Project description:ES derived Flk-1+ cells were separated by sorting into Hoxb6 positive and negative populations by Venus reporter. Gene expression was compared between these two groups. Duplicate analysis of Hoxb6-Venus positive and negative Flk-1+ cells.
Project description:ES derived Flk-1+ cells were separated by sorting into Hoxb6 positive and negative populations by RFP Hoxb6 reporter. Gene expression was compared between these two groups. Duplicate analysis of Hoxb6-RFP positive and negative Flk-1+ cells.
Project description:Screening for genes regulated by Etv2 within Flk-1+/PDGFRa+ ES derived mesoderm.Microarray analysis performed to screen for the candidate genes regulated by Etv2. TT2 ES cells differentiated on OP9 feeder cells were sorted using Flk-1 and PDGFRa antibodies.Gene expressions from these two populations were compared. Extract RNA from sorted Flk-1+/PDGFRa+ populations from Etv2Het vs KO cells. To obtain primitive mesoderm cells TT2 ES cells of corresponding genotypes were differentiated on OP9 cells for 4 days. Flk-1+/PDGFRa+ populations were sorted from Etv2 Het vs. KO cells for RNA extraction.
Project description:Screening for genes up in Etv2+ cells within Flk-1+ ES derived mesoderm Microarray analysis performed to screen for the candidate genes regulated by Etv2. Differentiated Flk-1+ mesoderm can be devided into Etv2+ or-. Etv2+ cells are assumed to be committed to hemato/endothelial cells. Comparison of two populations can reveal genes relevant in this commitment. Extract RNA from sorted Flk-1+/Etv2- vs Flk-1+/Etv2+ populations.Etv2-Venus KI ES cells were differentiated on OP9 for 4-5 days and Flk-1+ population was separated into Etv2-Venus+ or- cells. Total RNA was purified from each population for analysis.
Project description:This SuperSeries is composed of the following subset Series: GSE34537: Mesp1 induces a subset of hematopoietic-associated transcription factors in ES cell-derived Flk1+Tie2+ endothelium GSE34541: Identification of gene targets of Meis2 GSE34543: Identification of gene targets of Meis1 Refer to individual Series
Project description:The homeodomain protein Meis1 is essential for definitive hematopoiesis and vascular patterning in the mouse embryo. Our present study suggested it exerts two distinguishable effects in differentiating ES cells. First, it increases the numbers of hematopoietic progenitors and extends their persistence in culture. Second, Meis1 skews hematopoietic differentiation by suppressing erythroid while enhancing megakaryocytic progenitor differentiation. To identify the underlying transcriptional bases of these actions, we carried out microarray analysis to compare the various populations of cells developing in ES differentiation cultures in the presence and absence of Meis1 induction. ES cells with dox-inducible Meis1 (A2lox.Meis1) were differentiated as embryoid bodies (EBs) for 6 days before plating on OP9-GFP cell monolayers and cytokines, and treated with (+) or without (-) doxycycline (dox). Cells were purified by cell sorting on day 7 or 8 into various populations based on levels of CD41 expression: GFP-CD41-, GFP-CD41+ (day 7) and GFP-CD41-,GFP-CD41int, and GFP-CD41hi (day 8). Gene expression of these purified populations was determined by microarray analysis.
Project description:The homeodomain protein Meis1 is essential for definitive hematopoiesis and vascular patterning in the mouse embryo. Meis2, another member of the same family, shares 82% protein identities with Meis1. Our present study suggested Meis2 exerts two distinguishable effects in differentiating ES cells. First, it increases the numbers of hematopoietic progenitors and extends their persistence in culture. Second, Meis2 skews hematopoietic differentiation by suppressing erythroid while enhancing megakaryocytic progenitor differentiation. To identify the underlying transcriptional bases of these actions, we carried out microarray analysis to compare the various populations of cells developing in ES differentiation cultures in the presence and absence of Meis2 induction. ES cells with dox-inducible Meis2 (A2lox.Meis2) were differentiated as embryoid bodies (EBs) for 6 days before plating on OP9-GFP cell monolayers and cytokines, and treated with (+) or without (-) doxycycline (dox). Cells were purified by cell sorting on day 7 or 8 into various populations based on levels of CD41 expression: GFP-CD41-, GFP-CD41+ (day 7) and GFP-CD41-,GFP-CD41int, and GFP-CD41hi (day 8). Gene expression of these purified populations was determined by microarray analysis.
Project description:Hemogenic endothelium (HE) is the source of HSCs in the developing embryo. In this study we have identified the hemogenic endothelial progenitors and their precursors originating from differentiated H1 cells on OP9 stromal cells. RNA-seq of hemogenic endothelial progenitors and their precursors originating from differentiated H1 cells on OP9 stromal cells.
Project description:We previously demonstrated that hematopoietic stem cell (HSC)-like cells are robustly expanded from mouse embryonic stem (ES) cells by enforced expression of Lhx2, a LIM-homeobox domain (LIM-HD) transcription factor. Here we established an ES cell line which conditionally expressed Lhx2 by Tet-On system. The ES cells were differentiated into HSC-like cells by Lhx2 expression. Lhx2-regulated genes were identified by comparing the HSC-like cells with those cultured in the absence of Lhx2 expression. Mouse ES with inducible Lhx2 were differentiated on OP9 stromal cells into hematopoietic lineage. On day 5 of the differentiation induction,Lhx2 expression was started by the addition of doxycycline (dox) and the cells were cultured on OP9 stromal cells in the presence of IL-6 and SCF. On day 20, HSC-like cells were harvested and re-seeded onto OP9 stromal cells in the absence of Lhx2 expression by dox-removal for 3 days. these cells were compared with the original HSC-like cells.
Project description:Specification to primordial germ cells (PGCs) occurs under the mesoderm induction signals during gastrulation. Here, we found that Akt activation in embryonic stem (ES) cells generated self-renewing spheres during mesodermal differentiation induction and that the differentiation status of the sphere cells was in between ES cells and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, showing that the cells of the sphere commenced the differentiation to germ lineage. However, the spheres could not proceed to spermatogenesis after transplantation to testes. Meanwhile, the transfer of the spheres to the original feeder-free ES cell culture conditions induced chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, the reversion to the ES cell-like cell states was induced. These results indicate that the Akt signaling brings about a novel metastable and pluripotent state between ES cells and PGCs. Five samples were analyzed, which included the Akt-Mer-expressing ES cell (ESC) line #21 treated with or without 4OHT (4-hydroxytamoxifen), the #21 ESC-derived primordial germ cell (PGC)-like sphere cells and the ESC-like cells reverted from #21 PGC-like sphere cells. The PGC-like sphere cells derived from another Akt-Mer ESC line #42 was also examined.