Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Dynamic developmental signaling logic underlying lineage bifurcations during human endoderm induction and patterning from pluripotent stem cells [Expression data set]

ABSTRACT: The definitive endoderm germ layer is the provenance of multiple internal organs, including the lungs, liver, pancreas and intestines. Molecular events driving initial endoderm germ layer specification and subsequent anterior-posterior patterning of endoderm into distinct organ primordia remain largely cryptic. Through microarray analyses, we captured genome-wide transcriptional dynamics driving successive stages of endoderm development with the intent of identifying novel regulatory genes or diagnostic markers that respectively drive or mark endoderm committment. HES3 human embryonic stem cells (hESCs) were differentiated into highly homogeneous endodermal progenitor populations, and microarray analyses were conducted of six different populations at different tiers of the endodermal lineage hierarchy: undifferentiated hESCs, anterior primitive streak (day 1 of in vitro differentiation), definitive endoderm (day 3) and anterior foregut, posterior foregut or midgut/hindgut patterned endoderm populations (day 7). Additionally, we compared hESCs differentiated using two alternative endoderm induction protocols, serum-based or AFBLy-based differentiation (both day 3 of differentiation).

ORGANISM(S): Homo sapiens

SUBMITTER: Kyle Loh

PROVIDER: E-GEOD-52158 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

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.

Dataset Information

Dynamic developmental signaling logic underlying lineage bifurcations during human endoderm induction and patterning from pluripotent stem cells [Expression data set]

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets