Sexual dimorphism has been long noted in human neurobiology, apparent most notably in sex-biased distribution of multiple neurological disorders or diseases, from autism spectrum disorder to Parkinson's disease. With the advances in molecular biology, genetics and epigenetics have come into focus as key players in sexually dimorphic neural development; and yet, many studies in the field of neuroscience overlook the importance of sex for the human brain. For this project, human embryonic and neural stem cells were chosen for three main reasons. Firstly, they provide an easily obtainable, scalable and physiologically native model for the early stages of development. Secondly, neural stem cells populations are retained within the adult human brain, and are implicated to play a role in cognition and mental illness, and as such are of interest in themselves. Thirdly, stem cell lines are widely used in research, including clinical trials of transplantation treatments, and for this reason should be meticulously examined and characterized. Here, the morphology, behaviour, and expression of selected genes in four stem cell lines, two of female and two of male origin, was examined in side-by-side comparisons prior to and during neuronal differentiation using a variety of methods including light microscopy, time-lapse two-photon microscopy, quantitative real-time PCR and immunocytochemistry. The obtained results have shown previously uncharacterised differences between those cell lines, such as a higher rate of proliferation but a slower rate of neuronal differentiation in male cell cultures compared to female cells cultivated in the same conditions, and a sex-biased expression of several markers of neuronal maturation at late stages of differentiation, as well as diverse patterns of expression of X- and Y-linked genes involved in stem cell proliferation and neural development.

Células-tronco embrionárias (CTE) são importantes para estudos de desenvolvimento embrionário, diferenciação e manipulação genética. Além disso, essas células podem ser utilizadas na terapia celular e organogênese in vitro. Na pesquisa sobre terapia celular a partir de CTE oriundas de embriões humanos, considerações éticas, morais e religiosas têm sido feitas por pesquisadores e leigos. Portanto, um modelo animal como o suíno (Sus scrofa) será bastante válido por transpor tais barreiras, visto que o suíno possui parâmetros fisiológicos semelhantes aos humanos. Apesar do alto potencial biomédico das CTE, existem dificuldades na manutenção da pluripotência in vitro dessas células em suínos. Portanto, estudos que visam elucidar os mecanismos de manutenção da pluripotência de CTE in vitro são necessários para viabilizar o cultivo dessas células. Os objetivos do presente estudo foram (1) isolar células-tronco embrionárias suínas a partir de blastocistos produzidos in vitro e in vivo; (2) comparar dois sistemas de cultivo in vitro das massas celulares internas (MCI) isoladas, MEF ou Matrigel e (3) identificar e comparar a expressão dos fatores de transcrição Nanog, Sox2 e FoxD3 em CTE e blastocistos suínos produzidos in vitro e in vivo. Assim, blastocistos suínos foram produzidos in vitro a partir da maturação e fecundação in vitro de oócitos de ovários obtidos em matadouro. Os embriões foram cultivados in vitro por 7 dias, até atingirem o estágio de blastocisto. Blastocistos suínos também foram produzidos in vivo, através de superovulação seguida de inseminação artificial de marrãs com 150 dias de idade. Para a colheita dos embriões, foi realizada lavagem dos cornos uterinos post-mortem cinco dias após a ovulação. Tanto blastocistos produzidos in vitro quanto os produzidos in vivo foram submetidos à imunocirurgia para isolamento da MCI. Brevemente, a zona pelúcida foi digerida com solução de pronase e os embriões incubados com soro de coelho anti-suíno para remoção das células do trofoectoderma e soro complemento de cobaia. A MCI resultante foi cultivada em meio para células-tronco (GMEM acrescido de 15% SFB, 0,1 mM ß-mercaptoetanol, 1% aminoácidos não essenciais e 4 ng/mL de bFGF) sobre monocamada de fibroblastos fetais murinos (MEF) inativados por radiação ou sobre Matrigel. Não foi observada diferença entre os dois sistemas de cultivo in vitro (MEF e Matrigel) na adesão das MCI isoladas. Também não foi verificada diferença entre os grupos de blastocistos, produzidos in vitro e in vitro, nas taxas de adesão das MCI cultivadas. Contudo, nenhuma colônia de CTE suínas foi obtida. A análise da expressão gênica em blastocistos produzidos in vitro e in vitro demonstrou que os genes Nanog e Sox2 são menos expressos em blastocistos produzidos in vitro. Contudo, a expressão do gene FoxD3, demonstrada pela primeira vez em suínos no presente trabalho, se mostrou semelhante entre os dois grupos de embriões. Visto que nenhuma linhagem de CTE legítima foi isolada em suínos até o momento, sugere-se que esta espécie possua requerimentos diferentes dos já conhecidos para as espécies murina e humana. Portanto, novos estudos são necessários para o estabelecimento de protocolos mais efetivos para o isolamento de CTE de suínos. / Embryonic stem cells (ESC) represent a useful tool to study embryonic development, cell differentiation and genetic manipulation. Moreover, these cells can be applied in cell-based therapies and in vitro organogenesis. The research conducted with human ESC has generated many ethical, moral and religious considerations by scientists