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

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.

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

Chronic obstructive pulmonary disease (COPD) is one of the most common lung diseases and the third leading cause of death in the US, estimated to increase in magnitude in the future. Current treatment approaches are palliative in nature and restricted to controlling symptoms and reducing the risk of complications. Lung transplantation is an option for certain patients, but this option is limited by the shortage of donor organs and the possibility of rejection and the need for life-long immune-suppression. Therefore, current studies focus on cell based therapies for lung repair and regeneration. In addressing the issue of cell sourcing for such approaches, I tested the hypothesis that the efficiency of directed pulmonary differentiation of mouse embryonic stem cells (mESC) can be enhanced by employing certain micro-environmental cues, found in the developing lung. Such micro-environmental cues will provide appropriate physicochemical signals at the right time during the embryonic development and thus modulate fate decisions of progenitor cells during tissue assembly and maturation. In this study, I explored the effects of matrix stiffness on cell fate decisions in mESC, first into definitive endoderm and then into lung alveolar epithelial cells. I engineered bio-activated polyacrylamide (PA) gels with varying elastic moduli, mimicking those of physiologic tissues, and covalently modified the surfaces with fibronectin to provide optimal stem cell adhesion. My studies demonstrated, for the first time, a biphasic stiffness-dependent enhancement of endodermal differentiation of mESCs, with an optimum at ~ 20 kPa. This effect was qualitatively similar in three different mESC lines. By contrast, increasing matrix stiffness favored mESC differentiation towards a mesodermal phenotype. The enhanced endodermal differentiation of mESCs was abolished in the presence of a specific inhibitor of ROCK, suggesting that this process is mediated through cytoskeletal signaling. The subsequent differentiation of mESC-derived endodermal cells towards pulmonary epithelial cells was no longer dependent on the stiffness of the matrix. In this dissertation I demonstrate for the first time the feasibility of utilizing developmental and physiological / physicochemical cues, such as matrix stiffness, to selectively modulate and enhance mESC differentiation towards endodermal and pulmonary lineages. The impact of the results will be relevant for optimizing cell-based lung therapies and for effectively engineering lung and other endoderm-derived organs. / Bioengineering

Embryonic stem (ES) cells offer a valuable source for generating insulin-producing cells. However, current differentiation protocols often result in heterogeneous cell populations of various developmental stages. Here we show the activin A-induced differentiation of mouse ES cells carrying a homologous dsRed-IRES-puromycin knock-in within the Sox17 locus into the endoderm lineage. Sox17-expressing cells were selected by fluorescence-assisted cell sorting (FACS) and characterized at the transcript and protein level. Treatment of ES cells with high concentrations of activin A for 10 days resulted in up to 19% Sox17-positive cells selected by FACS. Isolated Sox17-positive cells were characterized by defini- tive endoderm-specific Sox17/Cxcr4/Foxa2 transcripts, but lacked pluripotency-associated Oct4 mRNA and protein. The Sox17-expressing cells showed downregulation of extraembryonic endoderm (Sox7, Afp, Sdf1)-, mesoderm (Foxf1, Meox1)- and ectoderm (Pax6, NeuroD6)-specific transcripts. The presence of Hnf4α, Hes1 and Pdx1 mRNA demonstrated the expression of primitive gut/foregut cell-specific markers. Ngn3, Nkx6.1 and Nkx2.2 transcripts in Sox17-positive cells were determined as properties of pancreatic endocrine progenitors. Immunocytochemistry of activin A-induced Sox17-positive embryoid bodies revealed coexpression of Cxcr4 and Foxa2. Moreover, the histochemical demonstration of E-cadherin-, Cxcr4-, Sox9-, Hnf1β- and Ngn3-positive epithelial-like structures underlined the potential of Sox17-positive cells to further differentiate into the pancreatic lineage. By reducing the heterogeneity of the ES cell progeny, Sox17-expressing cells are a suitable model to evaluate the effects of growth and differentiation factors and of culture conditions to delineate the differentiation process for the generation of pancreatic cells in vitro. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Tot i que es coneix l’involucrament de les cèl·lules pancreàtiques acinars en patologies exocrines (pancreatitis i càncer de pàncrees), la manca de models normals basats en cèl·lules ha limitat l’estudi de les alteracions que succeeixen en el programa de diferenciació pancreàtica. Hem demostrat prèviament que les cèl·lules mare embrionàries murines, que són pluripotents, poden adquirir un fenotip acinar in vitro. Això es va aconseguir, en part, amb una combinació de senyals que provenien del cultiu de pàncrees fetals que no era, però, específic del llinatge pancreàtic. L’objectiu d’aquest treball ha estat el de desenvolupar un protocol selectiu pel llinatge acinar basat en l’activació seqüencial de vies de senyalització que recapitulin el desenvolupament pancreàtic in vivo, a través de la formació definitiva de l’endoderm, l’especificació pancreàtica i acinar i l’expansió/diferenciació de progenitors acinars. El tractament de cossos embrionaris amb Activina A va promoure l’expressió de gens d’endoderm com està prèviament descrit. El tractament subsegüent amb àcid Retinoic, FGF10 i Ciclopamina, un inhibidor de la via de Hedgehog, va resutar en la inducció dels marcadors de progenitors pancreàtics Pdx1, Ptf1a i Cpa1 però també d’aquells expressats en el llinatge pancreàtic, que van ser reduïts amb la inhibició de BMPs. Les cèl·lules van ser a continuació cultivades en Matrigel utilitzant un sistema de cultiu en 3D en presència de fol·listatina, dexametasona i KGF comportant una inducció significativa dels nivells de mRNA i proteïna de marcadors acinars i una disminució de l’expressió dels de marcadors acinars. A més, es va veure que Amyl es secretava en el medi. Aquestes dades indiquen que l’activació selectiva del programa de diferenciació acinar en cèl·lules mare embrionàries es pot dur a terme mitjançant una inducció esgraonada de vies de senyalització involucrades en el desenvolupament pancreàtic exocrí proporcionant una eina potencial per estudiar la diferenciació pancreàtica i malalties relacionades amb el pàncrees. / Despite known involvement of pancreatic acinar cells in exocrine pathologies (pancreatitis and pancreatic cancer), the lack of normal cell-based models has limited the study of the alterations that occur in the acinar differentiation program. We have previously shown that mESC (murine embryonic stem cells), which are pluripotent, can acquire an acinar phenotype in vitro. This was achieved, in part, by a combination of signals provided by the culture of foetal pancreases which was, however, no specific for the acinar lineage. The aim of this work was to develop a protocol selective for the acinar lineage based on the sequential activation of signaling pathways that recapitulate pancreatic development in vivo, through the definitive endoderm formation, the pancreatic and acinar specification and the expansion/differentiation of acinar progenitors. Treatment of embryoid bodies with Activin A enhanced the expression of endodermal genes as previously described. Subsequent treatment with Retinoic acid, FGF10 and Cyclopamine, an inhibitor of the Hedgehog pathway, resulted in the enhancement of pancreatic progenitor markers Pdx1, Ptf1a and Cpa1 but also of those expressed in the hepatic lineage, which were reduced by BMPs inhibition. Cells were further cultured in Matrigel using a 3D culture system in the presence of follistatin, dexamethasone, and KGF leading to a significant enhancement of the mRNA and protein levels of acinar markers while decreasing the expression of endocrine ones. Moreover, active Amyl was released into the medium. These data indicate that the selective activation of the acinar differentiation program in ES cells can be achieved by stepwise induction of signaling pathways involved in pancreatic exocrine development providing a potential tool for studying pancreatic differentiation and pancreas-related diseases.