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<p dir="ltr">Embryonic development relies on the complex interplay of epigenetic regulation, timely expression of genes, signal transduction pathways, and diverse morphological changes. The heart is the first organ to form during mammalian embryonic development. The proper development of the heart is critical to supply nutrients and oxygen to other cell types of the organism. Most cells that comprise the heart originate from the mesoderm post-gastrulation. Cardiomyocytes are the predominant cell type and confer function to the heart via contractile activity. The development and proliferation of cardiomyocytes ceases shortly after birth, where cardiomyocytes only nucleate and increase in size. Consequently, cardiomyocyte insufficiency underlies most cardiovascular diseases, a leading cause of death globally.</p><p dir="ltr">Vascular endothelial zinc finger 1 (VEZF1) is a transcription factor expressed predominantly in mesoderm during development. Previous studies from our lab show that the loss of VEZF1 impairs the differentiation of embryonic stem cells into endothelial cells, a cell type derived from mesoderm. Other published studies also show that Vezf1 loss impairs cardiomyocyte growth in Zebrafish and hematopoietic cell differentiation. Our work here describes a detailed investigation of the role of Vezf1 in the differentiation of mesoderm and cardiomyocytes using mouse embryonic stem cell (ESC) differentiation as a mammalian model system. We initially developed an efficient method, known as the Wnt Switch method, to differentiate ESCs into cardiomyocytes. Our technique relies on the treatment of differentiating ESCs with small molecule inhibitors: i) CHIR99021, which induces mesoderm development via the activation of Wnt signaling in the first 48 hours of differentiation, followed by ii) XAV939, which inhibits Wnt signaling and drives mesoderm cells toward cardiomyocyte differentiation pathway. The Wnt Switch method significantly increases the efficiency of cardiomyocyte derivation (86%) from ESC compared to published methods (56%).</p><p dir="ltr">Interestingly, the Wnt Switch method showed that despite the external stimulation of Wnt signaling, Vezf1 KO cells are unable to differentiate into cardiomyocytes and show reduced expression of mesodermal genes 48 hrs post-differentiation. To better understand the stage-specific role of Vezf1 in cardiomyocyte development, we generated doxycycline-inducible Vezf1 knockdown clones that significantly reduce Vezf1 protein levels upon treatment with doxycycline. We found that the knockdown of Vezf1 prior to mesoderm induction significantly impaired ESC differentiation but had no significant effect on cardiomyocyte development after mesoderm induction. These data indicate that Vezf1 expression is crucial for proper mesoderm and, thus, mesodermal lineage development. Further, FACS analysis showed reduced mesoderm cell populations derived from Vezf1 null post-differentiation. We used high throughput sequencing methods to determine genome-wide Vezf1 binding by ChIP-SEQ and compared gene expression in WT and Vezf1 null cells using RNA-SEQ. The data indicated that VEZF1 binds near the promoters of numerous Wnt signaling genes after differentiation and that the expression of Wnt pathway genes decreases when Vezf1 is lost. Interestingly, supplementing WNT3A protein in culture media of Vezf1 null cells rescues the expression of Wnt target genes necessary for mesoderm formation.</p><p dir="ltr">Differentiating Vezf1 KO cells to endothelial or cardiomyocyte lineages also resulted in massive cell death. The surviving cells interestingly stained positive for alkaline phosphatase (AP) staining, indicating retention of the pluripotency in Vezf1 KO cells. Whereas, re-culturing of WT ESC in LIF media, after differentiating them for five days in the absence of LIF, results in cell death, Vezf1 KO cells proliferate and form AP-positive and SSEA-positive colonies. We further show the retention of pluripotency gene expression post-differentiation using RNA sequencing and RT-qPCR. Moreover, we show that the continued expression of pluripotency genes post-differentiation was not a consequence of reduced global DNA methylation in Vezf1 KO cells.