Thèses sur le sujet « SOX6, Cell differentiation »

SOX6 is a transcription factor (TF) belonging to the Sry-related HMG-box TFs family. It controls terminal differentiation and lineage specification of many cell types, by mediating cell cycle withdrawal and activation of lineage specific genes. In hematopoiesis, Sox6 sustains cell survival, proliferation and terminal maturation of murine erythroid cells. To explore the role of SOX6 in human erythropoiesis, we first overexpressed it in erythroleukemic BCR-ABL+ K562 cell line. In these cells, Sox6 overexpression induces a strong erythroid differentiation coupled with growth arrest. This last effect may be mediated by SOCS3 (Suppressor Of Cytokines Signalling 3), that is a direct transcriptional target of SOX6. SOCS3 represses two important pathways in erythropoiesis: the IGF1/IGF1R pathway, in response to insulin-like growth factor 1 (IGF1) and the Epo-Jak-STAT pathway, in response to erythropoietin. In particular, in K562 cells, proliferation depends on autocrine IGF1 signalling, induced by BCR-ABL. Cells overexpressing Sox6 or SOCS3 display a reduction in IGF1 expression, suggesting that SOX6 plays a pivotal role in blocking cell proliferation through SOCS3 upregulation and by inhibiting the BCR-ABL-dependent IGF1 signalling. Leukemias and myelodisplasias are characterized by a block in differentiation leading to an excess of proliferating immature cells. Since SOX6 is a potent inducer of growth arrest and differentiation, I investigated the effect of its ectopic expression in different model systems of leukemia, to enforce these cells to overcome their pathological conditions. Therefore, I used leukemic model systems that varies for their degrees of differentiation/maturation and to the presence/absence of specific genetic lesions (i.e. BCR-ABL fusion oncogene, JAK2V617F mutation). Sox6 overexpression blocks proliferation in all the tested BCR-ABL+ cell lines (erythroleukemic K562, megakaryoblastic MEG.01) and surprisingly even in the lymphoblastic SUPB15 and B-ALL BCR-ABL GFP+. The JAK2V617F mutation, typical of myelodysplastic syndromes, makes cells unresponsive to SOCS3. Remarkably, in JAK2V617F+ cell lines (thrombocythemic UKE1 and SET2; erythroleukemic HEL), Sox6 overexpression induces a block in cellular proliferation strictly dependent on the copy number of the JAK2V617F alleles, further confirming SOCS3 as a key effector of SOX6. A second aspect controlled by SOX6 is the activation of a set of specific erythroid genes that lead erythroblastic progenitors to undergo terminal differentiation. Indeed, Sox6 overexpression enhances erythroid differentiation in bipotent megakaryoblastic and erythroleukemic cells (K562 and HEL) and enforces megakaryoblastic cells towards an erythroid fate (MEG.01, SET2 and UKE1). Surprisingly, Sox6 overexpression is capable of downregulating genes essential for B-cells identity (i.e. PAX5), whereas it is not sufficient to activate an erythroid program in lymphoblastic cells (SUPB15 and B-ALL BCR-ABL GFP+). Taken together, these findings suggest that SOX6 plays a major role in activating cell cycle withdrawal, acting on SOCS3, and interferes with cell fate decision by downregulating specific cellular programs and enforcing, where possible, an erythroid lineage choice in leukemic and myelodysplastic cell lines. Finally, I explored the impact of SOX6 ectopic expression in B-ALL BCR-ABL GFP+ capability to engraft and generate leukemia in C57BL/6J mice. B-ALL BCR-ABL GFP+ SOX6+ cells are not capable to engraft recipient mice, suggesting that SOX6 overexpression in such cellular model interferes with the onset of leukemia. Taken together, these results strongly point to a pivotal role for SOX6 in suppressing cells proliferation both in vitro and in vivo, and pave the way for the identification of potential druggable targets to develop new therapeutical approaches for leukemic and myelodysplastic syndromes.

The main focus of our laboratory is clarifying the roles of DNA binding proteins belonging to the HMG box superfamily during embryonic development. In particular, I studied Sox18 and Sox7 in zebrafish and our data show that they act redundantly in vascular development. Mutations in SOX18 have been associated with Hypotrichosis-Lymphedema-Telangiectasia (HLT), a human syndrome combining defects in hair follicle, blood and lymphatic vessels development. Similarly, spontaneous mutations in Sox18 underlie cardiovascular and hair follicle defects in ragged mice, including symptoms of lymphatic dysfunction. On the other hand, mice null for Sox18 display only mild coat defects. This suggests that other Sox proteins may compensate for the absence of Sox18. zfsox18 and zfsox7 genes are co-expressed in endothelial cells and their precursors. Knock down of either genes had minimal effects on blood vessels, however their simultaneous knockdown selectively affects trunk/tail blood circulation: endothelial cells that fail to acquire the proper arteriovenous identity and multiple fusions are present between the major axial vessels. To identify the hypothetical targets of sox18 and sox7 I performed a genome wide analysis using Affymetrix chip. We compared the expression of control embryos and double knockdown at two different time points: during initial steps of blood vessels formation and around the time when circulation begins. Functional studies of the most interesting identified genes will follow.

<i>Sox1</i> is upregulated during ES cell differentiation into neural precursors, and its misexpression causes an EC cell line to differentiate into neurons. <i>Sox1 </i>expression during nervous system development is associated with proliferating cells of the CNS, expression being lost as cells exist mitosis and terminally differentiate. Its expression pattern is more restricted than that of most other markers for early neural cells such as nestin, making it a good marker gene for the study of neural development both <i>in vivo</i> and <i>in vitro</i> from ES cells. Here I have used gene targeting to generate a reporter ES cell line (46C) in which the <i>Sox1</i> open reading frame is replaced by the gene encoding enhanced green fluorescent protein linked to a selectable marker. The use of EGFP enables the observation of <i>Sox1</i> expression in live cells. The expression of the reporter faithfully recapitulates the normal expression of <i>Sox1</i> <i>in vivo </i>in mice generated from the 46C cells. This cell line has been used to analyse the differentiation of ES cells to neural fates, in particular to characterise a newly discovered, monoculture differentiation system. <i>Sox1-</i>EGFP expression is monitored by flow cytometry, which enables quantification of the differentiation process. The effect of proteins and inhibitors implicated in neural determination has been monitored quantitatively and over time using this system. Acquisition of neural fate occurs rapidly in the absence of any inducers or serum, and without formation of multicellular aggregates. BMP-4 completely blocks this neural fate specification, similarly to the situation in the frog and the chick. Collectively, the results indicate that restriction of ES cells pluripotency to neural fates occurs in a manner resembling default neural induction in amphibians.

The transcription factor Sox2 has a key role not only in maintaining stem state but also in specification of neural fate of embryonic cells. Multiple regulatory elements have been identified in the Sox2 locus (Uchikawa et al, 2003). In the developing embryo, these regulatory elements are activated differentially in time and space. We studied the activity of 25 defined regulatory elements of the Sox2 promoter in three different lines of chick ES cells. By transfection of plasmids encoding Enhanced Green Fluorescent Protein (EGFP) and the minimal promoter thymidine kinase (tk) coupled with individual Sox2 regulatory elements we find that the Sox2 enhancer N2 has the highest activity in proliferating chick cell lines compared with other enhancer regions. Under conditions that induce ES cells to differentiate into neurons the activity of the N2 enhancer increased along with an increase in levels of expression of Sox2 mRNA. Further analysis of the N2 enhancer sequence identified two subregions with 176 and 73 base pairs (bp) which are highly conserved between chick, mouse and man. Functional studies performed with the tk-EGFP reporter plasmids under the control of five regulatory sequences containing the mouse N2 enhancer in its full length, its two sub-regions (176 and 73 bp) or sequences composed of the full length of the mouse N2 from which each of the two sub-regions 176 bp and 73 bp has been deleted confirmed that the two sub-regions of the N2 enhancer account for its activity in both proliferating cES cells as well as their induced neural differentiation state. These findings suggest that N2 core regulatory regions encode conserved instructions required to direct expression of Sox2 both in embryonic stem cells induced to neural differentiation and in the neural plate of the embryo itself.

This study examined the role of heparan sulfate proteoglycans (HSPGs) in neural lineage differentiation of human mesenchymal stem cells (hMSCs). Several HSPGs were identified as potential new targets controlling neural fate specification and may be applied to the development of improved models to examine and repair brain damage. hMSCs were characterised throughout extended in vitro expansion for neural lineage potential (neurons, astrocytes, oligodendrocytes) and differentiated using terminal differentiation and intermediate sphere formation. Brain damage and neurological disorders caused by injury or disease affect a large number of people often resulting in lifelong disabilities. Multipotent mesenchymal stem cells have a large capacity for self-renewal and provide an excellent model to examine the regulation and contribution of both stem cells and their surrounding microenvironment to the repair of neural tissue damage.

Thesis (Ph.D.)--Kent State University, 2005.<br>Title from PDF t.p. (viewed Sept. 5, 2006). Advisor: Walter E. Horton. Keywords: chondrocytes; osteoarthritis; Sox9; Bcl-2; MEK-ERK 1/2. Includes bibliographical references (p. 91-106).

The aims of this PhD research were: to examine molecular mechanisms underlying the transcriptional regulation of the Sox2 gene during forebrain development; to examine the role of Sox2 for the proper neuronal differentiation of neural stem cells; and to examine the role of Sox2 in controlling the maintenance of neural stem cells (in vivo and in vitro). The aim of the first work (Chapter 1) was to investigate the transcription factors and the regulatory sequences that control transcription of the Sox2 gene in the developing brain and neural stem cells. Our laboratory previously identified Sox2 regulatory sequences able to drive expression of a reporter β-geo transgene to neural stem cells of the brain in transgenic mice. I focused on two mouse forebrain-specific enhancers able to recapitulate Sox2 telencephalic expression throughout forebrain development, also active in neural stem cells of the adult and embryonic brain (Sox2 5’ and 3’ enhancers). This work showed that Emx2 acts as a direct transcriptional repressor of both Sox2 telencephalic enhancers, acting in two different ways to repress their transcriptional activity: by directly binding to a specific site within these regulatory elements, thus preventing the binding of activators, or possibly by protein to protein interaction sequestring the activators, thus antagonizing their activity. By the study of double mutant mice (expressing reduced levels of Sox2 and Emx2) we further found that Emx2 deficiency counteracts (at least in part) the deleterious effects of Sox2 deficiency on neural stem cell proliferation ability in the postnatal hippocampus, and also rescued other brain morphological abnormalities of Sox2-deficient mutants. It is likely possible that a simultaneous decrease of Emx2 levels (a Sox2 repressor) may antagonize these defects, by restoring Sox2 levels. In the second line of my research (Chapter 2) we performed in vitro differentiation studies on neural stem cells cultured from embryonic and adult brains of Sox2 “knockdown” mutants (expressing reduced levels of Sox2) where Sox2 deficiency impairs neuronal differentiation. In particular, my contribution to this work was to evaluate the in vitro differentiation defects of Sox2 mutant neurospheres by immunofluorescence staining for different glial and neuronal markers. Strikingly, I observed that mutant cells produce reduced numbers of mature neurons (in particular GABAergic neurons), but generate normal glia. Most of the cells belonging to the neuronal lineage failed to progress to mature neurons showing morphological abnormalities. To evaluate if restoration of Sox2 levels is able to rescue the differentiation defects of mutant cells, I engineered Sox2-expressing lentiviral vector, which I used to infect neural cells at early or late differentiation stages. I found that, Sox2 overexpression is able to rescue the neuronal maturation defects of mutant cells only if administered at early stages of differentiation. Further, I observed that Sox2 suppresses the endogenous GFAP gene, a marker of glial differentiation. These results suggests that Sox2 is required in early in vitro differentiating neuronal cells, for maturation and for suppression of alternative lineage markers. The third research (Chapter 3) investigated neurogenesis and neural stem cells properties in mice carrying a conditional mutation in the Sox2 gene (Sox2flox). Here, Sox2 was deleted via a nestin-Cre transgene that leads to complete Sox2 loss in the central nervous system by 12.5 dpc. These studies showed that embryonic neurogenesis was not importantly defective, however shortly after birth, NSC and neurogenesis are completely lost in the hippocampus. The expression of cytokine-encoding genes, essential for stem cell niche, is also strongly perturbed and leads to impaired stem cell maintenance (in vivo and in vitro). In vitro, NSC cultures derived from Sox2-deleted forebrain become rapidly exhausted, losing their proliferation and self-renewal properties. In Sox2-deleted neurospheres, Shh is extremely downregulated. However, the conditioned medium from wild type NSC cultures or the administration of a Shh agonist efficiently rescue the proliferation defects. These results suggest that the effect of Sox2 on neural stem cells growth and maintenance is partially mediated by Shh secretion, and that the Shh gene must be a direct target of Sox2. To confirm this hypothesis, I infected Sox2-deleted NSC with a Sox2-IRES-GFP expressing lentivirus just prior to the beginning of the growh decline, and I observed that the re-expression of Sox2 induces the ability to re-express Shh and rescues the formation of neurosphere. These findings indicate that NSC control their status, at least in part, through non cell-autonomous mechanisms (such as activation of important cytochine-encoding genes) which depend on Sox2.

