagano et al 2000) as well as the spinal cord(Shihabuddin et al 2000), and can even be recovered from cadavers soon after death(Palmer et al 2001) Several studies have shown that neuronal stem cells can produce notonly mature neurons but also other tissues, including blood and muscle (Bjornsen et al

1999, Galli et al 2003, Clarke et al 2000, Galli et al 2000, Rietze et al 2001, Englund et

al 2002) Some animal studies have shown that adult neural stem cells can participate inrepair of brain damage after stroke via endogenous neuronal precursor (Arvidsson et al2002) as well as transplanted neural stem cells (Reiss et al 2002) Neural stemcells/neural progenitor cells may also show low immunogenicity thereby raising thepossibility for use of donor neural stem cells to treat degenerative brain conditions.Neural stem cells have also been used to investigate potential treatments for Parkinson’sdisease (Liker et al 2003, Kim et al 2003) Pluchino et al (2003) recently used adultneural stem cells to test potential treatment of multiple sclerosis lesions in the brain Using a mouse model of chronic multiple sclerosis, they injected neural stem cells eitherintravenously or intracerebrally into affected mice Donor cells entered damaged,demyelinated regions of the brain and differentiated into neuronal cells Remyelination

of brain lesions and recovery from functional impairment were also seen in the mice

Fetal stem cells

Fetal stem cells are primitive cell types in the fetus that eventually develop into thevarious organs of the body Research with fetal stem cells has thus far been limited toonly a few cell types because of the unavailability of abortuses These include neuralcrest stem cells, fetal hematopoietic stem cells and pancreatic islet progenitors (Beattie et

al 1997) Fetal neural stem cells are abundant in the fetal brain and have been shown todifferentiate into both neurons and glial cells (Brustle et al 1998, Villa et al 2000) Fetalblood, placenta and umbilical cord are rich sources of fetal hematopoietic stem cells.Several commercial enterprises trying to capitalize on the theoretical potential of fetalhematopoietic stem cells as a source of stem cells for cell replacement therapy havesurfaced in the last few years

Umbilical cord blood (UCB) contains circulating stem/progenitor cells, and thecellular contents of UCB are known to be quite distinct from those of bone marrow andadult peripheral blood Over the past two decades, the presence and characteristics ofhematopoietic stem cells in UCB have been clarified (Nakahata & Ogawa 1982,Broxmeyer et al 1989, Gluckman et al 1989) The frequency of UCB hematopoieticstem/progenitor cells equals or exceeds that of bone marrow and surpasses that of adultperipheral blood (Mayani & Lansdorp 1998) Compared with adult cells, UCBhematopoietic stem cells produce larger hematopoietic colonies in vitro, have differentgrowth factor requirements, are able to expand in long-term culture in vitro, and havelonger telomeres (Smith & Broxmeyer 1986, Salahuddin et al 1981, Gluckman 2000).UCB transplantation for various hematopoietic diseases has resulted in successfulhematopoietic reconstitution and a lower incidence of graft-versus-host disease thanexpected with conventional therapies (Wagner et al 1995, Kurtzberg et al 1996).Recently, it has been reported that UCB contains mesenchymal progenitor cells capable

of differentiating into marrow stroma, bone, cartilage, muscle, and connective tissues(Erices et al 2000) Furthermore, UCB provides no ethical problems for basic studies andclinical applications UCB cells can be collected without any harm to the newborn infant,and UCB hematopoietic stem cell grafts can be cryopreserved and transplanted to a hostafter thawing without losing their repopulating ability (Rubinstein et al 1995) For thesereasons, UCB could be a prominent source of cells for transplantation in variousdiseases It remains obscure, however, whether UCB contains stem/progenitor cellsleading to endodermal cells, including hepatocytes Although working with umbilicalcord blood appears to circumvent the majority of the ethical issues associated withresearch on fetal material, fetal stem cell research is in many ways underdeveloped and

is still in its infancy

Primordial germ cells

Primordial Germ Cells (PGCs) are diploid germ cell precursors that transiently exist inthe embryo before they enter into close association with the somatic cells of the gonadand become irreversibly committed as germ cells Human Embryonic Germ (hEG) cells,also a form of stem cells are isolates of PGCs from the developing gonadal ridge of 5 to

