Project description:Decreased levels of Cohesin proteins increases the amount of primitive hematopoietic stem and progenitor cells in an in vitro setting. We used microarrays to detail the changes in global transcription programme to understand the underlying mechanisms behind this phenotype. Of note, we discover that hematopoietic stem cell-specific genes are upregulated in cells with decreased Cohesin levels. CD34 positive cells were isolated from human cord blood (not older than 24h), lentivirally transduced with shRNA targeting different members of the Cohesin complex. In order to focus on immediate changes, transduced cells were sorted 36 hours post transduction and analyzed.
Project description:Epigenetic memory in induced pluripotent stem cells (iPSCs), with regards to their somatic cell type of origin, might lead to variations in their differentiation capacities. In this context, iPSCs from human CD34+ hematopoietic stem cells (HSCs) might be more suitable for hematopoietic differentiation than commonly used fibroblast-derived iPSCs. To investigate the influence of an epigenetic memory on the ex vivo expansion of iPSCs into erythroid cells, we compared iPSCs from human neural stem cells (NSCs) and human cord blood-derived CD34+ HSCs and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells (RBCs). Although genome-wide DNA methylation profiling at all promoter regions demonstrates an epigenetic memory of iPSCs with regards to their somatic cell type of origin, we found a similar hematopoietic induction potential and erythroid differentiation pattern. All human iPSC lines showed terminal maturation into normoblasts and enucleated RBCs, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the CD34+ HSC-derived iPSCs. More detailed methylation analysis of the hematopoietic and erythrocyte promoters identified similar CpG methylation levels in the CD34+ iPSCs and NSC iPSCs, which confirms their comparable erythroid differentiation potential. To investigate the influence of an epigenetic memory on the ex vivo expansion of iPSCs into erythroid cells, we compared iPSCs from human neural stem cells (NSCs) and human cord blood-derived CD34+ HSCs and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells (RBCs). RNA samples for microarray analysis were prepared using RNeasy columns (Qiagen, Germany) with on-column DNA digestion. 300ng of total RNA per sample was used as the input in the linear amplification protocol (Ambion), which involved the synthesis of T7-linked double-stranded cDNAs and 12hrs of in vitro transcription incorporating the biotin-labeled nucleotides. Purified and labeled cRNA was then hybridized for 18hrs onto HumanHT-12 v4 expression BeadChips (Illumina, USA) following the manufacturer's instructions. After the recommended washing, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and the accompanying software. The samples were exclusively hybridized as biological replicates. 8 samples were analyzed: CD34 1, Human CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) population 1, 1 replicate CD34 2, Human CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) population 2, 1 replicate CD34 OSiPS 1, Human Human two factors (POU5F1, SOX2) induced Pluripotent Cell (iPSC) reprogrammed from CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) induced Pluripotent Cell (iPSC) population 1, 1 replicate CD34 OSKMiPS 1, Human Human four factors (POU5F1, SOX2, KLF4, CMYC) induced Pluripotent Cell (iPSC) reprogrammed from CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) induced Pluripotent Cell (iPSC) population 1, 1 replicate CD34 OSiPS 2, Human Human two factors (POU5F1, SOX2) induced Pluripotent Cell (iPSC) reprogrammed from CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) induced Pluripotent Cell (iPSC) population 2, 1 replicate CD34 OSKMiPS 2, Human Human four factors (POU5F1, SOX2, KLF4, CMYC) induced Pluripotent Cell (iPSC) reprogrammed from CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) induced Pluripotent Cell (iPSC) population 2, 1 replicate H1, Human H1 embryonic stem cell (ESC), 1 replicate H9, Human H9 embryonic stem cell (ESC), 1 replicate
Project description:Transciptome analysis of CD34+ enriched human HSPC lentivirally transduced with cohesin WT or mutant CD34+ enriched HSPCs from cord blood were transduced with a constitutive lentiviral vector expressing cohesin WT or mutant tagged to GFP. After 72hrs cells were GFP+ sorted and subjected to downstream microarray protocol.
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.
Project description:Global gene expressions of human cord blood-derived 18Lineage-negative (18Lin-)CD34+CD38-CD133+GPI-80+ cells (CD34+ HSCs), 18Lin-CD34-CD133+GPI-80+ cells (CD34- HSCs) and 18Lin-CD34+CD133- cells (non-HSCs) were analyzed. Results provide an insight into the molecular mechanisms underlying the self-renewal, maintenance and differentiation of human cord blood-derived CD34+/- HSCs.