Dans le contexte de la physiologie comparée de la reproduction, les amphibiens sont peu étudiés. Le travail réalisé durant cette thèse visait à analyser des marqueurs de différenciation testiculaire chez l'urodèle Pleurodeles waltl, dont le déterminisme génétique du sexe (ZZ/ZW) peut être influencé par la température. Nos études ont d'abord porté sur le gène sox9 marqueur de la différenciation testiculaire chez les vertébrés supérieurs. Le gène cloné chez le pleurodèle montre une bonne conservation par rapport aux autres vertébrés. Son expression plus élevée dans la gonade mâle n'apparaît que tardivement suggérant qu'il n'est probablement pas impliqué dans les stades précoces de la différenciation testiculaire. En outre, son expression dans le mésonéphros rend difficile son utilisation comme marqueur de différenciation testiculaire. Nous avons ensuite étudié l'Amh, hormone testiculaire impliquée dans la régression des canaux de Müller chez de nombreux vertébrés. Son expression spécifique de la gonade, précocement plus élevée chez les larves ZZ que les ZW en font un excellent marqueur de la différenciation testiculaire. Le fait que les pleurodèles mâles voient les canaux de Müller persister malgré la présence d'Amh suggère que la fonction primaire de cette hormone était en relation avec la différenciation gonadique et que la fonction de régression des canaux de Müller n'est apparue que secondairement au cours de l'évolution. Ces marqueurs ont été mis à profit pour caractériser le phénotype gonadique lors d'inversions sexuelles ou lors de développements en absence de cellules germinales. Ils ont permis de montrer que les cellules germinales ne semblent pas jouer de rôle dans la différenciation gonadique du pleurodèle<br>In the context of comparative physiology of reproduction, amphibians are poorly studied. This work was dedicated to the analysis of testis differentiation markers in the newt Pleurodeles waltl, which shows a ZZ/ZW genetic mode of sex determination that can be affected by temperature. First, we studied sox9, a testis differentiation marker well characterized in many higher vertebrates. The gene cloned in Pleurodeles shows a good level of identity with other vertebrates. The testis-enriched expression appears late during the testis differentiation process indicating that it is probably not involved in the early steps of testis differentiation. Its use as a marker of testicular differentiation proved difficult since it is expressed not only in the gonads but also in the mesonephros. Then, we studied amh, a testis hormone responsible for müllerian duct regression in many vertebrates. Its early expression in the gonad, significantly higher in male than in female larvae makes it an excellent marker for testis differentiation. Since in Pleurodeles waltl, Müllerian ducts persist in males, it suggests that during the course of evolution, the function of Amh on the regression of Müllerian ducts appeared secondarily after its role in gonadal differentiation. These markers have been used to characterize the gonadal phenotype during sex reversal, or in gonads developed in the absence of germ cells. They showed that these cells do not seem to play a role in gonadal differentiation of Pleurodeles waltl

Cell based therapies require cells capable of self renewal and differentiation, and a prerequisite is the ability to prepare an effective dose of ex vivo expanded cells for autologous transplants. The in vivo identification of a source of physiologically relevant cell types suitable for cell therapies is therefore an integral part of tissue engineering. Bone marrow is the most easily accessible source of mesenchymal stem cells (MSCs), and harbours two distinct populations of adult stem cells; namely hematopoietic stem cells (HSCs) and bone mesenchymal stem cells (BMSCs). Unlike HSCs, there are yet no rigorous criteria for characterizing BMSCs. Changing understanding about the pluripotency of BMSCs in recent studies has expanded their potential application; however, the underlying molecular pathways which impart the features distinctive to BMSCs remain elusive. Furthermore, the sparse in vivo distribution of these cells imposes a clear limitation to their in vitro study. Also, when BMSCs are cultured in vitro there is a loss of the in vivo microenvironment which results in a progressive decline in proliferation potential and multipotentiality. This is further exacerbated with increased passage number, characterized by the onset of senescence related changes. Accordingly, establishing protocols for generating large numbers of BMSCs without affecting their differentiation potential is necessary. The principal aims of this thesis were to identify potential molecular factors for characterizing BMSCs from osteoarthritic patients, and also to attempt to establish culture protocols favourable for generating large number of BMSCs, while at the same time retaining their proliferation and differentiation potential. Previously published studies concerning clonal cells have demonstrated that BMSCs are heterogeneous populations of cells at various stages of growth. Some cells are higher in the hierarchy and represent the progenitors, while other cells occupy a lower position in the hierarchy and are therefore more committed to a particular lineage. This feature of BMSCs was made evident by the work of Mareddy et al., which involved generating clonal populations of BMSCs from bone marrow of osteoarthritic patients, by a single cell clonal culture method. Proliferation potential and differentiation capabilities were used to group cells into fast growing and slow growing clones. The study presented here is a continuation of the work of Mareddy et al. and employed immunological and array based techniques to identify the primary molecular factors involved in regulating phenotypic characteristics exhibited