9 week old fetuses of elective abortions Shamblott et al (1998) reported the successfulisolation and characterization of hEG cell lines hEG cells are pluripotent and are capable

of forming all 3 primordial germ layers (Shamblott et al 1998; 2001)

Embryonic stem cells

Embryonic stem cells on the other hand are derived from the isolated inner cell masses(ICM) of mammalian blastocysts The continuous in vitro sub-culture and expansion of

an isolated ICM on an embryonic fibroblast feeder layer (human or murine) leads to the

development of an embryonic stem cell line In nature however, embryonic stem cellsare ephemeral and present only in the ICM of blastocysts The cells of the ICM aredestined to differentiate into tissues of the three primordial germ layers (ectoderm,mesoderm and endoderm) and finally form the complete soma of the adult organism

Embryonic stem cells can be expanded in vitro very easily and under optimalculture conditions divide symmetrically to give two daughter cells Embryonic stem celllines express the telomerase gene, the protein product of which ensures that the telomereends of the chromosomes are retained at each cell division preventing the cells fromundergoing senescence These cells also retain a normal karyotype after continuouspassage in vitro thus making them truly immortal The earliest hESC lines derived in ourlaboratory have been maintained continuously in culture for over 300 populationdoublings, a figure which surpasses the theoretical Hayflick limit of 50 populationdoublings for normal cells (Bongso et al 1993; 1994, Richards et al 2002)

To qualify as a bona fide embryonic stem cell line, the following criteria must besatisfied, i) immortality and telomerase expression, ii) pluripotentiality and teratomaformation, iii) maintenance of stable karyotype after extended in vitro passage, iv)clonality, v) Oct-4 expression and vi) ability to contribute to chimera formation throughblastocyst injection hESCs have fulfilled all criteria with the exception of chimeracontribution (Ramalho-Santos et al 2002) For obvious ethical reasons, experimentsinvolving blastocyst injections and ectopic grafting in adult hosts cannot be performed inthe human

Animal embryonic stem cells

Embryonic stem cells were first derived from certain strains of mice (Evans & Kaufman1981) Embryonic stem cell lines have been established from the mouse, chicken,

hamster, rabbit, pig, bovine, fish (medaka) (Hong et al 1999), primate and humans.However, only mouse and chicken embryonic stem cells appear to have germ linecompetence and have the ability to contribute to chimera formation thus making themtrue embryonic stem cell lines Strikingly no group has yet been able to derive bona fiderat embryonic stem cell lines In general, rat rather than mouse physiology is believed to

be a closer parallel to human physiology Therefore, the rat model would in theory be acloser representative for the study of human disease For example, there is a rat modelfor hypertension but no similar mouse model The rat asthma model also mimics manyfeatures of human asthma and given the same level of cholesterol and triglycerides, therat atherosclerosis model demonstrates coronary artery disease and decreased survivalcomparable to that of humans (Bice et al 2000, Stoll & Jacob 2001) Therefore, if ratembryonic stem cell lines can be established they will perhaps benefit medical science inmore ways than mESCs have, through the use of knock-out technology

Several lines of evidence suggest that hESCs and mESCs do not representequivalent embryonic cell types In vitro differentiation of hESCs leads to the expression

of alpha-feto protein (AFP) and human chorionic gonadotropin (HCG), which aretypically produced by trophoblast cells in the developing human embryo, while mESCsare generally believed not to differentiate along this extra-embryonic lineage hESCsexpress the stage-specific embryonic antigens (SSEA)-3, SSEA-4, tumor rejectionantigen (TRA)-1-60, and TRA-1-81 surface antigens prior to differentiation but onlySSEA-1 upon differentiation, while mESCs only express SSEA-1 prior to differentiation.More strikingly, hESCs do not appear to have a perceivable LIF response unlike mESCs,which can be maintained, in the undifferentiated pluripotent state in vitro withexogenous LIF supplementation (Smith et al 1988, Willliams et al 1988) Transcription

profiling studies have shown that LIF and its cognate receptor are expressed at extremelylow levels in hESCs (Richards et al 2004)

