Transcriptome profiling of hESC-derived endoderm progenitors by single-cell RNA-seq
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE188362 | GEO | 2022/11/15
REPOSITORIES: GEO
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Project description:Transcriptome profiling of hESC-derived endoderm progenitors by RNA-seq. RNA-seq analysis during the stepwise progression of hESC toward the pancreatic progenitors, at five defined stages of differentiation and expansion: hESC, ADE, VFG.p3, VFG.p6. PE.
2022-11-15 | GSE185670 | GEO
Project description:Investigation of whole genome gene expression level changes in five hESC lines as a result of FGF2 withdrawal. Five hESC lines were grown in parallel with and without FGF2
2013-11-16 | E-GEOD-25479 | biostudies-arrayexpress
Project description:Investigation of whole genome gene expression level changes in five hESC lines as a result of FGF2 withdrawal.
2013-11-16 | GSE25479 | GEO
Project description:The aim of the study was to define the specific gene expression signature of pancreatic endoderm cells (PECs), by designing a strategy for generating putative PECs (PDX1+/NKX6-1+, and posterior foregut endoderm (PFG) cells (PDX1+/NKX6-1-). Cell line: PDX1-eGFP reporter cell line (PDXeG clone 170-3) generated using the HUES-4 embryonic stem cell line. Human embryonic stem cells (hESCs) were maintained on irradiated mouse embryonic fibroblasts (MEFs). Generation of a hESC-derived PDX1-eGFP reporter cell line: A PDX1-targeting vector was constructed by inserting an eGFP-pSV40-NeoR reporter cassette into the 5â² untranslated region of the PDX1 gene upstream of the PDX1 start codon (ATG), resulting in an GFP-tagged PDX1 allele. The GFP cassette was flanked by a 12.5kb homologous arm on the 5â direction and by a 3,5kb homologous arm on the 3 direction. The final targeting construct was verified by PCR, restriction analysis and sequencing. After approximately 2 weeks emerging clones were picked, expanded and analyzed by PCR. The Neomycin cassette was deleted by using a CRE expression vector (NLS-CRE-IRES-Puro) that was transiently co-transfected with a DsRED or GFP plasmid into selected targeted cloned. 24 hours post electroporation, GFP/DsRED positive single cells were FACS sorted and plated into 96-well plates. Clones were expanded and characterized for Neo excision by PCR. Differentiation protocol: hESCs cultured on MEFs were grown until 90% confluency and differentiated into definitive endoderm (DE) by using a modified version of the DâAmour protocol (D'Amour et al., 2005). The first day of differentiation, hESCs were cultured in the presence of 100ng/ml human Activin A (Peprotech) and 25ng/ml Wnt3a (R&D systems) in RPMI medium (Life Technologies). The four following days 100ng/ml human Activin A was added together with 1x B27-insulin in RPMI medium (Life Technologies). To generate posterior foregut cells, DE cells were treated with 64 ng/ml bFGF for additional 12 days. To generate pancreatic endoderm cells, DE cells were first treated with 2uM retinoic acid (RA, Sigma Aldrich) in DMEM/F12 medium containing 1x B27 +insulin (Life Technologies) for three days and then finally treated with 64 ng/ml FGF2 in combination with 100 ng/ml Noggin for the remaining 9 days.
2017-04-05 | E-MTAB-5088 | biostudies-arrayexpress
Project description:H1 hESC were exposed to BMP4 (10 ng/ml) in absence of FGF2 for 0, 3, 12, 24, 72, and 120 h under both physiological (4 % O2) and atmospheric (20 % O2) conditions to identify candidate genes and gene networks associated with specification from totipotent precursor cells to trophoblast. RNA from cells under both conditions was isolated at each time point. Approximately 32,000 probe sets (out of 40,391) provided a signal above background at one of the time points. Of these, 14,478 showed a ≥2-fold (plus or minus) change (p<0.05) under 4 % O2, and 18,580 under 20 % O2. The data indicate the potential usefulness of hESC grown in presence of BMP4 and absence of FGF2 for studying both the emergence of trophoblast from hESC precursors and for its subsequent differentiation over time, space and varied O2 concentrations. Keywords: Genes and gene networks associated with specification from totipotent precursor cells to trophoblast
2009-05-10 | GSE10469 | GEO
Project description:The foregut definitive endoderm is the precursor of many tissues including the liver, pancreas, thyroid, lungs, trachea and oesophagus. However, networks and pathways involved in the early development of the definitive endoderm of mammals are not well studied. To identify genes with potential roles in the early development of the foregut definitive endoderm in mouse embryos, we performed microarray analysis to compare the gene expression profile of foregut endoderm and non-endodermal tissues from early somite-stage mouse embryos.