by contrasting clonal populations. The subtractive immunization (SI) was used to generate novel antibodies against favourably expressed proteins in the fast growing clonal cell population. The difference between the clonal populations at the transcriptional level was determined using a Stem Cell RT2 Profiler TM PCR Array which focuses on stem cell pathway gene expression. Monoclonal antibodies (mAb) generated by SI were able to effectively highlight differentially expressed antigenic determinants, as was evident by Western blot analysis and confocal microscopy. Co-immunoprecipitation, followed by mass spectroscopy analysis, identified a favourably expressed protein as the cytoskeletal protein vimentin. The stem cell gene array highlighted genes that were highly upregulated in the fast growing clonal cell population. Based on their functions these genes were grouped into growth factors, cell fate determination and maintenance of embryonic and neural stem cell renewal. Furthermore, on a closer analysis it was established that the cytoskeletal protein vimentin and nine out of ten genes identified by gene array were associated with chondrogenesis or cartilage repair, consistent with the potential role played by BMSCs in defect repair and maintaining tissue homeostasis, by modulating the gene expression pattern to compensate for degenerated cartilage in osteoarthritic tissues. The gene array also presented transcripts for embryonic lineage markers such as FOXA2 and Sox2, both of which were significantly over expressed in fast growing clonal populations. A recent groundbreaking study by Yamanaka et al imparted embryonic stem cell (ESCs) -like characteristic to somatic cells in a process termed nuclear reprogramming, by the ectopic expression of the genes Sox2, cMyc and Oct4. The expression of embryonic lineage markers in adult stem cells may be a mechanism by which the favourable behaviour of fast growing clonal cells is determined and suggests a possible active phenomenon of spontaneous reprogramming in fast growing clonal cells. The expression pattern of these critical molecular markers could be indicative of the competence of BMSCs. For this reason, the expression pattern of Sox2, Oct4 and cMyc, at various passages in heterogeneous BMSCs population and tissue derived cells (osteoblasts and chondrocytes), was investigated by a real-time PCR and immunoflourescence staining. A strong nuclear staining was observed for Sox2, Oct4 and cMyc, which gradually weakened accompanied with cytoplasmic translocation after several passage. The mRNA and protein expression of Sox2, Oct4 and cMyc peaked at the third passage for osteoblasts, chondrocytes and third passage for BMSCs, and declined with each subsequent passage, indicating towards a possible mechanism of spontaneous reprogramming. This study proposes that the progressive decline in proliferation potential and multipotentiality associated with increased passaging of BMSCs in vitro might be a consequence of loss of these propluripotency factors. We therefore hypothesise that the expression of these master genes is not an intrinsic cell function, but rather an outcome of interaction of the cells with their microenvironment; this was evident by the fact that when removed from their in vivo microenvironment, BMSCs undergo a rapid loss of stemness after only a few passages. One of the most interesting aspects of this study was the integration of factors in the culture conditions, which to some extent, mimicked the in vivo microenvironmental niche of the BMSCs. A number of studies have successfully established that the cellular niche is not an inert tissue component but is of prime importance. The total sum of stimuli from the microenvironment underpins the complex interplay of regulatory mechanisms which control multiple functions in stem cells most importantly stem cell renewal. Therefore, well characterised factors which affect BMSCs characteristics, such as fibronectin (FN) coating, and morphogens such as FGF2 and BMP4, were incorporated into the cell culture conditions. The experimental set up was designed to provide insight into the expression pattern of the stem cell related transcription factors Sox2, cMyc and Oct4, in BMSCs with respect to passaging and changes in culture conditions. Induction of these pluripotency markers in somatic cells by retroviral transfection has been shown to confer pluripotency and an ESCs like state. Our study demonstrated that all treatments could transiently induce the expression of Sox2, cMyc and Oct4, and favourably affect the proliferation potential of BMSCs. The combined effect of these treatments was able to induce and retain the endogenous nuclear expression of stem cell transcription factors in BMSCs over an extended number of in vitro passages. Our results therefore suggest that the transient induction and manipulation of endogenous expression of transcription factors critical for stemness can be achieved by modulating the culture conditions; the benefit of which is to circumvent the need for genetic manipulations. In summary, this study has explored the role of BMSCs in the diseased state of osteoarthritis, by employing transcriptional profiling along with SI. In particular this study pioneered the use of primary cells for generating novel antibodies by SI. We established that somatic cells and BMSCs have a basal level of expression of pluripotency markers. Furthermore, our study indicates that intrinsic signalling mechanisms of BMSCs are intimately linked with extrinsic cues from the microenvironment and that these signals appear to be critical for retaining the expression of genes to maintain cell stemness in long term in vitro culture. This project provides a basis for developing an “artificial niche” required for reversion of commitment and maintenance of BMSC in their uncommitted homeostatic state.