Human embryonic stem cells

Human embryonic stem cell lines were first established in 1998 To date there are 78National Institute of Health (NIH) USA registered hESC lines, all of which have beenderived on MEFs The establishment of hESC lines is a highly efficient procedure, with

up to a 60% success rate from spare IVF blastocysts (Richards et al 2002, Thomson et al

1998, Reubonoff et al 2000) The quality of the donated embryos appears to be animportant determinant of success in deriving hESC lines Nevertheless, protocols forhESC line derivation have been reproduced in many labs and are relatively easy tofollow (Reubinoff et al 2000, Richards et al 2002, Cowan et al 2004)

History of human embryonic stem cell research

Pluripotent embryonal carcinoma (EC) lines were the first kind of stem cells that wererecognized in terminally differentiated tissues of spontaneously occurring murinetumours (teratocarcinomas) (Andrews 2002) They can be stimulated to differentiate invivo as well as in vitro Their similar characteristics and behaviour to embryonic stemcells served as a model to isolate comparable cells from mammalian embryos

The first report on the growth of ICMs and the isolation of stem cells fromhuman blastocysts was by Bongso et al 1993, 1994 In their study, 9 patients enrolled in

an IVF program donated 21 embryos for hESC production All 21 embryos at thepronuclear stage were co-cultured on human oviductal epithelial feeders to generateblastocysts The zona pellucida was then removed with pronase and zona-free blastocystscultured on irradiated human oviductal feeders as a whole embryo culture in the presence

of Chang’s medium supplemented with 1000 units/ml of hLIF Nineteen of the 21embryos produced “healthy” ICM lumps which were mechanically separated, trypsinisedand passaged further on fresh irradiated human feeders Nest-like embryonic stem cellscolonies were produced which were mechanically cut with hypodermic needles,disaggregated into single cells with trypsin-EDTA and seeded onto fresh irradiatedhuman feeders It was possible to retain the typical hESC morphology of high nuclear-cytoplasmic ratios, alkaline phosphatase positivity and normal karyotype for twopassages in 17 of the embryos (Bongso et al 1993;1994).

Later, primate embryonic stem cell lines were successfully produced from therhesus monkey (Thomson et al 1995) and the human (Thomson et al 1998) Irradiatedmurine embryonic fibroblast (MEF) feeders, immunosurgery to separate the ICM andpassaging of clumps of hESCs instead of disaggregation into single cells were used.Immunosurgery, mitomycin C treated MEFs and a similar ‘cut and paste’ method waslater used to derive and propagate hESC lines that would spontaneously differentiatedinto neuronal cells (Reubinoff et al 2000) Amit and Itskovitz (2002) confirmed that thewhole embryo culture worked as well as the immunosurgery protocol to produce hESClines Given the social nature of hESCs as known today, the disaggregration of ICM andhESC colonies with trypsin into single cells during early passage rather than a ‘cut andpaste approach’ may have been responsible for the hESCs differentiating after twopassages in the early reports of Bongso et al (1993, 1994)

Reliance on a xeno-support system such as MEF introduces considerabledisadvantages with respect to exploiting the therapeutic potential of hESCs A majordrawback is the risk of transmitting pathogens from the animal feeder cells orconditioned medium to hESCs The derivation of hESC lines on xeno-free supportsystems in the presence of xeno-free proteins thus needs to be urgently developed

Differentiation, transdifferentiation and stem cell plasticity

Differentiation is the process whereby an unspecialized early embryonic cell acquires thefeatures of a specialized cell such as that of a heart, liver or muscle Differentiation invitro can be spontaneous or controlled From a teleological perspective there appears to

be no limit to the types of cells that can be formed from hESC differentiation This is incontrast to the practical and theoretical constraints levied on somatic stem cells by virtue

of their position in embryonic development

In vitro, hESCs spontaneously differentiate in high-density cultures or whenculture conditions are sub-optimal to yield a mixed milieu of differentiated cell types.Cells and tissues representative of all 3 germ layers including neurons, cardiomyocytesand primitive endoderm have been identified in differentiating hESC cultures However,