and laymen alike. Therefore, an animal model like the pig (Sus scrofa) is valuable by overcoming such hurdles, since this species holds physiologic parameters similar to humans. In spite of the high biomedical potential of ESC, many difficulties have been faced to maintain these cells in a pluripotent state in vitro. For this reason, studies to elucidate the mechanisms of in vitro maintenance of undifferentiated ESC are needed to improve the culture of these cells. The objectives of this study were (1) to isolate ESC from in vitro and in vitro produced swine blastocysts, (2) to compare two in vitro culture conditions to maintain isolated inner cell masses (ICM), MEF or Matrigel and (3) to identify and to compare the expression of the pluripotency markers Nanog, Sox2 and FoxD3 at ESC and in vitro and in vitro produced swine blastocysts. In this manner, swine blastocysts were obtained by in vitro maturation and fertilization of oocytes from ovaries collected in abattoirs. Embryos were in vitro cultured for 7 days until blastocyst stage. In addition, in vitro produced blastocysts were obtained by superovulation followed by artificial insemination of gilts (150 days of age). Embryos were collected by post-mortem uterus flushing five days after ovulation. in vitro and in vitroproduced blastocysts were submitted to immunosurgery to isolate the ICM. Briefly, zona pellucida was digested with pronase solution and embryos were incubated with anti-swine rabbit serum to remove trophoectoderm cells and with guinea-pig complement serum. The resultant ICM was cultured in stem cells media (GMEM added by 15% SFB, 0.1 mM ß-mercaptoethanol, 1% non essential amino acids and 4 ng/mL of bFGF) over monolayer of irradiated murine fetal fibroblasts (MEF) or Matrigel. No difference was observed between the in vitro culture conditions (MEF and Matrigel) on isolated ICM adhesion. In addition, no difference was verified between in vitro and in vitro produced blastocysts on adhesion of cultured ICM. However, no swine ESC was obtained. Gene expression analysis of in vitro and in vitro produced blastocysts showed that Nanog and Sox2 are less expressed in in vitro produced blastocysts. However, the expression of FoxD3, demonstrated in this study for the first time, was similar between groups. Since no ESC lineage was obtained in swine until now, we believe this species have different requirements compared to murine and human. Therefore, more studies are necessary to establish protocols to isolate porcine ESC.

O estabelecimento de cultivo de células-tronco embrionárias (ESC) ainda não foi realizado com sucesso. Verificação de marcadores de pluripotência e diferenciação nos três folhetos germinativos são necessárias para validação de uma linhagem celular pluripotente. O objetivo deste estudo foi estabelecer e caracterizar o cultivo de ESC suínas usando Matrigel e comparar a expressão dos marcadores de pluripotência Oct-4, CD9 e α6-integrina em embriões. Blastocistos in vitro ou in vivo foram submetidos à imunocirurgia para cultura da massa celular interna, fixados para imunocitoquímica ou extração de RNA total para RT-PCR. Nenhuma colônia de ESC foi obtida usando co-cultivo em fibroblastos embrionários murinos (MEF) ou em Matrigel. Expressão de Oct-4, CD9 e α6-integrina foi detectada por PCR. Os produtos de PCR de CD9 e α6-integrina tiveram suas sequências nucleotídicas determinadas e comparadas com bases de dados públicas. O produto de CD9 foi idêntico à seqüência do CD9 suíno e o produto de α66integrina foi similar à humana e eqüina. Reação de Imunocitoquímica revelou a presença de Oct-4 no citoplasma de células da massa celular interna e do trofoblasto. CD9 e α6-integrina foram observados preferencialmente em células do trofoblasto. Não foi possível comparar a expressão dos marcadores de pluripotência entre ESC e embriões em suínos. Porém, este estudo descreve pela primeira vez a expressão de CD9 e α6-integrina em blastocistos suínos, os quais podem não estar relacionados com células pluripotentes embrionárias suínas. / Establishment of embryonic stem cell (ESC) culture in pigs has not been achieved. Verification of pluripotency markers and differentiation in the three embryonic layers are necessary for validation of a pluripotent cell line. The objective of this study was to establish and characterize porcine ESC culture using Matrigel and compare the expression of pluripotency markers Oct-4, CD9 and α6-integrin with embryos. In vitro or in vivo porcine blastocysts were submitted to immunosurgery for culture of inner cell mass, fixation for immunocytochemistry or total RNA extraction for RT-PCR. No ESC colonies were obtained using co-culture on mouse embryonic fibroblasts (MEF) or on Matrigel. Expression of Oct-4, CD9 and α6-integrin was detected by PCR. CD9 and α6-integrin PCR products had their nucleotide sequence assessed and compared with public nucleotide database. CD9 product was identical to CD9 porcine sequences and α6-integrin product was similar to human and equine α6-integrin. Immunocytochemistry revealed Oct-4 expression in cytoplasm of the inner cell mass and trophoblast cells. CD9 and α6-integrin were observed preferentially on trophoblast cells. It was not possible to compare expression of pluripotency markers between porcine ESC and embryos. However, this study describes for the first time expression of CD9 and α6-integrin in porcine blastocysts, which may not be related to pluripotent porcine embryonic cells.

The complexity of life exhibited in humans and other living creatures has drawn many to investigate the principles associated with organismal growth and development. A few broad questions: How do tissues develop into specified organs? How are these tissues maintained? Do they become different tissues? Scientific research has incessantly been seeking answers to these as well as a plethora of other questions. While on a quest to better understand developmental biology, investigators discovered unique populations of stem cells within a variety of tissues, which retain both varying degrees of developmental plasticity and their potential for self-regeneration. This thesis provides a brief review discussing the development and history of stem cells in medicine and associated research on these cells and their potential clinical applications. Substantial attention has been paid to pluripotent embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) which are able to be recapitulate ESC properties through the in vitro reprogramming of somatic cells. While, the ethical and legal issues have greatly hindered the use of ESCs this has made the benefit of iPSCs so great. An interconnected network of pluripotency-associated genes, integrates external signals and exerts control to maintain the state of pluripotency. Recent research has proven the pluripotency regulatory network to be flexible such that the underlying principles promise unprecedented opportunities for scientific study and regenerative medicine. Additional topics reviewed here include vast clinical applications of stem cells as well as their notable limitations.

Orientador: Fernanda da Cruz Landim / Banca: Claudia Barbosa Fernandes / Banca: Flávia Karina Dlella / Resumo: Células tronco embrionárias (CTE) são caracterizadas pelas capacidades de auto-renovação e diferenciação. No entanto, os mecanismos moleculares que regulam estes dois processos são mal compreendidos. CTE de camundongos foram originalmente isoladas a partir da massa celular interna (MCI) de blastocistos produzidos in vitro e in vivo e mantidas em cultura sem perda da pluripotência, originando tecidos das três camadas germinativas. Há profundo interesse em conhecer os processos envolvidos na proliferação e diferenciação das células embrionárias contribuindo, no futuro, para engenharia de tecidos e clonagem terapêutica. Objetivos: Comparar o potencial gerador de CTE de embriões bovinos produzidos in vitro em meio contendo alta e baixa concentração de soro, assim como avaliar a manutenção da pluripotência das células em cultivo através da ação de dois fatores, a LIF e o bFGF, utilizados isoladamente ou em conjunto, no cultivo in vitro de CTEbov. Foram utilizados blastocistos bovinos com 9 dias de desenvolvimento in vitro para remoção da massa celular interna (MCI) e posterior cultivo das mesmas em placas tratadas com monocamada de fibroblastos bloqueados. As colônias celulares semelhantes a células-tronco foram analisadas através da identificação da expressão in sito dos fatores de indiferenciação Oct-4, Nanog, SSEA-1, SSEA-3, SSEA-4, TRA-1- 60, TRA-1-81. Os blastocistos bovinos com 9 dias de idade também foram submetidos à marcação imunocitoquímica. Adicionalmente foi avaliado o potencial de diferenciação in vitro das CTEbov para linhagens celulares de origem endodermal, mesodermal e ectodermal. Em média, 525 embriões de cada um dos dois grupos (2,5% e 10% de soro fetal bovino, respectivamente) foram selecionados para o isolamento da MCI. Foram utilizados 300 blastocistos iniciais... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Embryonic stem cells (ESC) are characterized by their capacity for selfrenewal and differentiation. However, the molecular mechanisms which regulate these two processes are poorly understood. ESC of mice were originally isolated from the inner cell mass (ICM) of blastocysts in vitro and in vivo and maintained in culture without loss of pluripotency, yielding three germ layers of tissue. There is keen interest in learning about the processes involved in proliferation and differentiation of embryonic cells contribute in the future, tissue engineering and therapeutic cloning. To compare the potential generator ESC of bovine embryos produced in vitro in medium containing high and low concentrations of serum, as well as evaluating the maintenance of pluripotency of the cells in culture