2012-03-13 | GSE36453 | GEO
Project description:Methods for differentiating human pluripotent stem cells to pancreatic and liver lineages in vitro have been limited by the inability to identify and isolate distinct endodermal subpopulations specific to these two organs. Here we report that pancreatic and hepatic progenitors can be isolated using the surface markers CD177/NB1 glycoprotein and inducible T-cell costimulatory ligand CD275/ICOSL, respectively, from seemingly homogeneous definitive endoderm derived from human pluripotent stem cells. Anterior definitive endoderm (ADE) subpopulations identified by CD177 and CD275 show inverse activation of canonical and noncanonical WNT signaling. CD177+ ADE expresses and synthesizes the secreted WNT, NODAL and BMP antagonist CERBERUS1 and is specified toward the pancreatic fate. CD275+ ADE receives canonical Wnt signaling and is specified toward the liver fate. Isolated CD177+ ADE differentiates more homogeneously into pancreatic progenitors and into more functionally mature and glucose-responsive β-like cells in vitro compared with cells from unsorted differentiation cultures.
2020-04-27 | GSE113791 | GEO
Project description:HESC-H9 and iPSC lines 3.5, 3.6 and 3.12 were analyzed using Affymetrix microarray before and after Definitive Endoderm (DE) formation. DE was induced using the ActivinA differentiation protocol described by D'Amour et al., 2006 (PMID: 16258519) Clustering analysis of transcripts that were differentially regulated during DE formation indicated that iPSC lines 3.5 and 3.12 differentiate in manner that is highly similar to HESC-H9 cells iPSC line 3.6 had a more divergent transcriptional profile.
2010-12-25 | GSE25557 | GEO
Project description:HESC-H9 and iPSC lines 3.5, 3.6 and 3.12 were analyzed using Affymetrix microarray before and after Definitive Endoderm (DE) formation. DE was induced using the ActivinA differentiation protocol described by D'Amour et al., 2006 (PMID: 16258519) Clustering analysis of transcripts that were differentially regulated during DE formation indicated that iPSC lines 3.5 and 3.12 differentiate in manner that is highly similar to HESC-H9 cells iPSC line 3.6 had a more divergent transcriptional profile. Three induced pluripotent stem cell lines (iPSC) and one human embryonic stem cell line (hESC - H9) were collected as undifferentiated (UD) cells, and flash frozen. These cell lines were also subjected to definitive endoderm (DE) induction, collected and flash frozen. RNA was harvested from the frozen cell pellets and hybridized to the Affymetrix microarray chip. The three iPSC cell lines are iPSC 3.5, iPSC 3.6. and iPSC 3.12. In the UD state, iPSC 3.5 was analyzed in duplicate, while iPSCs 3.6 and 3.12 and H9s were analyzed in biological triplicate. All four cell lines were analyzed as biological triplicates for DE induction.
2010-12-25 | E-GEOD-25557 | biostudies-arrayexpress
Project description:Our understanding of how mesodermal tissue is formed, has been limited by the absence of specific and reliable markers of early mesoderm commitment. We report that mesoderm commitment from human embryonic stem cells (hESC) is initiated by Epithelial to Mesenchymal transition (EMT) as shown by gene expression profiling and by reciprocal changes in expression of the cell surface proteins, EpCAM/CD326 and NCAM/CD56. Molecular and functional assays reveal that CD326negCD56+ cells, generated from hESC in the presence of activin A, BMP4, VEGF and FGF2, represent a novel, multi-potent mesoderm-committed progenitor population. CD326negCD56+ progenitors are unique in their ability to generate all mesodermal lineages including hematopoietic, endothelial, mesenchymal (bone, cartilage, fat, fibroblast), smooth muscle and cardiomyocytes, while lacking the pluripotency of hESC. CD326negCD56+ cells are the precursors of previously reported, more lineage-restricted mesodermal progenitors. These findings present a novel approach to study how germ layer specification is regulated, and offer a unique target for tissue engineering. We used microarrays to compare gene expression profile of early mesodermal progenitors with undifferentiated hESC (H9 line). Mesoderm induction from hESC was initiatiated with combination of morphogens and growth factors including activin A, bone morphogenic protein 4, basic fibroblast growth factor and vascular endothelial growth factor. Proposed mesodermal progenitor population was isolated by FACS on day 3.5 of culture based on the presence of CD56 expression and the absenbce of CD326 expression.
2010-05-27 | E-GEOD-21668 | biostudies-arrayexpress