to fully appreciate the plasticity of hESCs one has to look at the teratomas formed inimmune compromised mice when undifferentiated hESCs are injected into these hosts toallow spontaneous differentiation and tumor formation Histological sections of non-malignant teratomas reveal complex, well-organized organ-like structures representative

of tissues from the ectoderm, endoderm and mesoderm Gut-like structures, bone andcartilage, neural rosettes and glandular epithelium with secretions are commonly found

in hESC formed teratomas

Several groups have reported controlled in vitro differentiation of hESCs.Typically, hESCs are induced to form embryoid bodies (EBs) by removal of the feederlayer and the disaggregation into single cells in suspension culture Alternatively, thehanging drop method is used to induce EB formation EBs and hESCs have been found

to differentiate in response to treatment with an array of protein-based cytokines andgrowth factors (Schuldiner et al 2000) However, in these studies homogenous

differentiation into specific cell types was not achievable, instead, the final population ofcells consisted of mixed cell types representative of two or three germ layers.

To date, several studies have been published on the targeted differentiation ofhESCs Kehat et al (2001) described a reproducible method based on spontaneousdifferentiation to derive cardiomyocytes while Mummery et al (2002) used co-culturetechniques with isolates of primitive visceral endoderm to induce cardiomyocyteformation in hESCs Reubinoff et al (2001) and Zhang et al (2001) described methodsfor the isolation of neural precursors from differentiating hESC cultures and showedincorporation of these precursor cells in animal hosts while Assady et al (2001) reportedthe ability of hESCs to differentiate into insulin-secreting cells More recently, hESCshave also been shown to be capable of differentiating into germ cell-like derivatives(Clark et al 2004)

Although these studies represent reproducible and convincing examples ofcontrolled in vitro hESC differentiation, in many respects much of this work will not beapplicable in a clinical setting due to the low efficiencies of the procedures and thedifficulties involved in isolating pure and specific precursor cell types Furthermore,none of these reports describe a truly efficient directed differentiation strategy.Currently, the best example of a directed differentiation strategy is probably that of bonemorphogenetic protein 4 (BMP4) induction of hESCs into the formation of trophoblastcells where up to 40% conversion of hESCs into trophoblasts was reported (Xu et al2002)

Nevertheless, these early reports of hESC differentiation lay an excellentframework for the establishment of true efficient directed differentiation strategies forthe large-scale derivation of differentiated specialized cell types from hESCs and thesubsequent functional testing of these cells in primate models

The possibility that cell fusion events might be an explanation for some remarkablereports of somatic stem cell transdifferentiation has been highlighted by some studies.Ying et al (2002) found that neural stem cells cocultured with embryonic stem cellscould contribute to non-neural tissues not by dedifferentiation but via fusion with the EScells while Terada et al (2002) carried out similar coculture experiments with bonemarrow cells and embryonic stem cells and found that the resulting embryonic stem-likecells, which could differentiate to many different cell types in vitro, were aneuploid.Several scientists believe that the in vivo environment might be permissive for cellfusion, and that cell fusion could be an alternative explanation for some of the reportedsomatic stem cell transdifferentiation events

The transdifferentiation phenomenon is not as straightforward as it seems Wecurrently have no understanding of the developmental mechanisms regulatingtransdifferentiation and its physiological significance Genuine rare transdifferentiationevents could be a reflection of an error rate in cell specification that was not previouslydetected or perhaps could represent a facultative repair mechanism in response to severetissue damage

The phenomenon of transdifferentiation is also intimately linked with the debate

on adult versus embryonic stem cells Despite several studies showing that manymultipotent adult stem cells are capable of forming a wider variety of cell types thanpreviously thought, it is unlikely that they can make the full range of cell types made byembryo-derived pluripotent stem cells

