Project description:The directed differentiation of induced pluripotent stem (iPS) and embryonic stem (ES) cells into definitive endoderm (DE) would allow the derivation of otherwise inaccessible progenitors for endodermal tissues. However, a global comparison of the relative equivalency of DE derived from iPS and ES populations has not been performed. Recent reports of molecular differences between iPS and ES cells have raised uncertainty as to whether iPS cells could generate autologous endodermal lineages in vitro. Here, we have shown that both mouse iPS and parental ES cells exhibited highly similar in vitro capacity to undergo directed differentiation into DE progenitors. With few exceptions, both cell types displayed similar surges in gene expression of specific master transcriptional regulators and global transcriptomes that define the developmental milestones of DE differentiation. Microarray analysis showed considerable overlap between the genetic programs of DE derived from ES/iPS cells in vitro and authentic DE from mouse embryos in vivo. Intriguingly, iPS cells exhibited aberrant silencing of imprinted genes known to participate in endoderm differentiation, yet retained a robust ability to differentiate into DE. Our results show that, despite some molecular differences, iPS cells can be efficiently differentiated into DE precursors, reinforcing their potential for development of cell-based therapies for diseased endodermal-derived tissues. Comparison of mouse ES cells, mouse iPS cells, and E8.25 Mouse embryos; in the undifferentiated state and definitive endoderm differentiated state. ckit+/Sox2dim =definitive endoderm (day 5 of differentiation in vitro); Sox2bright =undifferentiated ES or iPS cells (day 0 of differentiation); ENDM1+/Epcam+/SSlo=foregut endoderm sorted from mouse E8.25 embryos; Epcam+/ENDM1 negative =sorted comparison population from mouse E8.25 embryos.
Project description:Genomic imprinting is an epigenetic phenomenon resulting in parent-of-origin mono-allelic expression of a small subset of mammalian genes. Imprinted genes have been categorized into two groups: multi lineage (ML) imprinted genes that can show imprinted expression in both embryonic and extra-embryonic lineages, and genes that show extra-embryonic lineage (EXEL) specific imprinted expression restricted to tissues like the placenta and visceral yolk sac (VYS) endoderm. Many genes showing EXEL imprinted expression are silenced by lncRNAs that act over long distances. Thus the analysis of this form of gene silencing is likely to be pivotal for understanding new mechanisms of long range gene silencing by lncRNAs. It has been reported that ES cells differentiated into cystic embryoid bodies (cystic EBs) contain VYS endoderm and here we investigate if cystic EBs could serve as an in vitro model for the analysis of EXEL imprinted expression. Unexpectedly we found that cystic EBs lack EXEL imprinted expression, while retaining normal imprinted expression of ML genes. This shows that cystic EBs do not model VYS imprinted expression and also argues against previous claims that cystic EBs contain VYS endoderm. We further characterized cystic EBs by performing RNA-seq and whole genome bisulphite sequencing. By comparison to various embryonic tissues we found that cystic EBs more resemble embryonic liver, which contains definitive endoderm, than the extra-embryonic endoderm of the VYS. Examination of gene and retrotranssposone expression and DNA methylation in extraembryonic, embryonic tissues and ES cell diferentiated as cystic EBs
Project description:Genomic imprinting is an epigenetic phenomenon resulting in parent-of-origin mono-allelic expression of a small subset of mammalian genes. Imprinted genes have been categorized into two groups: multi lineage (ML) imprinted genes that can show imprinted expression in both embryonic and extra-embryonic lineages, and genes that show extra-embryonic lineage (EXEL) specific imprinted expression restricted to tissues like the placenta and visceral yolk sac (VYS) endoderm. Many genes showing EXEL imprinted expression are silenced by lncRNAs that act over long distances. Thus the analysis of this form of gene silencing is likely to be pivotal for understanding new mechanisms of long range gene silencing by lncRNAs. It has been reported that ES cells differentiated into cystic embryoid bodies (cystic EBs) contain VYS endoderm and here we investigate if cystic EBs could serve as an in vitro model for the analysis of EXEL imprinted expression. Unexpectedly we found that cystic EBs lack EXEL imprinted expression, while retaining normal imprinted expression of ML genes. This shows that cystic EBs do not model VYS imprinted expression and also argues against previous claims that cystic EBs contain VYS endoderm. We further characterized cystic EBs by performing RNA-seq and whole genome bisulphite sequencing. By comparison to various embryonic tissues we found that cystic EBs more resemble embryonic liver, which contains definitive endoderm, than the extra-embryonic endoderm of the VYS.