</p><p dir="ltr">Interestingly, our data show that Vezf1 is a transcriptional activator and binds to key pro-differentiation pathways like the MAPK signaling and WNT signaling pathways. The loss of Vezf1 correlates with reduced expression of genes in the pro-differentiation pathways. We show that CTCF, an insulator-binding protein, opportunistically binds to VEZF1 sites on genes in the pro-differentiation signaling pathways in VEZF1 KO cells. Therefore, we hypothesized that this opportunistic CTCF binding is the mechanism that drives the repression of pro-differentiation signaling genes or compensates for the loss of Vezf1 binding to support basal gene expression in the absence of VEZF1. Given the dire consequences of pluripotency in cancer stem cells, we investigated the expression of Vezf1 in cancers. We found that Vezf1 expression is reduced in many cancers and is correlated with poor prognosis. We also show that MAPK3, a prominent member of the MAPK signaling pathway, is reduced in these cancers, highlighting a strong correlation between Vezf1 expression and Mapk3 gene expression in cancers. The data extend our observation of pluripotency in ESCs to cancers. To gain further insights into the role of Vezf1 in cancer, we utilized F9 embryonic carcinoma cells. F9 cells have been reported to retain pluripotency expression post-differentiation. Interestingly, the ectopic and transient expression of Vezf1 in F9 cells significantly reduced the expression of pluripotency genes, suggesting that Vezf1 is sufficient to repress pluripotency gene expression in F9 carcinoma cells. These data highlight the significant role of Vezf1 in pluripotency gene repression and provide an excellent avenue for treating cancer relapse caused by the occurrence of cancer stem cells.</p><p dir="ltr">In conclusion, our research elucidates the critical role of Vezf1 in cardiomyocyte formation and pluripotency regulation during embryonic development. Understanding the molecular mechanisms underlying Vezf1-mediated pathways provides insights into developmental processes and holds promise for therapeutic interventions for cardiomyocyte regeneration and against cancers.</p>

Thesis (PhD (Physiology (Human and animal))-- University of Stellenbosch, 2007. / Definition: Stem cells are unspecialised cells with the capacity for long-term self-renewal and the ability to differentiate into multiple cell-lineages. The potential for the application of stem cells in clinical settings has had a profound effect on the future of regenerative medicine. However, to be of greater therapeutic use, selection of the most appropriate cell type, as well as optimisation of stem cell incorporation into the damaged tissue is required. In adult skeletal muscle, satellite cells are the primary stem cell population which mediate postnatal muscle growth. Following injury or in diseased conditions, these cells are activated and recruited for new muscle formation. In contrast, the potential of resident adult stem cell incorporation into the myocardium has been challenged and the response of cardiac tissue, especially to ischaemic injury, is scar formation. Following muscle damage, various growth factors and cytokines are released in the afflicted area which influences the recruitment and incorporation of stem cells into the injured tissue. Transforming Growth Factor-β (TGF-β) is a member of the TGF-β-superfamily of cytokines and has at least three isoforms, TGF-β1, -β2, and -β3, which play essential roles in the regulation of cell growth and regeneration following activation and stimulation of receptor-signalling pathways. By improving the understanding of how TGF-β affects these processes, it is possible to gain insight into how the intercellular environment can be manipulated to improve stem cell-mediated repair following muscle injury. Therefore, the main aims of this thesis were to determine the effect of the three TGF-β isoforms on proliferation, differentiation, migration and fusion of muscle progenitor cells (skeletal and cardiac) and relate this to possible improved mechanisms for muscle repair. The effect of short- and long-term treatment with all three TGF-β isoforms were investigated on muscle progenitor cell proliferation and differentiation using the C2C12 skeletal muscle satellite and P19 multipotent embryonal carcinoma cell-lineages as in vitro model systems. Cells were treated with 5 ng/mℓ TGF-β isoforms unless where stated otherwise. In C2C12 cells, proliferating cell nuclear antigen (PCNA) expression and localisation were analysed, and together with total nuclear counts, used to assess the effect of TGF-β on myoblast proliferation (Chapter 5). The myogenic regulatory factors MyoD and myogenin, and structural protein myosin heavy chain (MHC) were used as protein markers to assess early and terminal differentiation, respectively. To establish possible mechanisms by which TGF-β isoforms regulate differentiation, further analysis included determination of MyoD localisation and the rate of MyoD degradation in C2C12 cells. To assess the effect of TGF-β isoforms on P19 cell differentiation, protein expression levels of connexin-43 and MHC were analysed, together with the determination of embryoid body numbers in differentiating P19 cells (Chapter 6). Furthermore, assays were developed to analyse the effect of TGF-β isoforms on both C2C12 and P19 cell migration (Chapter 7), as well as fusion of C2C12 cells (Chapter 8). Whereas all three isoforms of TGF-β significantly increased proliferation of C2C12 cells, differentiation results, however, indicated that especially following long-term incubation, TGF-β isoforms delayed both early and terminal differentiation of C2C12 cells into myotubes. Similarly, myocyte migration and fusion were also negatively regulated following TGF-β treatment. In the P19 cell-lineage, results demonstrated that isoform-specific treatment with TGF-β1 could potentially enhance differentiation. Further research is however required in this area, especially since migration was greatly reduced in these cells. Taken together, results demonstrated variable effects following TGF-β treatment depending on the cell type and the duration of TGF-β application. Circulating and/or treatment concentrations of this growth factor could therefore be manipulated depending on the area of injury to improve regenerative processes. Alternatively, when selecting appropriate stem or progenitor cells for therapeutic application, the effect of the immediate environment and subsequent interaction between the two should be taken into consideration for optimal beneficial results.

Recently, laser light sources of different regimes have emerged as an essential tool in the biophotonics research area. Classic applications include, for example: manipulating single cells and their subcellular organelles, sorting cells in microfluidic channels and the cytoplasmic delivery of both genetic and non-genetic matter of varying sizes into mammalian cells. In this thesis several new findings specifically in the optical cell sorting as well as in the photo-transfection study fields are presented. In my optical cell sorting and guiding investigations, a new technique for enhancing the dielectric contrast of mammalian cells, which is a result of cells naturally engulfing polymer microspheres from their environment, is introduced. I explore how these intracellular dielectric tags influence the scattering and gradient forces upon these cells from an externally applied optical field. I show that intracellular polymer microspheres can serve as highly directional optical scatterers and that the scattering force can enable sorting through axial guiding onto laminin coated glass coverslips upon which the selected cells adhere. Following this, I report on transient photo-transfection of mammalian cells including neuroblastomas (rat/mouse and human), embryonic kidney, Chinese hamster ovary as well as pluripotent stem cells using a tightly focused titanium sapphire femtosecond pulsed laser beam spot. These investigations permitted advanced biological studies in femtosecond laser transfection: firstly, the influence of cell passage number on the transfection efficiency; secondly, the possibility to enhance the transfection efficiency via whole culture treatments of cells thereby, synchronizing them at the mitotic (M phase) as well as the synthesis phases (S phase) of the cell cycle; thirdly, this methodology can activate the up-regulation of the protective heat shock protein 70 (hsp70). Finally, I show that this novel technology can also be used to transfect mouse embryonic stem (mES) cell colonies and the ability of differentiating these cells into the extraembryonic endoderm.

The nucleus of epidermal keratinocytes (KCs) is a complex and highly compartmentalized organelle, whose structure is markedly changed during terminal differentiation and transition of the genome from a transcriptionally active state seen in the basal and spinous epidermal cells to a fully inactive state in the keratinized cells of the cornified layer. Here, using multicolor confocal microscopy, followed by computational image analysis and mathematical modeling, we demonstrate that in normal mouse footpad epidermis, transition of KCs from basal epidermal layer to the granular layer is accompanied by marked differences in nuclear architecture and microenvironment including the following: (i) decrease in the nuclear volume; (ii) decrease in expression of the markers of transcriptionally active chromatin; (iii) internalization and decrease in the number of nucleoli; (iv) increase in the number of pericentromeric heterochromatic clusters; and (v) increase in the frequency of associations between the pericentromeric clusters, chromosomal territory 3, and nucleoli. These data suggest a role for nucleoli and pericentromeric heterochromatin clusters as organizers of nuclear microenvironment required for proper execution of gene expression programs in differentiating KCs, and provide important background information for further analyses of alterations in the topological genome organization seen in pathological skin conditions, including disorders of epidermal differentiation and epidermal tumors.

La maladie coeliaque réfractaire de type II (MCRII) est une complication sévère de la maladie coeliaque caractérisée par l’émergence dans l’épithélium intestinal d’une population clonale de cellules innées lymphoïdes (IE-ILC) avec un phénotype inhabituel. Nos travaux montrent en effet que ces IE-ILC ont un phénotype mixte avec des caractéristiques à la fois de lymphocytes T (LT) et de cellules NK, puisqu’elles expriment des récepteurs NK et contiennent les chaines du complexe CD3 en intracellulaire et des réarrangements du récepteur T. En outre, des travaux précédents du laboratoire ont montré que l’interleukine 15 (IL-15), produite en excès par les entérocytes des patients MCRII, joue un rôle central en permettant la survie de ces lymphocytes anormaux et de ce fait leur accumulation progressive dans l’intestin. Le premier objectif de ma thèse a été de comprendre l’ontogénie des IE-ILC chez l’homme. Nous avons démontré qu’une population «polyclonale» d’IE-ILC, possédant des caractéristiques similaires à ceux des lymphocytes clonaux de MCRII, est présente dans l’épithélium intestinal des sujets sains. En outre, ces cellules prédominent dans l’épithélium intestinal des très jeunes enfants (<1ans) et des patients allo-greffés après une chimiothérapie. Nous avons montré que la différenciation des IE-ILC peut–être récapitulée in vitro à partir de cellules souches hématopoïétiques (CSH), en combinant un signal NOTCH et IL-15; le signal NOTCH initie un programme T qui est interrompu par l’IL-15, conduisant à la reprogrammation des cellules vers une différenciation NK. Nous avons aussi montré que l’effet de l’IL-15 résulte de l’induction de la sérine protéase Granzyme B, qui clive la protéine NOTCH en fragments dépourvus d’activité transcriptionnelle. Le deuxième objectif de mon travail a été d’identifier le site de la différenciation des IE-ILC. Thymus et épithélium intestinal expriment en effet des ligands de NOTCH ainsi que de l’IL-15. Cette partie du travail a été conduite chez la souris. Nous avons montré qu’une population d’IE-ILC similaire à celle observée chez l’homme est présente dans l’épithélium murin. L’étude de différentes souris mutantes nous a permis de démontrer sa dépendance stricte de l’IL-15. La réalisation de greffes de thymus, l’analyse de souris athymiques et le transfert de cellules de la moelle osseuse chez des souris immunodéficiences a permis d’exclure tout rôle du thymus dans la différenciation des IE-ILC, et de suggérer que ces cellules se différencient dans l’intestin avant la migration des lymphocytes T. Dans leur ensemble, ces résultats caractérisent une nouvelle population de cellules lymphoïdes innées et une voie originale de différenciation. Nos résultats éclairent un débat de longue date sur la différenciation extra-thymique des lymphocytes de l’épithélium. Nous montrons que la différenciation T peut être initiée dans l’épithélium intestinal mais que l’IL-15 bloque cette différenciation, ce qui conduit à la génération d’une population particulière de IE-ILC avec un phénotype mixte de LT et de NK. La présence des IE-ILC chez les très jeunes enfants et chez les patients greffés suggère un rôle possible dans les défenses de l’épithélium intestinal avant l’activation du système immunitaire adaptatif et la migration intraépithéliale des LT. / Refractory celiac disease type II (RCDII) is a rare but severe complication of celiac disease characterized by the appearance in gut epithelium of clonal population of innate lymphoid cells (IE-ILC) with atypical features. Our work shows that IE-ILC have mixed phenotype close both to T cells and NK cells. Indeed, IE-ILC express NK markers, intracellular CD3 chains and exhibit T cell rearrangement. Moreover, previous data from the laboratory have shown that interleukin 15 (IL-15), a cytokine overexpressed in the gut of RCDII patients, plays a key role in survival of clonal IE-ILC1 and therefore promotes their accumulation in the gut epithelium. The first objective of my thesis has been to understand the ontogeny of IE-ILC in humans. We have demonstrated that polyclonal IE-ILC sharing similar features with clonal IE-ILC are present in the gut of healthy control. In addition, IE-ILC are preponderant in the gut epithelium of young children (<1year) and of grafted patients after chemotherapy. We have shown that differentiation of IE-ILC can be recapitulated in vitro from hematopoietic stem cells (HSC) by combining both NOTCH signal and IL-15. Indeed, NOTCH signal initiates T cell program, which is blocked by IL-15, reprogramming these cells toward the NK lineage. Moreover, we have shown that Granzyme B, a serine protease induced by IL-15, cleaves the NOTCH protein into a transcriptionnally inactive peptide. The second objective of my work has been to determine the site of differentiation of IE-ILC. Thymus and gut epithelium express both NOTCH ligands and IL-15. This question has to be addressed in mouse. We have shown that the mouse gut epithelium contains the counterpart of human IE-ILC1. We first confirmed that mouse IE-ILC1 require IL-15 for their differentiation and or survival using IL-15 deficient mice. Thymus graft experiment, analysis of athymic mice (nude) and HSC transplantation in empty hosts suggested that IE-ILC1 differentiate in the gut epithelium before immigration of T cells from thymus. In summary, our results describe a novel subset of IE-ILC1 together with their novel unconventional mechanism of differentiation. Our data allow to revisit the long time controversy on extra-thymic T cell differentiation in the gut. We have demonstrated that T cell program can be initiated in the gut epithelium but IL15-induced Granzyme B blocks this program and permits the generation of unconventional IE-ILC with mixed T cell and NK cell features. Because IE-ILC1 are preponderant in the gut epithelium of young children and of patients with recent bone marrow transplantation, we suggest that IE-ILC1 can play a major role in gut defense before activation of the gut adaptative immune system activate and migration of thymus-derived T cells into the gut epithelium.

La maladie coeliaque réfractaire de type II (MCRII) est une complication sévère de la maladie coeliaque caractérisée par l’émergence dans l’épithélium intestinal d’une population clonale de cellules innées lymphoïdes (IE-ILC) avec un phénotype inhabituel. Nos travaux montrent en effet que ces IE-ILC ont un phénotype mixte avec des caractéristiques à la fois de lymphocytes T (LT) et de cellules NK, puisqu’elles expriment des récepteurs NK et contiennent les chaines du complexe CD3 en intracellulaire et des réarrangements du récepteur T. En outre, des travaux précédents du laboratoire ont montré que l’interleukine 15 (IL-15), produite en excès par les entérocytes des patients MCRII, joue un rôle central en permettant la survie de ces lymphocytes anormaux et de ce fait leur accumulation progressive dans l’intestin. Le premier objectif de ma thèse a été de comprendre l’ontogénie des IE-ILC chez l’homme. Nous avons démontré qu’une population «polyclonale» d’IE-ILC, possédant des caractéristiques similaires à ceux des lymphocytes clonaux de MCRII, est présente dans l’épithélium intestinal des sujets sains. En outre, ces cellules prédominent dans l’épithélium intestinal des très jeunes enfants (<1ans) et des patients allo-greffés après une chimiothérapie. Nous avons montré que la différenciation des IE-ILC peut–être récapitulée in vitro à partir de cellules souches hématopoïétiques (CSH), en combinant un signal NOTCH et IL-15; le signal NOTCH initie un programme T qui est interrompu par l’IL-15, conduisant à la reprogrammation des cellules vers une différenciation NK. Nous avons aussi montré que l’effet de l’IL-15 résulte de l’induction de la sérine protéase Granzyme B, qui clive la protéine NOTCH en fragments dépourvus d’activité transcriptionnelle. Le deuxième objectif de mon travail a été d’identifier le site de la différenciation des IE-ILC. Thymus et épithélium intestinal expriment en effet des ligands de NOTCH ainsi que de l’IL-15. Cette partie du travail a été conduite chez la souris. Nous avons montré qu’une