through the action of two factors, the LIF and bFGF, used singly or together, in vitro cultivation of ESCbov. We used bovine blastocysts to nine days of in vitro development to remove the inner cell mass (ICM) and further cultivation of the same plates treated with fibroblast monolayer blocked. The cell colonies similar to stem cells were analyzed by in situ identification of the expression of differentiation factors Oct-4, Nanog, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 . The bovine blastocysts with 9 days of age were also subjected to immunocytochemical labeling. Additionally it was evaluated the potential of differentiation in vitro of cell lines to ESCbov endodermal origin, mesodermal and ectodermal. The average 525 embryos from each of the two groups (2.5% and 10% fetal bovine serum, respectively) were selected for isolation of the ICM. Early blastocysts were used 300, 160 expanded blastocysts and 45 hatched blastocysts per group. However, only expanded blastocysts adhered to the monolayer of fibroblasts and developed into colonies similar to... (Complete abstract click electronic access below) / Mestre

Human embryonic stem cells are pluripotent cells that have indefinite replicative potential and ability to differentiate into derivatives of three germ layers. HESCs are conventionally derived and grown on mitotically inactivated mouse embryonic fibroblasts and there are some alternative feeder types of human origin that have been used to replenish hESCs while trying to prevent cross-species contamination. The trophic factors that are secreted by the feeders are found to be important for long-term pluripotency but there are also supportive culture systems for hESCs lacking feeder cells which might suggest that not only the interactions with the feeders affect the behaviour of hESCs but also the components of the niche may take part in the decision of self-renewal or differentiation. Extracellular matrix components are known to exert their stimulatory or inhibitory effects by localising cells into a specific microenvironment in natural niches but have been relatively little investigated for hESCs. The aim of this study was to investigate ECM components which might have a role in the maintenance of hESCs. I have first investigated human placental stromal fibroblasts and immortalised human placental stromal fibroblasts for the support hESC pluripotency as an anlternative feeder type to conventional mouse embryonic fibroblasts. Secondly, the matrices derived from hPSFs and ihPSFs were assessed for their ability to support hESC pluripotency. Tandem mass spectrometry was used to identify ECM components released by human feeders in order to characterise the range of extracellular matrix proteins that support the growth of self-renewing hESCs. The majority of the molecules was shared between the cell types irrespective of hPSF cell derived matrix was not being supportive for hESC pluripotency, with some ECM components being unique ihPSFs. Collagen VI, tenascin C and versican were tested for hESC attachment and as substrates for feeder-free culture system in order to develop an optimised feeder-free system. Furthermore, integrin receptor profile of different hESC lines was also determined in order to identify the mechanisms of substrate attachment. Integrin attachment was shown to be vital for hESC engagement to fibronectin and vitronectin in feeder-free systems. The components of the integrin signalling machinery were identified in hESCs and the significance of integrin-mediated signalling in hESC self-renewal was demonstrated by blocking integrin β1 on fibronectin and integrin aVβ5 on vitronectin. Moreover, intracellular signalling mediator c-Src was shown to involve in ECMregulated signalling by affecting the phosphorylation of Focal Adhesion Kinase. Inhibition of Src led to a decrease in the expression of pluripotency-associated markers. Finally, the effects of growth factor supplementation on the maintenance of pluripotency in defined feeder-free conditions were studied by withdrawal of growth factors and blocking FGF Receptors. FGF-2 was shown to be essential for long-term self-renewal while the effects on pluripotency deteriorated in the absence of both FGF-2 and Activin A. Taken together this project highlighted the importance of substrate attachment and growth factors on the regulation of hESC self-renewal.