Potential applications of hESCs

The most important potential use of hESCs is in transplantation medicine It is thisaspect of hESC research that attracts the most media attention as well as the most private

research funding In theory, it should be possible to coax hESCs to form a pletheora ofdifferentiated cell types These derivative cell types can in turn be used to develop cellreplacement therapies for a variety of human diseases Although in principle this impliesthat hESCs should have the potential to provide a source of tissues for replacement forall diseases in which native cell types are inactivated or destroyed, the diverse nature ofhuman diseases and technical shortcomings will limit the promise of hESC based cellreplacement therapy to a few common human diseases Diseases that affect multipleorgans are unlikely to be cured by any form of hESC replacement therapy Type Idiabetes and Parkinsonism emerge as prime candidates for hESC-based cell replacementtherapy The success of the Edmonton protocol and cadaver islet transplantation augurwell for the success of an analogous strategy using hESC derived islet progenitor cells(Shapiro et al 2000) A similar approach utilizing hESC derived dopaminergic neuronscould hypothetically be used to treat Parkinson’s disease.

In contrast, the value of hESCs as a developmental model in helping us gaininsight to human diseases with a genetic basis appears to be limitless For example,hESC lines are good in vitro models for studies on some early events of humandevelopment, the causes of early pregnancy loss and certain aspects of embryonicageing hESCs may also prove useful in the study of the toxicological effects of newdrugs Embryonic stem cells are very sensitive to culture conditions and differentiate orsenesce readily when the culture environment is sub-optimal The greater sensitivity ofembryonic stem cells compared to adult cells may be an advantage for drug screeningstudies especially for embryo toxicity and teratogenecity assays

Technical hurdles in hESC research

Treatment via stem cell research will be through 'cell based therapies' involving theadministration of a cell directly into the body Thus, several added precautions have to betaken before cell-based therapeutic products can be released into the market

Stringent tests have to be conducted to ensure that the specialized cell types,which are returned to the patient, are 100% pure No contaminating undifferentiatedhESCs should be present because any undetected transferred renegade hESC has thepotential to produce tumours Specialized cell types derived from hESCs and awaitingtransplantation to the patient must be rigorously tested in vitro and in animal or primatemodels in vivo to show that they can restore normal physiological function in diseasemodels

Several hurdles in the manipulation and differentiation of hESCs must also beovercome before the technology can be successfully transferred to the bedside Cellreplacement therapies require the growth of massive numbers of hESCs; so large-scalehESC culture strategies utilizing bioreactors and perfusion systems must be developed togenerate sufficient numbers of cells High efficiency directed differentiation strategies,safer and purer populations of hESCs and their differentiated progeny and clinicallycompliant xeno-free hESC cell lines must be produced

Lastly, the tissue rejection concerns with cell replacement therapy using ES cellshave to be overcome The derivation of a hESC line using a nuclear transfer embryo hasbeen recently achieved by Hwang et al (2004) This is one approach in obtainingrejection-free customised “tailor-made” hESC lines At present however, the widespreaduse of nuclear transfer technology to create such cell lines for individual patients seemsuntenable due to the extremely low success rates of a highly inefficient procedure, thepaucity of donor human oocytes and the unknown repercussions of absence of the sperm

imprinting mechanisms (Mann 2001) Instead, given the large number of supernumeraryIVF embryos available world wide, deriving and maintaining banks of HLA typed hESClines from different genetic and ethnic backgrounds might be a more feasible solution inovercoming the tissue rejection problem Alternatively, it has been suggested that hESCscould be made less reactive by genetically engineering histocompatibility complexesthrough the introduction or removal of the appropriate cell surface antigens, therebycreating a universal donor hESC line Yet unknown is whether hESCs are immuno-privileged because of their embryonic origin.

Adult stem cells versus embryonic stem cells

The general differences between the characteristics of adult and embryonic stem cells aresummarised in Table 1 The contention that somatic stem cells alone will provide for thedevelopment of long sought after cell based therapies and that somatic stem cells are anequivalent and perfect substitute for embryonic stem cells is a dated assertion.Substantial problems exist with the manipulation of adult stem cells Some of theseproblems are possibly technical and may be overcome in the near future but many mayreflect the inherent biology of somatic stem cells

