Project description:In order to investigate the role of CD34 antigen in haematopoietic commitment, we overexpressed the human CD34 cDNA in human CD34+ cells by retroviral gene transfer. Keywords: treatment comparison
Project description:In order to investigate the role of CD34 antigen in haematopoietic commitment, we silenced the CD34 gene expression in CD34+ stem/progenitor cells using a siRNA approach. Keywords: treatment comparison
Project description:In order to investigate the role of CD34 antigen in haematopoietic commitment, we silenced the CD34 gene expression in CD34+ stem/progenitor cells using a siRNA approach. Experiment Overall Design: To maximize siRNA transfection efficiency, we utilized the NucleofectorTM technology (Amaxa). CD34+ cells were transfected with a mixture of 4 siRNAs targeting CD34 mRNA and with a non-targeting siRNA as a negative control. The expression level of CD34 antigen on control cells (MOCK and negative control treated cells) and CD34siRNA treated cells was assessed by immunofluorescence analysis at 24 and 48h post-nucleofection.
Project description:In order to investigate the role of CD34 antigen in haematopoietic commitment, we overexpressed the human CD34 cDNA in human CD34+ cells by retroviral gene transfer. Experiment Overall Design: In a set of 10 independent experiments, gene transfer efficiency, assessed by flow cytometry analysis of ÎLNGFR positivity, ranged 15 to 30%. Transduced cells were than purified for NGFR expression at day 3 of liquid culture with the retroviral vector. Phenotypic analysis, performed on transduced/NGFR purified cells evidenced that the CD34 transgene was highly expressed at the protein level (80% of LCD34IÎN vs 30% of controls at 5 days post-infection) up to 14 days of liquid culture.
Project description:In this work, we compared gene expression profile (GEP) of CD34+ cells transduced with the retroviral vector LCALRIDN with CD34+ cells transduced with the empty vector control LXIDN and of CD34+ cells treated with CALR siRNA with control cells transfected with a Non-Targeting siRNA In order to explore the role of CALR in the proliferation and differentiation of hematopoietic stem/progenitor cells (HPSCs), we studied the effects of CALR overexpression in Cord Blood (CB) CD34+ cells. Assessment of cell differentiation unveiled that CALR enforced expression is able to skew the haematopoietic commitment towards the MK and erythroid cell lineages. Conversely, CALR silencing induced a marked repression of MK and erythroid differentiation. In order to characterize the effects of CALR mutations on human hematopoietic cells at the molecular level, we analyzed the coding RNA profiles of CB CD34+ overexpressing CALR. This analysis identified a list of differentially expressed genes. Among upregulated genes, we found genes involved in platelet aggregation, inflammation and erythroid differentiation, potentially related to the characteristics of the disease.
Project description:In this work, we compared gene expression profile (GEP) of CD34+ cells transduced with the retroviral vector LCALRIDN with CD34+ cells transduced with the empty vector control LXIDN and of CD34+ cells treated with CALR siRNA with control cells transfected with a Non-Targeting siRNA In order to explore the role of CALR in the proliferation and differentiation of hematopoietic stem/progenitor cells (HPSCs), we studied the effects of CALR overexpression in Cord Blood (CB) CD34+ cells. Assessment of cell differentiation unveiled that CALR enforced expression is able to skew the haematopoietic commitment towards the MK and erythroid cell lineages. Conversely, CALR silencing induced a marked repression of MK and erythroid differentiation. In order to characterize the effects of CALR mutations on human hematopoietic cells at the molecular level, we analyzed the coding RNA profiles of CB CD34+ overexpressing CALR. This analysis identified a list of differentially expressed genes. Among upregulated genes, we found genes involved in platelet aggregation, inflammation and erythroid differentiation, potentially related to the characteristics of the disease.
Project description:CD34+ Haematopoietic stem cells were differentiated ex vivo to generate ChIP-seq data for machine learning of rules underlying open chromatin dynamics.
Project description:CD34+ Haematopoietic stem cells were differentiated under two ex vivo protocols to generate ATAC-seq data for machine learning of rules underlying open chromatin dynamics.
Project description:The production of definitive haematopoietic stem/progenitor cells from human pluripotent stem cells (hPSCs) remains a significant challenge. Using reporter lines to track the endothelial (SOX17) to haematopoietic (RUNX1C) transition, we found that hPSC differentiated in growth factor supplemented serum free medium generated RUNX1C+CD34+ clonogenic cells that homed to the bone marrow, but did not engraft. Compared to repopulation-competent cord blood CD34+ cells, RUNX1C+CD34+ progenitors lacked HOXA gene expression, indicating incorrect mesoderm patterning. This deficiency was ameliorated by a timed pulse of WNT activation combined with ACTIVIN antagonism. Significantly, these HOXA+ cultures now formed complex SOX17+ vessels that produced fetal liver-like haematopoietic cells, similar to the human aorta-gonad-mesonephros (AGM). Comparison of transcriptional profiles of these nascent haematopoietic stem/progenitors with cells isolated from human AGM confirmed significant similarities, consistent with the assignment of our in vitro generated cells to the definitive human haematopoietic lineage. Our findings argue that HOXA codes established early in differentiation predict cellular potential and provide correct cell patterning for the specification of definitive haematopoietic lineages from hPSCs.
Project description:The production of definitive haematopoietic stem/progenitor cells from human pluripotent stem cells (hPSCs) remains a significant challenge. Using reporter lines to track the endothelial (SOX17) to haematopoietic (RUNX1C) transition, we found that hPSC differentiated in growth factor supplemented serum free medium generated RUNX1C+CD34+ clonogenic cells that homed to the bone marrow, but did not engraft. Compared to repopulation-competent cord blood CD34+ cells, RUNX1C+CD34+ progenitors lacked HOXA gene expression, indicating incorrect mesoderm patterning. This deficiency was ameliorated by a timed pulse of WNT activation combined with ACTIVIN antagonism. Significantly, these HOXA+ cultures now formed complex SOX17+ vessels that produced fetal liver-like haematopoietic cells, similar to the human aorta-gonad-mesonephros (AGM). Comparison of transcriptional profiles of these nascent haematopoietic stem/progenitors with cells isolated from human AGM confirmed significant similarities, consistent with the assignment of our in vitro generated cells to the definitive human haematopoietic lineage. Our findings argue that HOXA codes established early in differentiation predict cellular potential and provide correct cell patterning for the specification of definitive haematopoietic lineages from hPSCs.