Project description:These experiments treated cord blood-derived megakaryocytes with Cortistatin A for 0-8 h and analyzed resultant gene expression changes by microarray analysis.
Project description:The stable culture system derived from umbilical cord blood (UCB) was established, which differentiates into megakaryocytes (MKs)in vitro and the transcriptome data of UCB-derived MKs produced by our system was analyzed.
Project description:Ex vivo differentiation of megakaryocytes (Mk) was carried out using human cord blood (CB) CD34+ cells under the stimulation of recombinant human interleukin-3, stem cell factor, and thrombopoietin for 7 days, followed by thrombopoietin treatment alone for further 3 days. Total cellular RNA was extracted from Day-0 CD34+, Day-10 CD41+ and CD41- cells, respectively. Microarray was performed and the data was analyzed using the GeneChip Operating Software, Spotfire DecisionSite Software and Genomatix Application Software.
Project description:Characterize the genes regulated by MKL1/SRF complex in human megakaryocytes (MKs) derived from cord blood or cytapheresis : Using a knock down approach by small interference of MKL1 in MK progenitors, we observed a decrease in the percentage of cells with actin polymerization after adhesion on various substrates, an increase of mean ploidy level and apoptosis. Furthermore, MKL1 inhibition induced a major defect in pro-platelet formation and MKs migration. These results clearly demonstrate that MKL1 is involved in the cytoskeleton organization and maturation of MKs as well as in platelet formation. The goal of gene profiling was to identify new potential MKL1/SRF targets the expression level of which was down regulated after MKL1 inhibition: Human CD34+ cells isolated from cord blood or cytapheresis were transduced by a control lentivirus (designed as SCR; scramble) or by the lentivirus encoding for shRNA of MKL1 (designed as shMKL1) both containing a GFP expressed under the control of PGK promoter. The cells were than grown in serum free medium supplemented by thrombopoietin leading to a generation of megakaryocytes. At day 9 of culture (80% of MKs), the GFP positive cells were sorted, RNAs were extracted and submitted to hybridization as described in annex protocols. Two lists of genes (one for cord blood samples and one for cytapheresis samples) deregulated after the repression of MKL1 were done comparing the intensity of hybridization between SCR and shMKL1 samples. The common list of genes deregulated in MKs from cord blood and cytapheresis after the repression of MKL1 was than generated.
Project description:Cells obtained from adipose tissue are able to differentiate into megakaryocytes. We compared the gene expression profile of human adipose tissue derived megakaryocytes with that of megakaryocytes differentiated from human CD34 positive cord blood hematopoietic stem cells.