A study of the differentiation of Murine ES cells [Scl+/hCD4 ES cells (Chung et al., 2002)] into hemangioblasts

ABSTRACT: Molecular mechanisms that regulate the generation of hematopoietic and endothelial cells from mesoderm are poorly understood. To define the underlying mechanisms, we compared gene expression profiles between embryonic stem (ES) cell-derived hemangioblasts (Blast-Colony-Forming Cells, BL-CFCs) and their differentiated progeny, Blast cells. Bioinformatic analysis indicated that BL-CFCs resembled other stem cell populations. A role for Gata2, one of the BL-CFC-enriched transcripts, was further characterized by utilizing the in vitro model of ES cell differentiation. Our studies revealed that Gata2 was a direct target of BMP4 and that enforced GATA2 expression upregulated Bmp4, Flk1 and Scl. Conditional GATA2 induction resulted in a temporal-sensitive increase in hemangioblast generation, precocious commitment to erythroid fate, and increased endothelial cell generation. GATA2 additionally conferred a proliferative signal to primitive erythroid progenitors. Collectively, we provide compelling evidence that GATA2 plays specific, contextual roles in the generation of Flk-1+ mesoderm, the Flk-1+Scl+ hemangioblast, primitive erythroid and endothelial cells. Keywords: Comparison of induced differetiated state to original cell line. Generation of Hemangioblast and Blast Colony cell populations: Scl+/hCD4 ES cells (Chung et al., Development, 2002) were maintained on a layer of mitotically inactivated STO cells in DMEM media in the presence of LIF. Two days before differentiation, the cells were transferred to gelatinized tissue culture flasks and the media was changed to IMDM-based. ES cells were differentiated in liquid-differentiation media in the presence of FCS for 2.75 days. D2.75 EBs were recovered, dissociated in Trypsin/EDTA and stained with antibodies against Flk-1 and human CD4. Cells were then flow sorted on the basis of Flk-1 and Scl expression and the hemangioblast population was considered to be Flk-1+Scl+. D2.75 EB cells were replated as described (Chung et al., 2002) and individual Blast colonies were picked and pooled, and RNA was generated with TRIzol according to the manufacturers protocol. RNA (20 ug) was given to the Washington University Array core were it was reverse transcribed and cRNA synthesized. Replicated of the two populations (hCD4, Blast) were made. Pairwaise comparisons were performed to identify enriched genes.

ORGANISM(S): Mus musculus

SUBMITTER: kyunghee choi 

PROVIDER: E-GEOD-6835 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress