GENERAL INTRODUCTION Several types of stem cells have been discovered from germ cells, the embryo, fetus and adult Each of these has promised to revolutionize the future of regenerative medicine through the provision of cell replacement therapies to treat a variety of debilitating diseases The tremendous versatility of embryonic stem cells versus the unprecedented reports describing adult stem cell plasticity have ignited debates as to the choice of one cell type over the other for future applications However, the biology of these mysterious cells have yet to be understood through a lot more basic research before new therapies using stem cell differentiated derivatives can be applied Everyday, we read and listen to news reports about how stem cells promise to revolutionize medicine and change our lives with panaceas for every imaginable disease including 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 religious and genuine ethical concerns To further fuel the debate, embryonic stem cell research is often unjustly associated with reproductive cloning Stem cell research is politically charged, receives considerable media coverage, raises many ethical and religious debates and generates a great deal of public interest Stem cell research also opens the new field of ‘cell based therapies’ and as such several safety measures have also to be evaluated The hope that someday many debilitating human diseases may be treated with stem cell therapy is inspired by remarkable examples of whole organ and limb regeneration in animals as well as the historical success of bone marrow transplants that have improved the lives of many patients suffering from leukemia, immunological and other blood disorders Clearly, stem cell research leading to prospective therapies in reparative medicine has the potential 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 an effective cell-based therapy for widespread use is long and involves overcoming numerous technical, legislative, ethical and safety issues Embryos of most mammals are comprised of a special group of cells that have the potential to give rise to all the tissues and organs of the fetus and future adult This group 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 indefinitely without 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 human blastocysts hold the promise of revolutionizing the future of medicine by the creation of early developmental models for a multitude of human genetic diseases and through the development of cell and tissue replacement therapies Immense commercial interest as well as ethical controversy surrounds hESC research Several improvements in blastocyst culture techniques were a prerequisite for culturing and harvesting good quality blastocysts with large ICMs These breakthroughs not 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 lines from 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 if they are coaxed properly, ESCs have in theory the ability to turn into any of the cell types in the human body In contrast, adult stem cells, which are found in adult tissues and 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 This coupled with their restricted developmental potential are the main reasons why many scientists believe that embryonic stem cells are more promising and better alternatives for developing a wider range of cell based therapies In order for hESCs to retain their ability to form different cell types, they need to be grown on feeder cell supports The feeder 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 of mixed cell types This is often undesirable for the researcher as specific cell types are often difficult or near impossible to isolate from this mixed milieu Embryonic stem cells are a unique class of cell type for various reasons Most significantly, they can undergo self-renewal for extended periods of in vitro cultivation, have the ability to form teratomas when injected into severely combined immunodeficient (SCID) mice and can differentiate into a variety of cell types from all primitive germ layers in vitro and in vivo, thus distinguishing them from other adult stem cells hESCs differ in many ways from mouse embryonic stem cells (mESCs) Several lines of evidence suggest that hESCs and mESCs 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 to differentiation but only SSEA-1 upon differentiation, while mESCs only express SSEA1 prior to differentiation (Thomson et al 1998, Reubinoff et al 2000, Henderson et al 2002) The cytokine leukemia inhibitory factor (LIF) has an established facultative role in keeping mESCs undifferentiated and an exogenous supply of LIF in the culture medium is sufficient to keep mESCs undifferentiated for prolonged culture periods (Williams et al 1988, Smith et al 1988) hESCs on the other hand not appear to have a perceivable LIF response (Thomson et al 1998, Reubinoff et al 2000) The molecule or group of molecules involved in autocrine or paracrine signaling in keeping hESCs undifferentiated has also not been identified making the culture of undifferentiated hESCs heavily reliant on feeder layer support It has been over five years since the first hESC lines were established but our understanding of hESC biology is still limited for it to be exploited for clinical application This hopefully would change once hESC lines become widely and routinely available to all researchers Nevertheless, hESC research needs to be pursued aggressively if we are to quickly realize the full therapeutic potential of reparative cell therapy and several areas in particular warrant immediate attention Specifically, advances must be made to improve hESC culture techniques Purer and safer populations of functionally normal undifferentiated hESCs and differentiated hESC progenitor cell types need to be derived All current 78 NIH listed hESC lines approved for US government federal research funding have been derived and propagated on mouse embryonic fibroblast (MEFs) and in the presence of culture medium containing animal based ingredients The use of a feeder layer of animal origin and animal components in the culture media substantially elevates the risk of the crosstransfer of viruses and other pathogens to the hESCs Many studies have focused on the differentiation of MEF supported hESCs into a range of clinically useful cell types, while this is important, the development and refinement of a xeno-free culture system that decreases the risk of hESC contamination with adventitious agents while maintaining pure undifferentiated hESC populations amenable to expansion of cell numbers is critical before any clinical exploitation of hESC technology can occur New hESC lines need to be derived and bulk-cultured in current good manufacture practice (cGMP) conditions according to a xeno-free gold standard The establishment of new hESC lines in cGMP conditions necessitates the development of an effective cryopreservation protocol that minimizes or restricts the possibility of early passage hESC seed stock contamination with adventitious agents such as viruses and other pathogens during long-term liquid nitrogen storage Interestingly, hESC lines have heterogeneous genetic backgrounds unlike mESC lines that are from inbred mouse strains and appear to behave differently in culture For example, not all hESC lines are amenable to bulk and feeder-free culture protocols, doubling times differ considerably between different lines and the degree of spontaneous differentiation in vitro also appears to show much variation (Vogel 2002) Functional genomics data need to be gathered for a better understanding of the genetic pathways that regulate pluripotency, self-renewal and differentiation Identifying “master regulators” and as yet undiscovered genes that control hESC self-renewal and immortality will shed light on cancer genetics as well as have implications in ageing research In an effort to better understand the molecular cascades controlling the pluripotent phenotype in hESCs, transcriptional profiling using, microarray, serial analysis 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, Rao 2004a) Comparative analysis of the transcriptome profiles using these techniques will reveal several interesting candidate genes that are potentially important to the embryonic stem cell phenotype In principle, hESCs are capable of differentiating into all cell types in the adult human, therefore they have the potential to provide a source of tissues for replacement in diseases in which native cell types are inactivated or destroyed However, the wideranging diverse nature of human diseases and technical shortcomings will in most cases limit the promise of hESC replacement therapy to a few common human diseases In contrast, the value of hESCs as a developmental model in helping us gain insight to virtually all human diseases with a genetic basis appears limitless hESCs also hold promise in screening and toxicity testing assays in the pharmaceutical industry Established hESC lines provide a convenient tool for investigating cell differentiation in a way that is pertinent to human embryonic development, providing insights into the causes of birth defects and diseases such as cancer that involve aberrant cell proliferation and differentiation Perhaps even more powerful than generating healthy tissues from existing hESC lines for cell replacement therapy, would be the ability to generate diseased hESC lines with genetic defects through somatic cell nuclear transfer (SCNT) These diseased and genetically abnormal hESC lines could produce an unlimited quantity of diseased cell types that will be an invaluable resource and model to study the disease phenotype and its genetic basis Several technical hurdles need to be bypassed before the enormous implications of ES cell technology for understanding and curing 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 of dopaminergic neurons or perhaps insulin secreting beta cells of the pancreas The specialized neurons and beta cells derived from precursor hESCs can then be returned to patients suffering from Parkinson’s disease or diabetes to correct the defects of the malfunctioning 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 are often 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 products can be released into the market Firstly, stringent tests have to be conducted to ensure that the specialized cell types, which are returned to the patient, are totally pure No contaminating undifferentiated hESCs should be present because they have the potential to divide and replicate and produce a tumor if an undetected renegade hESC is accidentally 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 in the manipulation and differentiation of hESCs must be overcome before the technology can be successfully transferred to the bedside Cell replacement therapies require the growth of large numbers of hESCs Thus, large-scale hESC culture strategies using bioreactors need to be developed to generate sufficient numbers of cells High efficiency directed differentiation strategies via spontaneous, co-culture or genomics approaches, safer and purer populations of hESCs and their differentiated progeny, clinically compliant xeno-free hESC lines and xeno-free storage systems are very urgent areas that need investigation The studies in this thesis address some of these urgent issues, more specifically the derivation and propagation of xeno-free hESC lines, the xeno-free storage of hESCs and the understanding of the molecular genetics of hESCs that can help identify genes involved 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 damaged tissues, the degree of regeneration, however, varies considerably among species Such differences in regenerative capacity are perhaps indicative of specific mechanisms that control the different types of regeneration Several invertebrates like the Planarian flatworm and the Hydra regenerate tissues with speed and precision Planarians are spectacular examples of whole body regeneration by an invertebrate; a planarian sliced into 50 pieces will regenerate 50 new planarians from each piece The majority of higher vertebrates are incapable of any form of whole organ regeneration, even though they had all the necessary instructions and machinery to generate the tissue during embryonic development (Wolpert et al 1971, Brockes 1997) Of the higher vertebrates, mammals appear to have limited regenerative ability, a tradeoff perhaps for more proficient wound healing ability The most striking examples of whole organ regeneration in mammals are that of antler regeneration in Elks, and in humans, liver regeneration after partial hepatectomy (Kiessling & Anderson 2003) Most tissue repair events in mammals are dedifferentiation independent events resulting from the activation of pre-existing stem cells or progenitor cells In contrast, some vertebrates like the salamanders regenerate lost body parts through the dedifferentiation of specialized cells into new precursor cells These dedifferentiated cells then proliferate and later form new specialized cells of the regenerated organ Stem cells 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 involves complex dedifferentiation and redifferentiation events Following limb amputation, the wound is quickly covered by an epithelium that provides the necessary signals for the underlying tissues to dedifferentiate, proliferate, and form the blastema also known as the amphibian regeneration bud Blastema tissues then undergo redifferentiation to form muscle, bone and other mesodermal tissues to enable the reconstruction of the amputated limb Major cell signaling pathways activated in the blastema during this process are the fibroblast growth factor (FGF) and transforming growth factor (TGF) pathways (Tsonis 1996) 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cat no 11960-044), store at 40C L-Glutamine 200 mM, 100X liquid (GIBCO; cat no 25030-081), aliquot and store at –200C Penicillin-Streptomycin liquid containing 5000 units of penicillin and 5000 mg of streptomycin/ml (GIBCO; cat no 15070-63) aliquot and store at –200C MEM non-essential amino acids 10mM, (100X) (GIBCO; cat no.11140050), wrap in foil to protect from light and store at 40C Defined fetal bovine serum (Hyclone, Logan, UT; cat no SH30070-03) aliquot and store at –200C Qualified fetal calf serum (GIBCO; cat no 10099) aliquot and store at –200C Dulbecco’s phosphate buffered saline, PBS (GIBCO; cat no 14040-133), store at 40C Dulbecco’s phosphate buffered saline, Ca2+, Mg2+ free PBS (without Ca2+ and Mg2+) (GIBCO; cat no 14190-144), store at 40C Sterile water, tissue culture grade (GIBCO; cat no 15230-162), store at 40C 10 Porcine gelatin (Sigma, St Louis, MO; cat no G1890), store at 40C 11 Insulin-transferrin-selenium growth supplement (GIBCO; cat no 41400045), store at 40C 205 12 0.05% Trypsin-EDTA (1X) (GIBCO; cat no 25300-062), aliquot and store at –200C 13 β-Mercaptoethanol (GIBCO; cat no 21985-023), store at 40C 14 Dispase (GIBCO; cat no 17105-041), store at 40C 15 Collagenase IV (GIBCO; cat no.17104-019) 16 Knockout™ D-MEM (GIBCO; cat no 10829-018) 17 KnockoutTM Serum Replacement (GIBCO; cat no.10828-028) 18 Basic fibroblast growth factor (bFGF), human recombinant (GIBCO; cat no.13256-029) 19 1M HEPES solution (GIBCO; cat no 15630-080), store at 40C 20 Ethylene glycol (Sigma; cat no E9129), store at room temperature 21 Dimethyl sulfoxide, hybridoma tested, x 10 ml in flamed sealed ampules (Sigma; cat no D2650), store at room temperature and protect from light 22 Mitomycin C, cell culture grade (Sigma; cat no M4287), store at 40C 23 1-well dish, 60-mm diameter, well area: 2.89 cm2 (BD, Franklin Lakes, NJ; cat no 353652) 24 4-well plate, well area: 1.39 cm2 (BD; cat no 353653) 25 4-well culture slides, well area: 1.7 cm2 (BD; cat no 354114) 26 75 cm2 canted neck, vented tissue culture flask (BD; cat no 353136) 27 175 cm2 canted neck, vented tissue culture flask (BD; cat no 353112) 28 15-ml conical centrifuge tubes, high-clarity polypropylene (BD; cat no 352196) 29 50-ml conical centrifuge tubes, high-clarity polypropylene (BD; cat no 352070) 206 30 1-ml individually wrapped serological pipet (BD; cat no 357522) 31 5-ml individually wrapped serological pipet (BD; cat no 357543) 32 10-ml individually wrapped serological pipet (BD; cat no 357551) 33 25-ml individually wrapped serological pipet (BD; cat no 357525) 34 500-ml Stericup-GP filter unit (Millipore, Billerica, MA; cat no SCGP U05 RE) 35 Sterivex-GP 2000 filling bell filter unit (Millipore; cat no SVGP B10 10) 36 33-mm Millex-GP filter unit (Millipore; cat no SLGP 033 RS) 37 Nunc System 100 cryogenic vials with silicon gasket (Nalgene, Rochester, NY; cat no 5000-1012) 38 Glass capillaries, 1.0-mm OD (Clark Electromedical Industries, Kent, UK; cat no GC100T-15) 39 Sterile 30G hypodermic needles (BD; cat no 511252/511256) 40 Vector Red Alkaline Phosphatase Substrate Kit I, SK-5100 (Vector Labs, Inc., Bulingame, CA; cat no SK-5100) 41 KaryoMAX Colcemid solution, liquid (10µg/ml) in PBS (Invitrogen; cat no 15212-012) 42 TRIzolTM reagent (Invitrogen; cat no 15596-026) 43 Ambion DNA-freeTM reagent (Ambion; cat no 1906) 44 SuperScriptTM III first-strand synthesis system for RT-PCR (Invitrogen; cat no 18680051) 45 TaqManTM probes were purchased from Applied BioSystems (ABI) Assay on DemandTM and Assay by DesignTM service 207 ... in the body There are four classes of pluripotent stem cells in humans, other primates and mice These are embryonic stem cells, embryonic germ cells, embryonic carcinoma cells and recently the. .. stem cells versus embryonic stem cells The general differences between the characteristics of adult and embryonic stem cells are summarised in Table The contention that somatic stem cells alone... cells make up the human body viz., germ cells, somatic cells and stem cells Somatic cells include the bulk of the cells that make up the human adult and each of these cells in their differentiated