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Targeted differentiation of human embryonic stem cells (hESCs) towards definitive endoderm (DE) is the first step in generating hepatic or pancreatic cell types with potential for clinical application. Characterisation and efficiency of DE differentiation is improving, however the specific effects of the different exogenous growth factors used, and the changing presence and activity of endogenous factors, are still not well understood. One such endogenous factor, the TGFβ ligand Nodal, is known to drive patterning and differentiation of the primitive streak and DE in the developing mouse embryo. The effect of Nodal signalling during hESC DE differentiation is unknown, and the common use of a related exogenous ligand Activin A may also serve to upregulate rather than simply mimic it. In order to explore this, Activin A differentiation of hESCs in defined culture conditions was analysed. The expression of characteristic mesendoderm and DE markers increased during Activin A treatment, which was significantly enhanced by the inclusion of exogenous Wnt3a. A maintained presence of the pluripotency factor Nanog was observed in most cells expressing markers of DE. The levels of Nodal and its co-receptor Cripto, which were raised during the early stage of Activin A treatment, were also marginally enhanced by Wnt3a, and evidence of Nodal endocytosis further suggested an active signalling presence. RNA interference (RNAi) of Nodal negatively affected both pluripotency maintenance during normal pluripotent culture, and the capacity to differentiate towards DE. Use of a Cripto blocking antibody also inhibited differentiation towards DE. The results strongly suggested the presence of Nodal signalling, as well as possible roles for Nanog, Wnt-related signalling, and Nodal signalling during Activin A-mediated DE differentiation. The results contribute to current understanding of how DE differentiation in hESCs is regulated. They also identify clear targets for further investigation, which would lead to improved characterisation and differentiation of DE from hESCs.

Pluripotent embryonic stem cells hold a great promise as an unlimited source of tissue for treatment of chronic diseases such as Type 1 diabetes and chronic liver disease. Various attempts have been made to produce cells that can serve as precursors for pancreas and liver. By using all-trans-retinoic acid, basic fibroblast growth factor, dibutyryl cAMP, and cyclopamine, an attempt has been made to produce definitive endoderm and subsequently cells that can serve as pancreatic and hepatocyte precursors from mouse embryonic stem cells. By using retinoic acid and basic-FGF, in the absence of embryoid body formation, mouse embryonic stem cells were differentiated at different culture periods. Four protocols of varying lengths of culture and reagents and their cells were analyzed by quantitative PCR, immunohistochemistry and static insulin release assay for markers of trilaminar embryo, pancreas and hepatocytes. Inclusion of DBcAMP and extension of culture time resulted in cells that display features of definitive endoderm by expression of Sox 17 and FOXA2 and minimal expression of primitive endoderm and other germ cell layers such as ectoderm and mesoderm. These cells produced insulin and C-peptide and secreted insulin in a glucose responsive manner. However, they seem to lack mature insulin secretion mechanism. There was a production of hepatocyte markers (AFP-2 and transthyretin) but there was insufficient data to assess for convincing production of hepatocytes. In summary, one of the protocols produced cells that displayed characteristics of definitive endoderm and they may serve as pancreatic endocrine precursors.

Pluripotent embryonic stem cells hold a great promise as an unlimited source of tissue for treatment of chronic diseases such as Type 1 diabetes and chronic liver disease. Various attempts have been made to produce cells that can serve as precursors for pancreas and liver. By using all-trans-retinoic acid, basic fibroblast growth factor, dibutyryl cAMP, and cyclopamine, an attempt has been made to produce definitive endoderm and subsequently cells that can serve as pancreatic and hepatocyte precursors from mouse embryonic stem cells. By using retinoic acid and basic-FGF, in the absence of embryoid body formation, mouse embryonic stem cells were differentiated at different culture periods. Four protocols of varying lengths of culture and reagents and their cells were analyzed by quantitative PCR, immunohistochemistry and static insulin release assay for markers of trilaminar embryo, pancreas and hepatocytes. Inclusion of DBcAMP and extension of culture time resulted in cells that display features of definitive endoderm by expression of Sox 17 and FOXA2 and minimal expression of primitive endoderm and other germ cell layers such as ectoderm and mesoderm. These cells produced insulin and C-peptide and secreted insulin in a glucose responsive manner. However, they seem to lack mature insulin secretion mechanism. There was a production of hepatocyte markers (AFP-2 and transthyretin) but there was insufficient data to assess for convincing production of hepatocytes. In summary, one of the protocols produced cells that displayed characteristics of definitive endoderm and they may serve as pancreatic endocrine precursors.

