Academic literature on the topic 'Definitive endoderm'

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.

The definitive endoderm is one of the three germ layers of the embryo that are generated during gastrulation and gives rise to the lungs, liver, pancreas and the gut. Understanding the development of the definitive endoderm is crucial for future in vitro based approaches to regenerative medicine for diabetes or liver regeneration. However, progress in the understanding of definitive endoderm development has been limited by the lack of genetic markers specific to this tissue. To address this, our lab previously performed gene expression profiling of the definitive endoderm using Serial Analysis of Gene Expression. From this study, a number of genes expressed specifically in this tissue, including Nephrocan and Peptide YY, were uncovered. In an attempt to uncover novel genes expressed in the definitive endoderm, an extended study of this gene expression profiling was done, and two ESTs, Endy and NAPS, were further identified. Through whole mount in situ hybridization analysis of the early mouse embryo, expression of Endy and NAPS was seen in different cell populations of the developing definitive endoderm. In addition to the SAGE analysis, an ES cell differentiation system for definitive endoderm was set up, as a tool to study some of the early signaling pathways leading to the development of the definitive endoderm. Differentiation of mES cells using Activin induced expression of the novel definitive endoderm markers, Nepn and Pyy, in a temporal manner. Inhibition of TGFβ signaling during differentiation resulted in a significant down regulation of these genes. Furthermore, differentiation of mES cells mutant for TGFβ signaling factor Foxh1 revealed an expression pattern for Nepn and Pyy that was inconsistent with what is observed in vivo. Lastly, the ES cell differentiation system was also used to test expression of Endy and NAPS as little molecular information existed for these ESTs. Collectively, the characterization of these markers in vivo, and manipulations of the ES cell differentiation system to definitive endoderm will facilitate the creation of more accurate fate maps of the definitive endoderm, and address some of the questions regarding early lineage decisions during specification and patterning of this tissue.

During the course of development, specification of the three embryonic germ layers, ectoderm, mesoderm and definitive endoderm (DE), is a critical process by which pluripotent cells acquire the temporal and spatial information needed to form specialised tissues. Of these initial germ layers, the DE arises during gastrulation, which latterly gives rise to the liver, pancreas, lung and epithelial lining of the digestive tract. Elucidation of the molecular mechanisms that govern DE specification not only facilitates our understanding of developmental biology but also aids in the differentiation of human pluripotent stem cells to specific cell types for disease modelling and regenerative therapies. DE formation is largely driven by the cooperation of Activin/Nodal and Wnt/β-catenin signalling, however recent evidence has additionally implicated PI3K/Akt signalling in modulating this process. Although it has been previously reported that PI3K activation acts to antagonise the in vitro differentiation of DE, the molecular mechanisms responsible for this effect remains unclear. To address this issue, this study utilises pluripotent human embryonic stem cells (hESCs) as an in vitro model to interrogate the molecular underpinnings of DE formation through a fully defined differentiation protocol. Modulation of PI3K activity was found to reciprocally downregulate the activation of Smad2/3, which was mitigated in the presence of the PI3K inhibitor LY294002 (LY). Suppression of PI3K/Akt signalling prolongs the activation of Smad2/3 in response to Activin, promoting their nuclear accumulation and the enhancement of transcriptional activity, resulting in the upregulation of mesendoderm and DE gene expression. Activation of PI3K negatively impacts the activity of Smad2/3 via phosphorylation of the Smad2/3 linker T220/T179 residue, which is fully independent of Erk and CDK activity. Phosphorylation of this residue induces the recruitment of the E3 ubiquitin ligase Nedd4L to activated Smad2/3, which in turn promotes their ubiquitin-mediated degradation and attrition of activity. Inhibition of mTORC2 activity by both inhibitor supplementation and genetic manipulation, rather than modulation of Akt or mTORC1 activity, recapitulates the LY-mediated reduction of T220/T179 phosphorylation and increases the duration of Smad2/3 transcriptional activity, promoting a more robust mesendoderm and endoderm differentiation. These findings reveal a new and novel connection between the PI3K/mTOR and TGFβ/Activin pathways, which will greatly impact our understanding of both cell fate determination and the preservation of normal cellular functions. Notably, identification of mTORC2 as a key player in the regulation of this differentiation provides new avenues through which hESCs differentiation protocols can be improved for both regenerative and biomedical applications.

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.<br>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.

Bioengineering<br>Ph.D.<br>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.<br>Temple University--Theses

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<br>Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

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.<br>Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.