Most of the literature describing the plasticity of somatic stem cells derives fromstudies in rodent models Not all of this work may be directly applicable to human stemcell biology Stem cells in adult human tissues are known to be notoriously difficult toisolate and characterize In addition, few somatic stem cell types have been confirmed toexist in human tissues and those that can be isolated with relative ease are unfortunatelydifficult to scale-up in culture and their true latent plasticity has also not been establishedclearly These difficulties, coupled with an innate reduced plasticity and cell fusionrather than transdifferentiation properties has marred progress in the field Although in

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the long term, research on hESCs may be the best in realizing the therapeutic potential ofstem cells, somatic stem cell research and ES cell research can complement each other inmany ways and thus both directions should be actively pursued.

hESC research and the future

As it often happens in science, stem cell research has raised as many new questions as ithas answered, the field is advancing but several difficult hurdles in the science still need

to be overcome Additionally, legislation and restrictions on hESC research in somecountries are slowing progress Socially, scientists have a responsibility to dispelmisconceptions about hESC research Myths that hESCs are derived from abortedfetuses and have the potential to form the whole human being need to be dismissed Thepublic needs to be reassured of the soundness of the science, that there are regulatoryframeworks that can govern hESC research and that punitive measures can be put inplace to censure rogue scientists attempting to clone whole human beings

In the US, federal funds can only be used for work on a few hESC lines createdbefore August 2001 under a partial ban decreed by the US President These hESC linesare inadequate for the long term because only a few of these lines are available toresearchers (Brivanlou et al 2003) Some of the lines are hard to cultivate, are not fullycharacterized and all of them have been exposed to mouse feeder layers making themundesirable for therapeutic endeavors This has prompted scientists in the US to rely onprivate funding to circumvent these legislative issues and derive new hESC lines toproceed with their work (Cowan et al 2004)

Political and religious disagreements about stem cells and their use areeverywhere, but nowhere is there a more bewildering array of positions than in Europe,where four different models are emerging The first model, developing in the UnitedKingdom, permits the generation and use of hESCs as well as therapeutic cloning, withcertain restrictions The second, currently seen in the Netherlands, permits the generationand use of hESCs but forbids therapeutic cloning The third, seen in Germany, forbidsthe generation of hESCs and therapeutic cloning, but allows, under exceptional

conditions, the use of existing hESC lines for research only The fourth, evident inIreland and Austria, forbids all generation and use of hESCs and therapeutic cloning aswell (Lanza & Rosenthal 2004) At the opposite end of the spectrum, hESC research incountries like the UK, Singapore, Israel, South Korea, China and Japan enjoys generousgovernment support.

A recurrent statement in stem cell biology today is the importance ofstandardizing culture conditions Culture conditions have a profound effect on stem cellself-renewal, differentiation and possibly on stem cell plasticity A re-acquisition ofplasticity in somatic stem cell transdifferentiation may arise because of in vitro cultureconditions that actively promote reactivation and dedifferentiation

For future work on hESCs, it is important to identify a common set of molecularmarkers and to understand how different hESC lines from diverse genetic backgroundsderived in different labs differ from each other Indeed there appears to be severalsignificant detectable differences at the molecular level between different hESC lines(Richards et al 2004) Some hESC lines seem predisposed to differentiate along aparticular cell lineage and form for example cardiomyocytes readily, when undergoingspontaneous differentiation, while other hESC lines may form neural precursors morereadily (Abeyta et al 2004) The karyotypic stability of hESC lines over hundreds ofpopulation doublings in vitro and the frequency of occurrence of aneuploidy need to beaccurately determined

It is imperative that hESC lines be created for research as well as for clinicalapplication Furthermore, the existing pool of hESCs may not be truly representative ofthe general normal human population because all current hESC lines have been derivedfrom infertile couples The study of hESC lines harboring genetic and other

Another area of future research entails the delivery of stem cells to the tissues inwhich they are needed Current practice involves either the injection of stem cellsdirectly into the targeted tissue, or injection of the stem cells into the bloodstreamwithout any guarantee that they will actually home in the appropriate tissues “Targeteddelivery” would ensure that the therapeutic stem cells are introduced only to organs andtissues that need them Research should also be aimed at identifying and understandingthe in vivo somatic stem cell niche In particular, a more thorough understanding of howniche cells influence stem cell-fate decisions will lead to the development of betterisolation and expansion techniques.

Both embryonic and adult stem cells have enormous potential to further ourunderstanding of basic developmental processes but the exceptional properties of hESCsmake them uniquely powerful tools for the development of cell-based therapies inreparative medicine as well as invaluable models for the study of early humanembryogenesis

Although there are less moral objections associated with adult stem cell work, theassertion that both adult and embryonic stem cells are equivalent is tenuous Attempts

have been made to hype adult stem cells at the expense of hESCs but hESCs clearly havegreater differentiation potential over adult stem cells by virtue of their position inembryonic development It is this fact and the ability to culture hESCs easily in vitro inlarge numbers that makes hESCs the current best hope for the development of cellreplacement therapies Nevertheless, research on adult stem cells and embryonic stemcells should be energetically pursued in tandem as some diseases may benefit from one,and some from the other.

It is important to identify the exact nature of the pluripotent state in hESCs andhEGCs, how it is acquired, maintained, propagated and which genes confer pluripotency.Furthermore, we will need to unravel how epigenetic modifications permit a switch inpatterns of gene expression that are central to plasticity and transdifferentiation in adultstem cells Finally, understanding the fundamental mechanisms by which cell fate isdetermined during embryonic development will prove informative for the in vitromanipulation of stem cells

CHAPTER 1

Derivation and Propagation of Human Embryonic Stem Cell Lines on Xeno-Free Support Systems and in the Presence of Xeno-Free Proteins

hESC lines have been successfully derived from the ICM of blastocysts and can bemaintained in vitro for prolonged periods of time (Thomson et al 1998, Reubinoff et al2000) Unlike their murine counterparts, hESCs lose pluripotency and differentiaterapidly when grown on uncoated plastic tissue-culture dishes, even in the presence ofculture medium supplemented with leukemia inhibitory factor (Thomson et al 1998,Reubinoff et al 2000) Currently, the culture of ICMs and hESCs still requires MEFs,either as a feeder layer (Thomson et al 1998, Reubinoff et al 2002) or as a source ofconditioned medium when the cells are grown on laminin- or Matrigel-coated plastic (Xu

et al 2000)

Reliance on a xenosupport system introduces considerable disadvantages withrespect to exploiting the therapeutic potential of hESCs In particular, a major drawback

is the risk of transmitting pathogens from the animal feeder cells or conditioned medium

to the hESCs The use of MEFs and other components of animal origin in the culturemedia such as FCS for hESC support substantially increase the risk of contaminatingthese lines with infectious animal agents like retroviruses and severely limits thepotential of these lines for clinical usage All 78 NIH-registered hESC cell linesapproved for US federal government research funding have been derived on MEF feederlayers and have also been exposed to xenoproteins Commercially available serum-freeembryonic stem cell media preparations such as KNOCKOUTTM from Invitrogen alsocontain animal ingredients (Andrews 2002) This makes these lines undesirable forclinical application although they may suffice for most basic research studies Therefore,new hESC lines derived in xeno-free, cGMP and GTCP conditions need to beestablished for clinical investigation and application

Previous reports by Bongso et al (1993, 1994) have shown that a human adultfallopian tubal (AFT) epithelial feeder layer was successful in supportingundifferentiated growth of human ICMs and hESC-like cells in the presence of Chang'smedium supplemented with human LIF However, at the time of that work, no attemptwas made to use human fetal feeders with conditioned or non-conditioned medium.Recently, Xu and colleagues (2002) reported that conditioned media from immortalizedadult human foreskin fibroblasts and retinal epithelial cells could not supportundifferentiated hESC growth in a feeder-free culture system dependent on MEF-conditioned medium Thus, all published reports on hESC derivation and culture appear

to be reliant on MEFs either as a direct attachment substrate or as an obligatory source ofconditioned media

In this chapter, the growth of two MEF-supported hESC lines, HES-3 and HES-4(ES Cell International Pte Ltd, Singapore) were evaluated on feeder layers derived fromhuman fetal muscle (FM), fetal skin (FS), and AFT epithelial cells with culture mediumcontaining FCS At the same time, the growth of HES-3 and HES-4 on feeder-freematrices with conditioned culture media prepared independently from the three humanfeeders and with MEF-conditioned medium was evaluated Having confirmed that bothfetal and adult human feeders support HES-3 and HES-4, the establishment of a newhESC line from the ICM stage was evaluated on human FM fibroblasts in the presence

of xeno-free culture medium containing human serum as a substitute for FCS.Furthermore, to examine if there were differences in support between feeder sources, apanel of in-house derived and commercial human fetal and adult fibroblast feeders wasevaluated for their ability to support prolonged undifferentiated hESC growth

Materials and methods

A Preamble- general overview of hESC culture protocol

All steps in the preparation and handling of cells were undertaken with special attention

to quality control Daily montiroing was carried out, especially for the first few weeks.The use of high quality commercial reagents and disposable plasticware in all procedureshelps ensure the establishment and maintenance of healthy hESC cultures

The culture techniques in this chapter incorporate several modifications to a laborintensive, specialized subculture routine requiring manual colony manipulation, slicingand microdissection under stereo optics based on work described in Reubinoff et al(2000) The key objective of this subculture routine is the passage of cells in clusters ofapproximately 200-400 cells and the elimination of differentiated cells from thesubculture A major drawback of this “cut and paste” method is the difficulty ingenerating sufficient numbers of cells for clinical application and some large-scaleexperiments Nevertheless, this technique will be particularly useful for the expansion ofearly passage hESC cell stock and for propagating hESC lines that are not amenable tobulk culture protocols (Richards et al 2002, Vogel 2002) In addition, manual colonyslicing results in the growth of larger colonies, thus making colony selection andharvesting for experiments easier It is also possible when harvesting hESCs forexperiments to slice into the peripheral regions of the hESC colony to avoid harvestingfeeder cells and degenerating cells at the colony core

Enzymatic dispersion and the bulk-culture of hESC lines on human feeders ispossible This passaging protocol involves the enzymatic dispersion and disaggregation

of hESC colonies into small clumps of approximately 20 to 30 cells each with eithertrypsin-EDTA (Cowan et al 2004), collagenase I (Pera et al 2003) or collagenase IV (Xu

et al 2002) Bulk-cultured hESCs form smaller rounder colonies and need to be passaged

at shorter time intervals of 5 to 6 days It is possible to generate millions of hESCs overfour to five serial passages with the bulk culture protocol However in some hESC lines,the bulk culture passaging protocol often leads to an acceleration of growth and anoverall decrease in colony differentiation (Pera et al 2003) Also, a recent study reportedkaryotypic instabilities in mid to late passage hESC lines subcultured on MEFs using theenzymatic bulk culture approach (Draper et al 2004) It remains to be seen to what extentculture conditions or passaging protocols can influence and affect the karyotypic stability

of these lines

B Feeder layer culture media

Human feeder layer establishment medium (xeno-free) contained 50% Dulbecco’smodified Eagles medium (DMEM) (Invitrogen, Carlsbad, CA), 50% human serum (HS),1X antimycotics (Invitrogen) and 2 mmol/l L-glutamine (Invitrogen) Human feedermaintenance medium (xeno-free) contained 90% high-glucose DMEM, 10% HS, 2mmol/l L-glutamine, 50 IU/ml penicillin and 50 µg/ml streptomycin (Invitrogen) MEFculture media comprised 90% high-glucose DMEM, 10% fetal calf serum, 2 mmol/l L-glutamine, 50 IU/ml penicillin and 50 µg/ml streptomycin Human serum was obtainedfrom patients undergoing IVF cycles

C hESC culture media

H1 medium contained 80% (vol/vol) DMEM, 20% (vol/vol) Hyclone defined FCS(Hyclone, Logan, UT), 1X L-glutamine, 1X penicillin–streptomycin, 1X nonessentialamino acids (Invitrogen), 1X insulin-transferrin-selenium G supplement (Invitrogen) and

1 mM β-mercaptoethanol (Invitrogen) For the derivation and establishment of the new