Project description:The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocyes from ESCs. Here we report that a high density of human embryonic stem cell (ESC)-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells (HLCs) with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells (iPSCs). Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and Activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, as its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model, and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells. Total RNA were isolated from ORMES6 ESC, differentiated cells at IVDS2 and 3, and cells in the central foci (IVDS2-C) and peripheral (IVDS2-P) area of ESC colonies at IVDS2. Each condition was repeated twice and used ORMES6 ESC as control.
Project description:The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocyes from ESCs. Here we report that a high density of human embryonic stem cell (ESC)-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells (HLCs) with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells (iPSCs). Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and Activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, as its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model, and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells.
Project description:The integration of cell metabolism with signalling pathways, transcription factor networks and epigenetic mediators is critical in coordinating molecular and cellular events during embryogenesis. Induced pluripotent stem cells (IPSCs) are an established model for embryogenesis, germ layer specification and cell lineage differentiation, advancing the study of human embryonic development and the translation of innovations in drug discovery, disease modelling and cell-based therapies. The metabolic regulation of IPSC pluripotency is mediated by balancing glycolysis and oxidative phosphorylation, but there is a paucity of data regarding the influence of individual metabolite changes during cell lineage differentiation. We used <sup>1</sup>H NMR metabolite fingerprinting and footprinting to monitor metabolite levels as IPSCs are directed in a three-stage protocol through primitive streak/mesendoderm, mesoderm and chondrogenic populations. Metabolite changes were associated with central metabolism, with aerobic glycolysis predominant in IPSC, elevated oxidative phosphorylation during differentiation and fatty acid oxidation and ketone body use in chondrogenic cells. Metabolites were also implicated in the epigenetic regulation of pluripotency, cell signalling and biosynthetic pathways. Our results show that <sup>1</sup>H NMR metabolomics is an effective tool for monitoring metabolite changes during the differentiation of pluripotent cells with implications on optimising media and environmental parameters for the study of embryogenesis and translational applications.
Project description:Patients with dyskeratosis congenita (DC) and related telomeropathies resulting from premature telomere dysfunction suffer from multi-organ failure. In the liver, DC patients present with nodular hyperplasia and cirrhosis. We model DC liver pathologies using isogenic human induced pluripotent stem (iPS) cells harboring a causal DC mutation in DKC1, or a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-corrected control allele. Differentiation of these iPS cells into hepatocytes or hepatic stellate cells reveals a dominant phenotype in the parenchyma. Generation of genotype admixed hepatostellate organoids indicates that DC hepatocytes elicit a pathogenic hyperplastic response in stellate cells independent of stellate cell genotype. Phenotypic rescue was achieved via suppression of AKT activity, a central regulator of mTORC1, MYC, and DC hepatocyte-driven hyperplasia. Thus, isogenic, iPS-derived admixed hepatostellate organoids offer insight into the liver pathologies in telomeropathies and provide a framework for evaluating emerging therapies.