Pluripotent embryonic stem cells hold a great promise as an unlimited source of tissue for treatment of chronic diseases such as Type 1 diabetes and chronic liver disease. Various attempts have been made to produce cells that can serve as precursors for pancreas and liver. By using all-trans-retinoic acid, basic fibroblast growth factor, dibutyryl cAMP, and cyclopamine, an attempt has been made to produce definitive endoderm and subsequently cells that can serve as pancreatic and hepatocyte precursors from mouse embryonic stem cells. By using retinoic acid and basic-FGF, in the absence of embryoid body formation, mouse embryonic stem cells were differentiated at different culture periods. Four protocols of varying lengths of culture and reagents and their cells were analyzed by quantitative PCR, immunohistochemistry and static insulin release assay for markers of trilaminar embryo, pancreas and hepatocytes. Inclusion of DBcAMP and extension of culture time resulted in cells that display features of definitive endoderm by expression of Sox 17 and FOXA2 and minimal expression of primitive endoderm and other germ cell layers such as ectoderm and mesoderm. These cells produced insulin and C-peptide and secreted insulin in a glucose responsive manner. However, they seem to lack mature insulin secretion mechanism. There was a production of hepatocyte markers (AFP-2 and transthyretin) but there was insufficient data to assess for convincing production of hepatocytes. In summary, one of the protocols produced cells that displayed characteristics of definitive endoderm and they may serve as pancreatic endocrine precursors. / Surgery, Department of / Medicine, Faculty of / Graduate

The purpose of this study was to rationally design animal component free, chemically defined serum free media (ACF-CD-SFM) for MRC-5 and Vero cells while adhering to the Quality by Design guidelines. This was achieved by using the Modified Vero Serum Free Medium (MVSFM) as the basal formulation and supplementing it with various combinations of growth factors (LONG® EGF, LONG® R3 IGF-I, rTransferrin, bFGF, TGF-3 and PDGF-AA), lipids (linoleic acid, cholesterol, and dexamethasone), lipid precursors (ethanolamine and phosphoethanolamine) and vitamins (all-trans retinoic acid, -tocopherol and ascorbic acid). Media development was achieved by conducting a series of steps using different experimental methodologies with the end goal of satisfying the requirements of each cell line. MRC-5 and Vero cells were each cultured in specific media containing unique concentrations of supplements that were prepared according to the different statistical design methodologies. The original objective was to create a SFM, however due to the stringent nutritious requirements of anchorage dependent cell lines, only a reduction to 0.5% FBS was achieved. For MRC-5 cells, the one-factor-at-a-time (OFAT) generated the Prototype + 0.5% FBS medium. The Definitive Screening Design (DSD) gave rise to the Delta 1 + 0.5% FBS, which was the optimum medium formulation for MRC-5 cells as it had comparable cell yields to DMEM + 10 % FBS. This result was confirmed by the Genetic Algorithms-Hill Climbing (GA-HC) method. In the case of Vero cells, the OFAT and the DSD confirmed that MVSFM + 0.5 % FBS was the most optimal formulation. The morphology in both media for both cell lines was comparable to that in DMEM-10% FBS. It was concluded that the DSD method successfully achieved a reduction of the serum concentration from 10% to 0.5% FBS. / October 2017