Fong Shu Pan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 147-167). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / LIST OF PUBLICATIONS --- p.ii / ABSTRACT --- p.iii / ABSTRACT [IN CHINESE] --- p.vii / TABLE OF CONTENT --- p.ix / LISTS OF FIGURES --- p.xv / LIST OF TABLES --- p.xxi / LIST OF ABBREVATIONS --- p.xxii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Embryonic stem (ES) cells --- p.1 / Chapter 1.2 --- Stem cell plasticity --- p.5 / Chapter 1.2.1 --- Differentiation and trans-differentiation of lineage-restricted stem cells --- p.5 / Chapter 1.2.1.1 --- Multilineage differentiation in-vitro --- p.5 / Chapter 1.2.1.2 --- Trans-differentiation --- p.6 / Chapter 1.2.2 --- Prospective applications of stem cells --- p.7 / Chapter 1.2.2.1 --- Basic research on development --- p.7 / Chapter 1.2.2.2 --- Study of human disease --- p.7 / Chapter 1.2.2.3 --- Cancer research --- p.7 / Chapter 1.2.2.4 --- Drug screening --- p.8 / Chapter 1.2.2.5 --- Cell therapy --- p.8 / Chapter 1.3 --- Neuro-degenerative diseases and cell therapy --- p.9 / Chapter 1.3.1 --- Neuro-degenerative diseases --- p.9 / Chapter 1.3.2 --- Neuro-regeneration --- p.10 / Chapter 1.3.3 --- Cell sources for neuro-regenerative therapy --- p.11 / Chapter 1.3.3.1 --- Comparison of stem cells --- p.11 / Chapter 1.3.3.2 --- Stem cells in neuro-regenerative therapy --- p.12 / Chapter 1.4 --- In-vitro derivation into neural lineage --- p.17 / Chapter 1.4.1 --- In-vitro induction strategies available --- p.17 / Chapter 1.4.1.1 --- Chemical agents --- p.18 / Chapter 1.4.1.1.1 --- Retinoic acid (RA) --- p.18 / Chapter 1.4.1.1.2 --- Ascorbic acid --- p.19 / Chapter 1.4.1.2 --- Growth factors/cytokines --- p.19 / Chapter 1.4.1.2.1 --- Neurotrophins --- p.20 / Chapter 1.4.1.2.2 --- Stimulants --- p.20 / Chapter 1.4.1.2.3 --- Signalling molecules --- p.21 / Chapter 1.4.1.3 --- Culture Selection --- p.23 / Chapter 1.4.1.3.1 --- Conditions --- p.23 / Chapter 1.4.1.3.2 --- Medium --- p.23 / Chapter 1.4.1.4 --- Transfection of regulator genes using viral vector --- p.24 / Chapter 1.4.1.5 --- Stromal cell-derived inducing activity (SDIA) --- p.26 / Chapter Chapter 2 --- Aims --- p.28 / Chapter 2.1 --- Hypothesis and study objectives --- p.28 / Chapter 2.1.1 --- Soliciting an optimal method for ES cell propagation --- p.28 / Chapter 2.1.2 --- Pursuing alternative SDIA --- p.29 / Chapter Chapter 3 --- Materials and Methods --- p.33 / Chapter 3.1 --- Chemicals and Reagents --- p.33 / Chapter 3.1.1 --- Cell Culture --- p.33 / Chapter 3.1.2 --- Immunohistochemistry and staining --- p.35 / Chapter 3.1.3 --- Molecular Biology --- p.36 / Chapter 3.2 --- Consumable --- p.37 / Chapter 3.3 --- Cell lines --- p.39 / Chapter 3.3.1 --- Feeder cells --- p.39 / Chapter 3.3.1.1 --- Primary mouse embryonic fibroblasts --- p.39 / Chapter 3.3.1.2 --- STO --- p.39 / Chapter 3.3.1.3 --- L Cells --- p.40 / Chapter 3.3.1.4 --- L-Wnt-3A Cells --- p.40 / Chapter 3.3.1.5 --- C17.2 --- p.40 / Chapter 3.3.2 --- ES cells --- p.41 / Chapter 3.3.2.1 --- ES-D3 --- p.41 / Chapter 3.3.2.2 --- ES-E14TG2a --- p.41 / Chapter 3.4 --- In-house prepared solutions --- p.42 / Chapter 3.4.1 --- "Stock solution of Insulin, Transferrin, Selentine (ITS) Supplement" --- p.42 / Chapter 3.4.2 --- Enriched Knock-Out Dulbecco's Modified Eagle's Medium (KO DMEM) --- p.42 / Chapter 3.4.3 --- Mitomycin C solution --- p.42 / Chapter 3.4.4 --- Gelatin solution 0.1% --- p.42 / Chapter 3.4.5 --- p-mercaptoethanol solution --- p.43 / Chapter 3.4.5.1 --- (3-mercaptoethanol solution 0.1M --- p.43 / Chapter 3.4.5.2 --- P-mercaptoethanol solution 0.1M --- p.43 / Chapter 3.4.5.3 --- p-mercaptoethanol solution 0.1M for preparation of culture medium --- p.43 / Chapter 3.4.6 --- ALL-trans retinoic acid --- p.43 / Chapter 3.4.6.1 --- ALL-trans retinoic acid stock solution 0.01M --- p.43 / Chapter 3.4.6.2 --- ALL-trans retinoic acid working solution lμM --- p.43 / Chapter 3.4.7 --- Paraformaldehyde solution 4% (PFA) --- p.44 / Chapter 3.4.8 --- TritoxX-100 solution --- p.44 / Chapter 3.4.8.1 --- Tritox X-100 solution 3% --- p.44 / Chapter 3.4.8.2 --- Tritox X-100 solution 0.3% --- p.44 / Chapter 3.4.9 --- Popidium iodide solution lug/mL (PI) --- p.44 / Chapter 3.4.10 --- Geneticin solution --- p.45 / Chapter 3.4.10.1 --- Geneticin solution 50mg/mL --- p.45 / Chapter 3.4.10.2 --- Geneticin solution 5mg/mL --- p.45 / Chapter 3.4.11 --- Poly-L-ornithine solution --- p.45 / Chapter 3.4.12 --- Laminin solution --- p.45 / Chapter 3.4.13 --- Maintenance medium for cell feeders --- p.46 / Chapter 3.4.14 --- Mitomycin C inactivation medium --- p.46 / Chapter 3.4.15 --- Freezing medium --- p.46 / Chapter 3.4.16 --- Propagation medium for ES cells --- p.47 / Chapter 3.4.16.1 --- Serum-based propagation medium for ES cells --- p.47 / Chapter 3.4.16.2 --- Serum-free propagation medium for ES cells --- p.47 / Chapter 3.4.16.3 --- Serum-free induction medium for ES cells --- p.48 / Chapter 3.4.16.3.1 --- Serum-free induction medium 1 --- p.48 / Chapter 3.4.16.3.2 --- Serum-free induction medium II --- p.48 / Chapter 3.4.16.3.3 --- Serum-free induction medium III --- p.48 / Chapter 3.5 --- Equipments --- p.49 / Chapter 3.6 --- Methods --- p.50 / Chapter 3.6.1 --- Cell Culture --- p.50 / Chapter 3.6.1.1 --- Preparation of round cover-slips --- p.50 / Chapter 3.6.1.2 --- Gelatinization of tissue culture wares --- p.51 / Chapter 3.6.1.3 --- Poly-L-ornithine and laminin coating --- p.51 / Chapter 3.6.1.4 --- Thawing frozen cells --- p.51 / Chapter 3.6.1.5 --- Passage of adherent culture --- p.52 / Chapter 3.6.1.6 --- Cell count --- p.52 / Chapter 3.6.1.7 --- Cytospin --- p.53 / Chapter 3.6.1.8 --- Cell viability test --- p.53 / Chapter 3.6.1.9 --- Cryopreservation --- p.53 / Chapter 3.6.1.10 --- Preparation of primary mouse embryonic fibroblast (PMEF) --- p.54 / Chapter 3.6.1.11 --- Mitomycin C inactivation of feeder cells --- p.55 / Chapter 3.6.1.12 --- Gamma irradiation of various feeders --- p.55 / Chapter 3.6.1.13 --- Preparation of CM from feeder cells --- p.56 / Chapter 3.6.1.14 --- Propagation of ES cells in serum-based medium --- p.56 / Chapter 3.6.1.15 --- Propagation of ES cell in serum-free medium --- p.56 / Chapter 3.6.1.16 --- Neural differentiation using all-trans retinoic acid --- p.57 / Chapter 3.6.1.17 --- Stromal cells-derived inducing activity --- p.58 / Chapter 3.6.1.18 --- BrdU labeling of the cell products --- p.59 / Chapter 3.6.2 --- Molecular analysis --- p.60 / Chapter 3.6.2.1 --- RNA extraction --- p.60 / Chapter 3.6.2.2 --- RNA quantitation --- p.60 / Chapter 3.6.2.3 --- Reverse Transcription of the First Strand complementary DNA --- p.61 / Chapter 3.6.2.4 --- Polymerase chain reaction --- p.61 / Chapter 3.6.2.5 --- RNA Integrity Check --- p.66 / Chapter 3.6.2.6 --- Electrophoresis and visualization of gene products --- p.66 / Chapter 3.6.3 --- Immunofluoresent staining --- p.66 / Chapter 3.6.4 --- In-vivo studies --- p.69 / Chapter 3.6.4.1 --- Induction of cerebral ischaemia in mice --- p.69 / Chapter 3.6.4.2 --- Transplantation --- p.69 / Chapter 3.6.4.3 --- Assessment of learning ability and memory --- p.70 / Chapter 3.6.5 --- Histological analysis --- p.70 / Chapter 3.6.5.1 --- Animal sacrifice for brain harvest --- p.70 / Chapter 3.6.5.2 --- Cryosectioning --- p.71 / Chapter 3.6.5.3 --- Paraffin sectioning --- p.71 / Chapter 3.6.5.4 --- Haematoxylin and eosin staining --- p.72 / Chapter 3.7 --- Data analysis --- p.73 / Chapter Chapter 4 --- Results --- p.74 / Chapter 4.1 --- ES cell maintenance --- p.74 / Chapter 4.1.1 --- Serum effect --- p.74 / Chapter 4.1.2 --- Feeder effect --- p.79 / Chapter 4.1.3 --- Serum-free and feeder-free condition --- p.86 / Chapter 4.1.4 --- Overall effect --- p.89 / Chapter 4.2 --- ES cell Induction --- p.91 / Chapter 4.2.1 --- Retinoic acid --- p.91 / Chapter 4.2.2 --- Stromal cell-derived inducing activity --- p.96 / Chapter 4.2.2.1 --- Molecular characterization of candidate stromal cells --- p.96 / Chapter 4.2.2.2 --- Direct contact co-culture --- p.98 / Chapter 4.2.2.3 --- Non-contact co-culture --- p.100 / Chapter 4.2.2.4 --- Cultures in CM --- p.109 / Chapter 4.3. --- ES cell Differentiation --- p.115 / Chapter 4.4 --- In vivo study of ES cell-derived cell products --- p.117 / Chapter 4.4.1 --- Animal preparation --- p.117 / Chapter 4.4.2 --- Cell preparation --- p.117 / Chapter 4.4.3 --- Cell implantation --- p.117 / Chapter 4.4.4 --- Behaviour Monitoring --- p.121 / Chapter 4.4.5 --- Histology of cell-implanted brain --- p.125 / Chapter Chapter 5 --- Discussion --- p.129 / Chapter Chapter 6 --- Conclusion --- p.144 / References --- p.147

Mammalian DNA methylathion is a chemical reaction catalyzed by DNA methyltransferases (DNMTs) and involves the addition of a methyl group from the methyl donor SAM to the carbon 5 position of cytosine (C) in a CpG dinucleotide. Specifically, DNA methylation is essential for normal development and is involved in numerous key mechanisms such as genomic imprinting, X-chromosome inactivation, suppression of repetitive elements and may be involved in the regulation of single-copy gene expression. In the human genome the majority of CpGs are methylated whereas regions with high density of CpG sites, termed CpG islands and often co-localized within gene promoters, are typically free of this mark. Recently, a new modified cytosine, 5-hydroxymhetylcytosine (5-hmC), was identified and found at significant levels in mouse brain and both mouse and human embryonic stem (ES) cells. The conversion of 5-mC to 5-hmC is catalyzed by the ten-eleven translocation (TET) proteins of the 2-oxoglutarate (2OG)-and Fe(II)-dependent oxygenase superfamily. Many studies were conducted since the identification of 5-hmC and significant levels of 5-mC hydroxylation were found in many other mouse and human tissues. Importantly, many of the techniques used for 5-mC detection, such as bisulphite sequencing and methyl-sensitive restriction digestion, are incapable of distinguishing between 5mC and 5hmC implying the necessity not only to develop techniques specific for 5-hmC characterization but also reevaluation of previously published 5mC data. The biological function of 5-hmC is unknown however many recent studies have suggested a role for 5-hmC as an intermediate of either passive or active demethylation. The majority of studies of 5- hmC and TETs have used mouse ES cells as model system. Therefore, very little is known about 5-hmC patterns and TET expression within and between normal tissues. During my PhD, I used the recently developed 5-hmC-specific antibody for tiling microarrays and 5hmC-qPCR to examine both global 5hmC content and locus-specific patterns of 5hmC in several normal human tissues and breast cancer. I found that global 5-hmC content is highly variable between tissues compared to global 5-mC content. Moreover, TETs genes are highly expressed in most of tissues tested. Importantly, both global 5-hmC content and TETs genes are rapidly and significantly reduced as consequence of adaptation of cells from normal human tissue to cell culture. Using the 5hmC-specific antibody for tiling microarrays and 5-hmC-qPCR to profile locus-specific patterns of 5hmC, I found that 5-hmC patterns are tissue-specific in human samples. In addition, comparing array data to RNA-seq data, 5- hmC was found to co-localize at gene bodies of active genes. Moreover, despite the global 5-hmC reduction in cell lines, 5-hmC content remains enriched in some specific loci. In summary, my results show that tissue type is a major modifier of both global and locus-specific 5hmC at genes in normal human tissues. Furthermore, I also show that both TET gene expression and 5hmC content are significantly reduced and 5-hmC profiles reprogrammed during the passage from tissues to cell culture.

