Project description:Homodimerization of Mpl can also be accomplished in the absence of Tpo, by binding of a synthetic ligand (Chemical inducer of dimerization, CID) to a constitutively expressed fusion protein F36VMpl consisting of a ligand binding domain (F36V) and the intracellular signaling domain of Mpl. In contrast to Tpo stimulation, F36VMpl dimerization in human CD34+ progenitor cells generates robust erythropoiesis. Microarray gene expression profiling of progenitors demonstrated that F36VMpl dimerization, but not Tpo, results in upregulation of critical erythroid genes. CD34+ cord blood cells were transduced with F36VMpl-GFP (GFP reporter gene) and cultured on MS-5 stroma for 7 days in the presence of CID, Tpo, Epo or no factors (no CID, negative control). CD34+GFP+ cells were sorted on day 7 and subjected to microarray (n=3 independent experiments).
Project description:Homodimerization of Mpl can also be accomplished in the absence of Tpo, by binding of a synthetic ligand (Chemical inducer of dimerization, CID) to a constitutively expressed fusion protein F36VMpl consisting of a ligand binding domain (F36V) and the intracellular signaling domain of Mpl. In contrast to Tpo stimulation, F36VMpl dimerization in human CD34+ progenitor cells generates robust erythropoiesis. Microarray gene expression profiling of progenitors demonstrated that F36VMpl dimerization, but not Tpo, results in upregulation of critical erythroid genes.
Project description:analysis of differential gene expression in CD34+ derived neutrophil progenitors from umbilical cord blood, in response to Mek1 activation
Project description:Expansion for hematopoietic cells from umbilical cord blood is a strategy for use this cell source in clinic transplants, however, it is important to know about the genomic changes that can occur in expanded cells. In order to detect global expression profiles changes in hematopoietic stem and progenitors cells generated in vitro, we analyzed hematopoietics populations obtained by FACS in fresh from umbilical cord blood. HSC (fHSC) was defined as CD34+ CD38- CD71- CD45RA- Lin- and were cocultured with stromal cell line OP-9 plus FL, SCF, IL3, IL6, TPO, GMCSF and G-CSF by 7 days, after time we repurified HSC population by FACS using same immunophenotype (ivHSC). In other hand, fresh erythroid progenitors cells (fEPC) were identified as CD34+CD38+CD71+CD45RA- Lin- and fresh myeloid progenitors cells (fMPC) were identified as CD34+CD38+CD71-CD45RA+Lin-. In vitro progenitors cells (ivEPC and ivMPC) were obtained by culturing fHSC in Stemspan serum-free media plus SCF, TPO, IL6, FL and IL3 by 10 days, after time cells were repurified by FACS using same immunophenotype for fresh progenitors. In vitro generated cells were compared with their corresponding fresh population cells.
Project description:Global gene expression analysis of FACS-purified CD31+CD146+ vascular progenitors (VP) derived from (a) human embryonic stem cells (VP-hESC), (b) adult fibroblast derived induced pluripotent stem cells (VP-AdF-iPSC), (c) stromal primed cord blood CD34+ myeloid progenitor derived iPSC (VP-sp-CB-iPSC), (d) corresponding starting fibroblasts and myeloid progenitors, (e) human umbilical vein endothelial cells, and (f) human dermal microvascular endothelial cells. Total RNA was harvested from (a) adult fibroblasts, (b) human myeloid progenitors: Non-nucloefected CD34+ CB cells that were expanded with growth factors from Day -3 until Day 0 and harvested, (c) FACS-purified CD31+CD146+ vascular progenitors (VP) that were differentiated from human embryonic stem cells (hESC), (d) VP differentiated from low passage stromal-primed episomal iPSC derived from growth-factor activated cord blood myeloid progenitors, (e) VP differentiated from iPSC derived from adult fibroblasts, (f) human umbilical vein endothelial cells, and (g) human dermal microvascular endothelial cells.. Illumina HumanHT-12 V4.0 expression beadchips were used for all analyses in this series. Three or independent samples of each sample type were each run on individual microarrays.