Gastrulation is one of the most critical events of embryogenesis, generating the three primary germ layers (definitive endoderm, mesoderm, and ectoderm) and establishing the embryonic body plan. The definitive endoderm, which generates the lungs, liver, pancreas, and digestive tract, has become a tissue of particular interest in recent years. Understanding definitive endoderm formation and patterning will greatly aid progress in the in vitro differentiation of embryonic stem cells to definitive endoderm for use in treatment of diseases such as diabetes and hepatitis as an alternative for whole organ replacement. Gene targeting studies have demonstrated a critical role for the Nodal signaling pathway and the forkhead transcription factors Foxh1 and Foxa2 in specification of a group of cells referred to as the anterior primitive streak (APS). However, the transcriptional targets of Foxh1 and/or Foxa2 other than Nodal that regulate specification of this group of cells are currently unknown. Fate mapping and lineage tracing experiments have shown the APS to be the source of the definitive endoderm. However, many questions regarding specification and patterning of the definitive endoderm remain. The study of this tissue has been hampered by the lack of genetic markers specific for the definitive endoderm as many of the current markers, including Cerl, Foxa2, and Sox17, are also expressed in the visceral endoderm, an extraembryonic tissue. To further investigate the role of Foxh1 in specification of the anterior primitive streak and to address the lack of genetic markers for the definitive endoderm we performed expression profiling on post-implantation mouse embryos using Affymetrix™ GeneChips®. From this analysis we identified and characterized a novel marker of the mouse definitive endoderm. Examination of this, and other, novel endoderm markers in Foxh1 and Foxa2 deficient mouse embryos revealed that contrary to current models of definitive endoderm formation, we find some definitive endoderm is formed in these mutants. Specifically, specification of the midgut and hindgut definitive endoderm is largely unaffected, while foregut formation is severely affected. These results suggest that the formation of the midgut and hindgut definitive endoderm populations is independent of the anterior primitive streak and separate from the foregut definitive endoderm. This represents a major insight into the mechanisms regulating endoderm formation and patterning.

Gastrulation is one of the most critical events of embryogenesis, generating the three primary germ layers (definitive endoderm, mesoderm, and ectoderm) and establishing the embryonic body plan. The definitive endoderm, which generates the lungs, liver, pancreas, and digestive tract, has become a tissue of particular interest in recent years. Understanding definitive endoderm formation and patterning will greatly aid progress in the in vitro differentiation of embryonic stem cells to definitive endoderm for use in treatment of diseases such as diabetes and hepatitis as an alternative for whole organ replacement. Gene targeting studies have demonstrated a critical role for the Nodal signaling pathway and the forkhead transcription factors Foxh1 and Foxa2 in specification of a group of cells referred to as the anterior primitive streak (APS). However, the transcriptional targets of Foxh1 and/or Foxa2 other than Nodal that regulate specification of this group of cells are currently unknown. Fate mapping and lineage tracing experiments have shown the APS to be the source of the definitive endoderm. However, many questions regarding specification and patterning of the definitive endoderm remain. The study of this tissue has been hampered by the lack of genetic markers specific for the definitive endoderm as many of the current markers, including Cerl, Foxa2, and Sox17, are also expressed in the visceral endoderm, an extraembryonic tissue. To further investigate the role of Foxh1 in specification of the anterior primitive streak and to address the lack of genetic markers for the definitive endoderm we performed expression profiling on post-implantation mouse embryos using Affymetrix™ GeneChips®. From this analysis we identified and characterized a novel marker of the mouse definitive endoderm. Examination of this, and other, novel endoderm markers in Foxh1 and Foxa2 deficient mouse embryos revealed that contrary to current models of definitive endoderm formation, we find some definitive endoderm is formed in these mutants. Specifically, specification of the midgut and hindgut definitive endoderm is largely unaffected, while foregut formation is severely affected. These results suggest that the formation of the midgut and hindgut definitive endoderm populations is independent of the anterior primitive streak and separate from the foregut definitive endoderm. This represents a major insight into the mechanisms regulating endoderm formation and patterning.