The potential of embryonic stem (ES) cells to generate any developmental or adult cell type holds much promise for regenerative medicine and in vitro modelling of development and disease. Haematopoietic stem cells (HSCs) regenerate all lineages of the blood throughout adult life and are essential for the treatment of a vast number of haematalogic disorders. Current sources of HSCs for clinical use and research, including adult bone marrow, peripheral blood stem cells and umbilical cord blood, are limited by the number of HSCs they contain and by the availability of a suitable donor. A system that generates a reliable source of HSCs from ES cells would therefore be an ideal alternative. While much progress has been made in the generation of downstream lineages of the haematopoietic system, progress in the derivation of HSCs capable of long-term self-renewal and multilineage reconstitution from ES cells has been limited. Understanding of the developmental steps leading to HSC emergence in the embryo has been advancing in recent years. In particular, precursors of HSCs (preHSCs) have been isolated from the mouse embryo, characterised and matured into HSCs ex vivo using the specialised conditions of aggregate culture systems (Taoudi et al 2008, Rybtsov et al 2011). We hypothesised that application of the aggregate culture system in the differentiation of ES cells could provide a missing link in the in vitro generation of HSCs. Here I have developed a novel ES cell differentiation system that employs the specialised conditions of the aggregate culture system, after an initial stage of mesoderm differentiation. I show that this system creates an environment for efficient haematopoietic and endothelial progenitor formation and generates cells of a preHSC type I (VE-Cadherin+CD45-CD41lo) and preHSC type II (VE-Cadhein+CD45+) surface phenotype. Notably, the system gives rise to cells that achieve low-levels of haematopoietic repopulation in sublethally irradiated NSG mice. The low-level repopulating cells persist for over 4 months in animals and show both myeloid and lymphoid potential. I identify genes that are expressed in cells of a preHSC II surface marker-phenotype from the E11.5 dorsal aorta, but not in cells of this phenotype from the E11.5 Yolk sac or differentiated ES cells. I also show that enforced expression of Notch downstream target Hes1 in Flk1+ mesoderm during ES cell differentiation does not improve levels of ES-derived repopulation.

The governance of cell fate decisions during development is a fundamental biological problem. An important aspect of this is how cells exit a multipotent state and choose their fates in a correct manner and proportion. To tackle an aspect of this problem, I have focused on 2 multipotent models: one infinite self-renewal pluripotency in an artificial environment, and the other, bipotent progenitors in the context of the mouse embryo. The first model aimed to explore the effects of chromatin-associated factors on the ability of pluripotent mouse Embryonic Stem Cells (ESCs) to self-renew, via monitoring gene expression heterogeneity of key genes. The second model focused on Neural Mesodermal Progenitors (NMPs), a bipotent cell population found in the Caudal Lateral Epiblast (CLE) of mammalian embryos, which contributes to the spinal cord and paraxial mesoderm. The aim here was to derive NMPs in vitro which exhibit similar gene expression patterns and function like their mouse embryo counterpart and study their renewal and differentiation in detail. The first multipotent model explores the effects of chromatin remodelling on cell fate decisions, specifically investigating the consequences of inhibiting the histone acetyltransferase Kat2a on the ESCs fate. I found first, that the effect of Kat2a inhibition depends on the pluripotent state of the cells; cells in a ground state exhibit a resistance to Kat2a inhibition and maintain their pluripotency, whereas cells in a naïve state experience destabilization of their pluripotency gene regulatory network and shift towards differentiation. Second, that Kat2a inhibition in the naïve state results in a decline in the gene expression noise strength contributed by the promoter activation operation, which suggests that when ESCs become lineage-primed their transcriptional noise is constrained. In the bipotent model, the NMPs are identified as cells coexpressing Sox2 and T/Brachyury, a criterion used to derive NMP-like cells from ESCs in vitro. Comparison between the different NMPs protocols stresses that Epiblast Stem Cells (EpiSCs) are an effective source for deriving a multipotent population resembling the embryo Caudal Epiblast (CE), that generates NMPs. Furthermore, self-organization of this CE-like population, resulted in axially organized aggregates. Exploiting the mouse embryo CLE as a reference shows that EpiSCs derived NMPs, monolayers and aggregates, consist of a high proportion of cells with the embryo's NMP signature. Importantly, studying this system in vitro sheds light on the sequence of events which lead to NMP emergence in vivo. On this basis, I conclude that understanding the initial state of cells at a crossroads is important to reveal the limitations it imposes on the cells fate exploration, hence makes it possible to mimic more precisely the fate decision process in vitro.