Project description:Endodermal cells differentiated from human induced pluripotent stem cells can produce multiple functional epithelial cells of vital organs and tissues that can be used for disease modeling as well as cell therapy. However, the detailed molecular mechanism of how hiPSCs differentiate towards definitive endoderm is not fully understood. By analyzing single-cell RNA sequencing data of definitive endoderm differentiation from human embryonic stem cells, transcription factor 7 (TCF7) was identified as a gene that transiently increases then decreases during differentiation of definitive endoderm.
Project description:The directed differentiation of induced pluripotent stem (iPS) and embryonic stem (ES) cells into definitive endoderm (DE) would allow the derivation of otherwise inaccessible progenitors for endodermal tissues. However, a global comparison of the relative equivalency of DE derived from iPS and ES populations has not been performed. Recent reports of molecular differences between iPS and ES cells have raised uncertainty as to whether iPS cells could generate autologous endodermal lineages in vitro. Here, we have shown that both mouse iPS and parental ES cells exhibited highly similar in vitro capacity to undergo directed differentiation into DE progenitors. With few exceptions, both cell types displayed similar surges in gene expression of specific master transcriptional regulators and global transcriptomes that define the developmental milestones of DE differentiation. Microarray analysis showed considerable overlap between the genetic programs of DE derived from ES/iPS cells in vitro and authentic DE from mouse embryos in vivo. Intriguingly, iPS cells exhibited aberrant silencing of imprinted genes known to participate in endoderm differentiation, yet retained a robust ability to differentiate into DE. Our results show that, despite some molecular differences, iPS cells can be efficiently differentiated into DE precursors, reinforcing their potential for development of cell-based therapies for diseased endodermal-derived tissues.
Project description:Pluripotent hESCs can differentiate into the three primary embryonic lineages (endoderm, mesoderm, ectoderm) as well as extraembryonic tissues. Definitive endoderm is the first step into the pathway to endoderm dreived tissues (pancreas, liver, gut, lung). We used microarrays to detail the changes in microRNA expression during the transition from pluripotent hESCs into definitive endoderm.
Project description:Pluripotent hESCs can differentiate into the three primary embryonic lineages (endoderm, mesoderm, ectoderm) as well as extraembryonic tissues. Definitive endoderm (DE) is the first step into the pathway to endoderm dreived tissues (pancreas, liver, gut, lung). We used microarrays to detail the changes in microRNA expression during the transition from pluripotent hESCs into definitive endoderm.
Project description:Pluripotent hESCs can differentiate into the three primary embryonic lineages (endoderm, mesoderm, ectoderm) as well as extraembryonic tissues. Definitive endoderm is the first step into the pathway to endoderm dreived tissues (pancreas, liver, gut, lung) We used microarrays to detail the changes in microRNA expression during the transition from pluripotent hESCs into definitive endoderm
Project description:Pluripotent hESCs can differentiate into the three primary embryonic lineages (endoderm, mesoderm, ectoderm) as well as extraembryonic tissues. Definitive endoderm (DE) is the first step into the pathway to endoderm dreived tissues (pancreas, liver, gut, lung). We used microarrays to detail the changes in microRNA expression during the transition from pluripotent hESCs into definitive endoderm. hESCs (H9) were differentiated in the presence of Activin A and Wnt3A under low serum conditions to induce DE formation. Samples were collected at day 0 (hESCs), and day 4 (DE).