Adult haematopoiesis occurs in the context of a supportive stromal cell niche. The bone marrow, spleen and thymus all contain specific, but relatively poorly defined, stromal cells, which are important for maintenance of quiescence and directed differentiation. Even less is known about the haematopoietic niche during haematopoietic development. The formation of red blood cells (erythropoiesis) occurs during haematopoiesis, and is also controlled by a variety of stromal cells. This thesis examined the visceral endoderm, a group of cells that surrounds the developing epiblast and is required for primitive erythropoiesis (early blood production). We attempted to determine which factors in the visceral endoderm were responsible for induction of primitive erythropoiesis, and whether they would be useful as blood induction factors in embryonic stem cell differentiation. Thus, I attempted to immortalise the visceral endoderm using an immortalising agent (SV40Tag), driven off of a previously identified visceral endoderm gene, Indian hedgehog. We modified a bacterial artificial chromosome so that SV40Tag was driven off of the Indian hedgehog gene. The modified bacterial artificial chromosome was used in both pronuclear injections of mouse blastocysts and the electroporation of embryonic stem cells. After neither attempt produced a visceral endoderm cell line, we examined a visceral endoderm-like cell line, END2, for the presence of the blood inducing factors. We demonstrated the ability of END2 conditioned media to apparently increase expression of blood transcripts in differentiating embryonic cells indicating the presence of blood inducing factors. Expression profiles of END2 cells were compared to a previously completed embryonic stem cell differentiation profile to identify enriched genes. Two genes, angiopoietin-like 7 and Bc064033, were tested for an ability to induce blood in differentiating embryonic stem cells. When neither protein was capable of inducing blood, the END2 cells were examined for the presence of other known blood inducing factors and similarity to in vivo visceral endoderm. The END2 cells were found to produce bone morphogenetic protein 4, a potent inducer of blood in embryonic stem cell differentiation, which complicated the search for additional factors. Examination of END2 cells also indicated a lack of visceral endoderm markers such as alpha fetoprotein, indicating that the END2 cells may not be as visceral endoderm-like as expected from the current literature. The previously identified Indian hedgehog gene was also examined for its blood induction abilities in vivo. Indian hedgehog knockout mice were examined for the effect gene removal had on both primitive and definitive erythropoiesis. Levels of primitive erythrocytes were unaffected in the Indian hedgehog knockout mice, but levels of definitive erythrocytes were found to be significantly decreased. Further examination of Indian hedgehog knockout fetal livers also showed that they had decreased numbers of haematopoietic stem cells. The haematopoietic stem cells were fully capable when cultured and generated appropriate numbers of progenitor cells, indicating a non-intrinsic cause for this defect. Levels of hedgehog target genes that are usually highest in the stromal compartment were also found to be most significantly decreased in Indian hedgehog knockout fetal livers. Another hedgehog gene, desert hedgehog, was also shown to be expressed in the fetal liver and may act with Indian hedgehog to regulate stromal production in the fetal liver.

Gastrulation is one of the most critical events of embryogenesis, generating the three primary germ layers (definitive endoderm, mesoderm, and ectoderm) and establishing the embryonic body plan. The definitive endoderm, which generates the lungs, liver, pancreas, and digestive tract, has become a tissue of particular interest in recent years. Understanding definitive endoderm formation and patterning will greatly aid progress in the in vitro differentiation of embryonic stem cells to definitive endoderm for use in treatment of diseases such as diabetes and hepatitis as an alternative for whole organ replacement. Gene targeting studies have demonstrated a critical role for the Nodal signaling pathway and the forkhead transcription factors Foxh1 and Foxa2 in specification of a group of cells referred to as the anterior primitive streak (APS). However, the transcriptional targets of Foxh1 and/or Foxa2 other than Nodal that regulate specification of this group of cells are currently unknown. Fate mapping and lineage tracing experiments have shown the APS to be the source of the definitive endoderm. However, many questions regarding specification and patterning of the definitive endoderm remain. The study of this tissue has been hampered by the lack of genetic markers specific for the definitive endoderm as many of the current markers, including Cerl, Foxa2, and Sox17, are also expressed in the visceral endoderm, an extraembryonic tissue. To further investigate the role of Foxh1 in specification of the anterior primitive streak and to address the lack of genetic markers for the definitive endoderm we performed expression profiling on post-implantation mouse embryos using Affymetrix™ GeneChips®. From this analysis we identified and characterized a novel marker of the mouse definitive endoderm. Examination of this, and other, novel endoderm markers in Foxh1 and Foxa2 deficient mouse embryos revealed that contrary to current models of definitive endoderm formation, we find some definitive endoderm is formed in these mutants. Specifically, specification of the midgut and hindgut definitive endoderm is largely unaffected, while foregut formation is severely affected. These results suggest that the formation of the midgut and hindgut definitive endoderm populations is independent of the anterior primitive streak and separate from the foregut definitive endoderm. This represents a major insight into the mechanisms regulating endoderm formation and patterning. / Medicine, Faculty of / Medical Genetics, Department of / Graduate