Gene expression profiles of cultured megakaryocytes
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE112820 | GEO | 2021/04/05
REPOSITORIES: GEO
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Project description:Cord blood CD34+ hematopoietic precursors (3 donors) were control or HES6 shRNA (HES6 knockdown) transduced and cultured for four days in media containing SCF, TPO and EPO to drive differentiation towards megakaryocytes and erythroid cells. After four days four subpopulations were sorted for RNA isolation to research the effect of HES6 knockdown on gene expression. We sorted megakaryocytes (CD41+) and early (CD71+ CD235-) and late (CD71+ CD235+) erythroblasts (CD41-) and CD34- precursor cells (CD41- CD71- CD235- CD34-).
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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.
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Project description:Normal donors BM CD34+ cells were differentiated in thrombopoietin-treated liquid suspension cultures. Briefly, CD34+ cells (80,000 /mL) were resuspended in a serum-free medium in the presence of 100 ng/mL of human recombinant thrombopoietin (TPO; Genzyme, Boston, MA). Every 3 days, viable cells were scored by Trypan blue dye exclusion and cultures were amplified with fresh serum-free medium. Each well was then supplemented with 100 ng/mL TPO. After 14 to 16 days of liquid culture, CD34-derived megakaryocytes (MKs) were purified by means of an anti-CD41a monoclonal antibody (MoAb) (Dako, Milan, Italy) directed against the glycoproteic *IIb-*3 complex and immunobeads (MPC 450 Dynabeads; Dynal, Oslo, Norway), as previously described (27). The purity of MKs was determined for each isolation by indirect immunofluorescence, using an anti-CD41b MoAb which reacts with a different epitope of *IIb-*3 subunit (Immunotech Inc., Westbrook, ME), followed by a goat anti-mouse IgG, covalently linked to fluorescein (Becton Dickinson, San Jose, CA). Essential Throbocytemia BM CD34+ cells were differentiated in thrombopoietin-treated liquid suspension cultures. Briefly, CD34+ cells (80,000 /mL) were resuspended in a serum-free medium in the presence of 100 ng/mL of human recombinant thrombopoietin (TPO; Genzyme, Boston, MA). Every 3 days, viable cells were scored by Trypan blue dye exclusion and cultures were amplified with fresh serum-free medium. Each well was then supplemented with 100 ng/mL TPO. After 14 to 16 days of liquid culture, CD34-derived megakaryocytes (MKs) were purified by means of an anti-CD41a monoclonal antibody (MoAb) (Dako, Milan, Italy) directed against the glycoproteic *IIb-*3 complex and immunobeads (MPC 450 Dynabeads; Dynal, Oslo, Norway), as previously described (27). The purity of MKs was determined for each isolation by indirect immunofluorescence, using an anti-CD41b MoAb which reacts with a different epitope of *IIb-*3 subunit (Immunotech Inc., Westbrook, ME), followed by a goat anti-mouse IgG, covalently linked to fluorescein (Becton Dickinson, San Jose, CA). Keywords: other
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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.
2009-09-14 | E-TABM-640 | biostudies-arrayexpress
Project description:Normal donors BM CD34+ cells were differentiated in thrombopoietin-treated liquid suspension cultures. Briefly, CD34+ cells (80,000 /mL) were resuspended in a serum-free medium in the presence of 100 ng/mL of human recombinant thrombopoietin (TPO; Genzyme, Boston, MA). Every 3 days, viable cells were scored by Trypan blue dye exclusion and cultures were amplified with fresh serum-free medium. Each well was then supplemented with 100 ng/mL TPO. After 14 to 16 days of liquid culture, CD34-derived megakaryocytes (MKs) were purified by means of an anti-CD41a monoclonal antibody (MoAb) (Dako, Milan, Italy) directed against the glycoproteic *IIb-*3 complex and immunobeads (MPC 450 Dynabeads; Dynal, Oslo, Norway), as previously described (27). The purity of MKs was determined for each isolation by indirect immunofluorescence, using an anti-CD41b MoAb which reacts with a different epitope of *IIb-*3 subunit (Immunotech Inc., Westbrook, ME), followed by a goat anti-mouse IgG, covalently linked to fluorescein (Becton Dickinson, San Jose, CA). Essential Throbocytemia BM CD34+ cells were differentiated in thrombopoietin-treated liquid suspension cultures. Briefly, CD34+ cells (80,000 /mL) were resuspended in a serum-free medium in the presence of 100 ng/mL of human recombinant thrombopoietin (TPO; Genzyme, Boston, MA). Every 3 days, viable cells were scored by Trypan blue dye exclusion and cultures were amplified with fresh serum-free medium. Each well was then supplemented with 100 ng/mL TPO. After 14 to 16 days of liquid culture, CD34-derived megakaryocytes (MKs) were purified by means of an anti-CD41a monoclonal antibody (MoAb) (Dako, Milan, Italy) directed against the glycoproteic *IIb-*3 complex and immunobeads (MPC 450 Dynabeads; Dynal, Oslo, Norway), as previously described (27). The purity of MKs was determined for each isolation by indirect immunofluorescence, using an anti-CD41b MoAb which reacts with a different epitope of *IIb-*3 subunit (Immunotech Inc., Westbrook, ME), followed by a goat anti-mouse IgG, covalently linked to fluorescein (Becton Dickinson, San Jose, CA).
2008-06-17 | E-GEOD-997 | biostudies-arrayexpress
Project description:Physical factors can have major influences on the proliferation and differentiation fate of hematopoietic stem cells in culture. Recently, we demonstrated that cord blood CD34+ cells undergo accelerated and increased megakaryocyte differentiation when incubated at 39°C. In this study, we investigated in detail the impacts of mild hyperthermia on the kinetics of megakaryocyte differentiation, maturation, polyploidization and cell viability. The qualitative and quantitative effects on Mk differentiation were found to be rapidly induced, and optimization of the culture length at 39°C led to greater Mk yields. Mild hyperthermia did not promote endomitosis of cord blood-derived Mk, but rather led to a small reduction in the proportion of polyploid Mk. Moreover, it had little impact on viability but induced a significant shift in the proportion of cells undergoing apoptosis at 37°C to necrosis at 39°C. Finally, our results suggest that the effects on megakaryocyte differentiation could be the consequences of aberrant expression of key megakaryocyte transcription factors induced by mild hyperthermia. Experiment Overall Design: To define potential differences between megakaryocytes (Mk) derived at 37°C or 39°C, we compared their gene expression repertoire by micro-gene chip technology (Affymetrix GeneChip HG133A). Two independent experiments were carried out on Mk at 37 or 39°C. CD41a+ Mk issued from CB CD34+ cells were cultured for 7-days with TPO at 100 ng/ml and purified by positive magnetic selection using a CD41a-FITC monoclonal antibody (Immunotech, Marseille, France) and MACS columns according to manufacturerâs instructions (Miltenyi, Auburn, CA, USA) with a purity of >95%.
2008-06-16 | E-GEOD-9218 | biostudies-arrayexpress