Les adipocytes sont les unités fonctionnelles du tissu adipeux (TA). Les adipocytes blancs du TA blanc assurent le stockage et la libération de l'énergie au sein de l'organisme, principalement sous forme d'acides gras1. A l'opposé, les adipocytes bruns du TA brun ont une grande capacité à consommer les acides gras par l'activité de la protéine UnCoupling Protein 1 (UCP1)2. Enfin, il a été observé des adipocytes UCP1+ dans le TA blanc, notamment en réponse à une exposition au froid3. Ces adipocytes sont appelés adipocytes beiges et sont issus de deux processus : d'une part via l'adipogenèse à partir des cellules souches/stromales mésenchymateuses du TA (ASC), et d'autre part par la conversion des adipocytes blancs en beiges4. Ce processus de conversion est réversible, ce qui montre le caractère très plastique de ces cellules. Le but de la thèse a été de caractériser les mécanismes moléculaires impliqués dans les processus d'adipogenèse et de plasticité cellulaire. Pour ce faire, nous avons utilisé des modèles innovants de culture d'ASC humaines et réalisé des expériences in vivo chez la souris. Compte tenu de la localisation péri-vasculaire et péricytaire des ASC in vivo5,6, nous nous sommes intéressés à l'utilisation du milieu Endothelial Growth Medium 2 (EGM2) pour leur expansion in vitro, comme une alternative aux méthodes de culture Standard (milieu de type Eagle's medium supplémenté en sérum de veau fœtal). Nos travaux ont montré que le TGFß1 contenu dans le sérum de culture altérait le caractère immature des ASC par leur engagement dans des voies de différenciation de type ostéoblastique, chondroblastique et vasculaire musculaire lisse. Aussi, grâce à sa faible quantité en sérum, et donc en TGFß1, le milieu EGM2 permet de conserver l'immaturité des ASC en culture ainsi que leurs fortes capacités à se différencier en adipocytes, notamment vers le phénotype beige. D'autre part, nous montrons que les ASC qui présentaient un fort potentiel à générer des adipocytes beiges sur-exprimaient la protéine SOX2. Nos résultats montrent que l'expression de SOX2 est positivement corrélée d'une part à la formation des adipocytes beiges et d'autre part à l'activation des adipocytes bruns in vivo chez la souris exposée au froid. [...]<br>Adipocytes are the functional units of adipose tissue (AT). Within white AT, white adipocytes contribute to both storage and release of energy within the organism, mainly in the form of fatty acids1. On the other hand, brown adipocytes, from brown AT, have a high capacity to consume fatty acids. This results from the activity of the UnCoupling Protein 1 (UCP1)2. Finally, UCP1+ adipocytes have been described in white AT, notably in response to cold exposure3. These adipocytes are named beige adipocytes and are generated through two pathways: on one hand via adipogenesis from adipose-derived mesenchymal stem/stromal cells (ASC), and on the other hand by conversion of white-to-beige adipocytes4. Being a reversible process, beige conversion highlights the plasticity of these cells. The aim of the thesis was to characterize the molecular mechanisms involved in both processes, by using culture models of human ASC and in vivo mice models. Given the perivascular and pericyte localization of ASC in vivo5,6, we investigated the use of Endothelial Growth Medium 2 (EGM2) for their in vitro expansion as an alternative to Standard culture conditions (Eagle's medium supplemented with fetal calf serum). Our results showed that the TGFß1 contained in serum of culture medium altered the relative immature state of ASC. Indeed, TGFß1 induces their commitment toward osteoblastic, chondroblastic or vascular smooth muscle lineage. Also, the small amount of serum in EGM2 medium, and thus low TGFß1 concentration, preserves ASC immaturity in culture, as well as their strong capacities to differentiate into adipocytes, including beige phenotype. We showed that ASC with high potential to generate beige adipocytes over-expressed SOX2 protein. Our results also showed that expression of SOX2 was positively correlated to both formation of beige adipocytes and to brown adipocytes activation in vivo in cold-exposed mice. In addition, using two types of human ASC models in vitro, we observed that SOX2 was overexpressed during adipogenesis, and even more when cells were differentiated into beige adipocytes. Thus, SOX2 appears to be a key factor involved in AT browning potential and adipocyte plasticity in vivo and in vitro. This thesis has allowed the access to a better understanding of the impact of culture conditions on the biology of ASC and highlighted molecules involved in the plasticity of adipocytes