population d’IE-ILC similaire à celle observée chez l’homme est présente dans l’épithélium murin. L’étude de différentes souris mutantes nous a permis de démontrer sa dépendance stricte de l’IL-15. La réalisation de greffes de thymus, l’analyse de souris athymiques et le transfert de cellules de la moelle osseuse chez des souris immunodéficiences a permis d’exclure tout rôle du thymus dans la différenciation des IE-ILC, et de suggérer que ces cellules se différencient dans l’intestin avant la migration des lymphocytes T. Dans leur ensemble, ces résultats caractérisent une nouvelle population de cellules lymphoïdes innées et une voie originale de différenciation. Nos résultats éclairent un débat de longue date sur la différenciation extra-thymique des lymphocytes de l’épithélium. Nous montrons que la différenciation T peut être initiée dans l’épithélium intestinal mais que l’IL-15 bloque cette différenciation, ce qui conduit à la génération d’une population particulière de IE-ILC avec un phénotype mixte de LT et de NK. La présence des IE-ILC chez les très jeunes enfants et chez les patients greffés suggère un rôle possible dans les défenses de l’épithélium intestinal avant l’activation du système immunitaire adaptatif et la migration intraépithéliale des LT. / Refractory celiac disease type II (RCDII) is a rare but severe complication of celiac disease characterized by the appearance in gut epithelium of clonal population of innate lymphoid cells (IE-ILC) with atypical features. Our work shows that IE-ILC have mixed phenotype close both to T cells and NK cells. Indeed, IE-ILC express NK markers, intracellular CD3 chains and exhibit T cell rearrangement. Moreover, previous data from the laboratory have shown that interleukin 15 (IL-15), a cytokine overexpressed in the gut of RCDII patients, plays a key role in survival of clonal IE-ILC1 and therefore promotes their accumulation in the gut epithelium. The first objective of my thesis has been to understand the ontogeny of IE-ILC in humans. We have demonstrated that polyclonal IE-ILC sharing similar features with clonal IE-ILC are present in the gut of healthy control. In addition, IE-ILC are preponderant in the gut epithelium of young children (<1year) and of grafted patients after chemotherapy. We have shown that differentiation of IE-ILC can be recapitulated in vitro from hematopoietic stem cells (HSC) by combining both NOTCH signal and IL-15. Indeed, NOTCH signal initiates T cell program, which is blocked by IL-15, reprogramming these cells toward the NK lineage. Moreover, we have shown that Granzyme B, a serine protease induced by IL-15, cleaves the NOTCH protein into a transcriptionnally inactive peptide. The second objective of my work has been to determine the site of differentiation of IE-ILC. Thymus and gut epithelium express both NOTCH ligands and IL-15. This question has to be addressed in mouse. We have shown that the mouse gut epithelium contains the counterpart of human IE-ILC1. We first confirmed that mouse IE-ILC1 require IL-15 for their differentiation and or survival using IL-15 deficient mice. Thymus graft experiment, analysis of athymic mice (nude) and HSC transplantation in empty hosts suggested that IE-ILC1 differentiate in the gut epithelium before immigration of T cells from thymus. In summary, our results describe a novel subset of IE-ILC1 together with their novel unconventional mechanism of differentiation. Our data allow to revisit the long time controversy on extra-thymic T cell differentiation in the gut. We have demonstrated that T cell program can be initiated in the gut epithelium but IL15-induced Granzyme B blocks this program and permits the generation of unconventional IE-ILC with mixed T cell and NK cell features. Because IE-ILC1 are preponderant in the gut epithelium of young children and of patients with recent bone marrow transplantation, we suggest that IE-ILC1 can play a major role in gut defense before activation of the gut adaptative immune system activate and migration of thymus-derived T cells into the gut epithelium.