Cardiovascular diseases are among the leading causes of death worldwide and particularly in the developed World. The search for new therapeutic approaches for improving the functions of the damaged heart is therefore a critical endeavour. Myocardial infarction, which can lead to heart failure, is associated with irreversible loss of functional cardiomyocytes. The loss of cardiomyocytes poses a major difficulty for treating the damaged heart since terminally differentiated cardiomyocytes have very limited regeneration potential. Currently, the only effective treatment for severe heart failure is heart transplantation but this option is limited by the acute shortage of donor hearts. The high incidence of heart diseases and the scarcity donor hearts underline the urgent need to find alternative therapeutic approaches for treating cardiovascular diseases. Pluripotent embryonic stem (ES) cells can differentiate into functional cardiomyocytes. Therefore the engraftment of ES cell-derived functional cardiomyocytes or cardiac progenitor cells into the damaged heart to regenerate healthy myocardial tissues may be used to treat damaged hearts. Stem cell-based therapy therefore holds a great potential as a very attractive alternative to heart transplant for treating heart failure and other cardiovascular diseases. A major obstacle to the realisation of stem cell-based therapy is the lack of donor cells and this in turn is due to the fact that, currently, the molecular mechanisms or the regulatory signal transduction mechanisms that are responsible for mediating ES cell differentiation into cardiomyocytes are not well understood. Overcoming this huge scientific challenge is absolutely necessary before the use of stem cell-derived cardiomyocytes to treat the damaged heart can become a reality. Therefore the aim of this thesis was to investigate the signal transduction pathways that are involved in the differentiation of stem cells into cardiomyocytes. The first objective was the establishment and use of cardiomyocyte differentiation models using H9c2 cells and P19 stem cells to accomplish the specific objectives of the thesis. The specific objectives of the thesis were, the investigation of the roles of (i) nitric oxide (ii) protein kinase C (PKC), (iii) p38 mitogen-activated protein kinase (p38 MAPK) (vi) phosphoinositide 3-kinase (PI3K) and (vi) nuclear factor-kappa B (NF-kB) signalling pathways in the differentiation of stem cells to cardiomyocytes and, more importantly, to identify where possible any points of convergence and potential cross-talk between pathways that may be critical for differentiation to occur. P19 cells were routinely cultured in alpha minimal essential medium (α-MEM) supplemented with 100 units/ml penicillin /100 μg/ml streptomycin and 10% foetal bovine serum (FBS). P19 cell differentiation was initiated by culturing the cells in microbiological plates in medium containing 0.8 % DMSO to form embryoid bodies (EB). This was followed by transfer of EBs to cell culture grade dishes after four days. H9c2 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% FBS. Differentiation was initiated by incubating the cells in medium containing 1% FBS. In both models, when drugs were employed, they were added to cells for one hour prior to initiating differentiation. Cell monolayers were monitored daily over a period of 12 or 14 days. H9c2 cells were monitored for morphological changes and P19 cells were monitored for beating cardiomyocytes. Lysates were generated in parallel for western blot analysis of changes in cardiac myosin heavy chain (MHC), ventricular myosin chain light chain 1(MLC-1v) or troponin I (cTnI) using specific monoclonal antibodies. H9c2 cells cultured in 1% serum underwent differentiation as shown by the timedependent formation of myotubes, accompanied by a parallel increase in expression of both MHC and MLC-1v. These changes were however not apparent until 4 to 6 days after growth arrest and increased with time, reaching a peak at day 12 to 14. P19 stem cells cultured in DMSO containing medium differentiated as shown by the timedependent appearance of beating cardiomyocytes and this was accompanied by the expression of cTnI. The differentiation of both P19 stem cells and H9c2 into cardiomyocytes was blocked by the PI3K inhibitor LY294002, PKC inhibitor BIM-I and the p38 MAPK inhibitor SB2035800. However when LY294002, BIM-I or SB2035800 were added after the initiation of DMSO-induced P19 stem cell differentiation, each inhibitor failed to block the cell differentiation into beating cardiomyocytes. The NF-kB activation inhibitor, CAPE, blocked H9c2 cell differentiation into cardiomyocytes. Fast nitric oxide releasing donors (SIN-1 and NOC-5) markedly delayed the onset of differentiation of H9c2 cells into cardiomyocytes while slow nitric oxide releasing donors (SNAP and NOC-18) were less effective in delaying the onset of differentiation or long term differentiation of H9c2 cells into cardiomyocytes. Akt (protein kinase B) is the key downstream target of PI3K. Our cross-talk data also showed that PKC inhibition and p38 MAPK inhibition respectively enhanced and reduced the activation of Akt, as determined by the phosphorylation of Akt at serine residue 473. In conclusion, PKC, PI3K, p38 MAPK and NF-kB are relevant for the differentiation of stem cells into cardiomyocytes. Our data also show that the PKC, PI3K and p38 MAPK signalling pathways are activated as very early events during the differentiation of stem cells into cardiomyocytes. Our data also suggest that PKC may negatively regulate Akt activation while p38 MAPK inhibition inhibits Akt activation. Our fast NO releasing donor data suggest that nitric oxide may negatively regulate H9c2 cell differentiation.

碩士<br>國立臺灣海洋大學<br>生物科技研究所<br>96<br>The generation of functional neuronal subtypes in the vertebrate central nervous system involves several steps. Early vertebrate neural development started from epiblast which is the pluripotent descendents of inner cell mass(ICM) of the blastocyst. The epiblast cells give rise to three embryonic germ layers(ectoderm,mesoderm and endoderm). Subsequently, ectodermal precursors forms early neuroepithelium containing very early neural stem cells. Then, neuroectoderm forms the neural plate and neural tube which become the source of the central nervous system. Recently, it has been demonstrated that Pax6 , Neurogenin, Sox1 and Nestin genes play an important role in early development of murine neuroectoderm. Among these genes, Sox1 is the earliest gene to be expressed in neuroectoderm and plays essential role in neural specification. However, the function of Sox1 gene in the development of human is still unknown. In order to understand the functional role of Sox1 in human neural specification, I generated lenti-viral Sox1 RNAi clones with GFP reporter and transducted them into hESCs to knowdown the expression of Sox1. In addition, heterogeneity among progenitor cells in the vertebrate nervous system has been demonstrated and it is believed that celluar heterogeneity is an important step toward developmental diversity. Therefore, I also investigated the expression profile of Oct4,Pax6 and Sox1 gene of single hESCs and their differentiated derivatives. My results show Sox1 siRNA knockdown has no obvious effect on altering characteristics of hESCs and their potentials in neural differentiation. Furthermore, results of the single cell analysis indicated the expression pattern of Oct4,Pax6 and Sox1 genes are not uniform in individual cells of early neural differentiation. This may imply a diversified neural cell types are emerging at these developmental stages.

碩士<br>國立臺灣海洋大學<br>生物科技研究所<br>95<br>Abstract Sox1 is one of the earliest transcription factors expressing in embryonic neuroepithelial cells and its expression correlates with the formation of the neural tube during early development. Additionally, it was demonstrated that Sox1 also expressing in the proliferating neural progenitors of both embryonic telencephalon and adult lateral ventricle at which active neurogenesis is evident. Taken together, these data indicate that Sox1 expressing cells may persistently contribute to neurogenesis from early development to adulthood. Sox1 is the earliest specific marker of neural progenitor and it is down-regulated when the cells exit from mitosis and differentiate into neurons and glia. In order to investigate the survival and differentiation potential of neural progenitor cells derived from embryonic stem cells. We transfected a constitutive expressed red fluorescence protein under the control of human elongation factor 1α（EF1α） promoter into the 46C mES cells, which carry GFP knocked into Sox1 gene for in vivo tracking of the engrafted cells. After antibiotics screening, we obtained a stable constitutive expressed red fluorescence protein 46C mES cells. After further differentiation in vitro, we isolated Sox1 expressing neural progenitor cells form three different time point. These isolated Sox1-expressing cells from different time point were then transplant into normal and ischemia lesioned cortex of rat. One week post-transplantation, we observed only the early stage NPCs migrated from transplantation site in lesioned cortex, whereas the late stage NPCs stay at the transplantation site in normal cortex. Three weeks post-transplantation, we observed engrafted early-stage NPCs migrated along the corpus callosum to the lesion site. Furthermore, these cells give rise to glia lineage which express glia marker, Glial fibrillary acidic protein. Taken together, our result showed that NPCs derived from different in vitro differentiation stage of ES cells processed different properties, in terms of , their in vivo response to brain injury, this finding together with the procedures created in the current research, provide an important first-step for selection of specific neural cell types from ES cells for stem cell based therapy.

碩士<br>國立陽明大學<br>生化暨分子生物研究所<br>98<br>Aberrant expression and function of epidermal growth factor receptor (EGFR) is reported in most lung cancer cases. Sox2, a core transcription factor regulating self-renewal of stem cells, is highly expressed in lung cancer. We discovered that EGFR and its ligands (TGF-??nand EGF) induce Sox2 expression in lung cancer. Ectopic expression of c-Myc, a key effector of EGFR signaling, induced Sox2 expression; knockdown of c-Myc decreased Sox2 level in lung cancer, suggesting that EGFR induces Sox2 via a c-Myc dependent pathway. Ectopic expression of Sox2 promoted cell proliferation and anchorage-independent cell growth; knockdown of Sox2 attenuated oncogenic properties of lung cancer. In addition, Sox2-silencing induced autophagic death of lung cancer; overexpression of Sox2 prevented cell from starvation-induced autophagy. These data demonstrates that Sox2 induces oncogenesis of lung cancer. Overexpression of Sox2 promoted tumor growth in xenograft mouse model; knockdown of Sox2 inhibited tumor formation in vivo. These results support the notion that Sox2 enhances tumorigenesis of lung cancer. Through a cDNA microarray analysis for Sox2 target genes, we identified that Sox2 regulates EGFR. Immunoblotting showed that Sox2 induced EGFR expression, suggesting that an EGFR-Sox2-EGFR positive feedback loop exist in lung cancer. In addition, ectopic expression of Sox2 in lung cancer induces mesenchymal-epithelial trans-differentiation and promotes cell-matrix adhesion. Thus, Sox2 may serve as an important effector of EGFR-mediated oncogenesis and provide a novel prognostic biomarker and therapeutic target for lung cancer.