Project description:Gene expression analysis of Normal CD34+ Cord Blood and UKE1 cell lines treated with hairpins targeting ASXL1. Two independent studies where 1) CD34+ cord blood from normal donors were treated with either A) GFP Vector or B) ASXL1 specific short hairpin and 2) UKE1 cell lines treated with either A) GFP Vector or B) ASXL1 specific short hairpin.
Project description:AML1-ETO expression in normal human umbilical cord blood CD34+ cells leads to long-term proliferation of an early self-renewing primitive progenitor cell with multilineage potential and stem cell ability, but these cells do not induce leukemia in immunodeficient mice. This comparative microarray study was initiated to determine the differences in the transcriptome of AML-ETO-expressing CD34+ cells after extended culture in vitro, using normal cord blood cells expanded for 6-8 weeks in vitro and subsequently purified for the CD34+ population as the control comparison. Keywords: Disease state analysis; comparison of changes in transcriptome due to long-term AML1-ETO expression in normal human hematopoietic CD34+ progenitor cells
Project description:To identify target genes of mutant ASXL1 (ASXL1-MT) and HHEX in hematopoietic cells, we performed RNA-seq using RUNX1-ETO expressing cord blood cells transduced with vector or ASXL1-MT together with vector or HHEX.
Project description:AML1-ETO expression in normal human umbilical cord blood CD34+ cells leads to long-term proliferation of an early self-renewing primitive progenitor cell with multilineage potential and stem cell ability, but these cells do not induce leukemia in immunodeficient mice. This comparative microarray study was initiated to determine the differences in the transcriptome of AML-ETO-expressing CD34+ cells after extended culture in vitro, using normal cord blood cells expanded for 6-8 weeks in vitro and subsequently purified for the CD34+ population as the control comparison. Experiment Overall Design: We have established a culture system whereby we retrovirally transduce human CD34+ cells, obtained from cord blood, with the leukemia fusion gene AML1-ETO. Cells expressing this fusion protein are able to proliferate long-term in vitro in a cytokine dependent manner. AML1-ETO-expressing cord blood cells have a large population of primitive self-renewing CD34+ cells with continued abnormal differentiation. We grow these cells in serum-free conditions using the BIT supplement from Stem Cell Technologies. For the current experiments we used cell cultures that had been proliferating in vitro for 8-12 weeks, in a cytokine cocktail of SCF, TPO, FLT3L, IL-6 all at 20 ng/mL and IL-3 at 10 ng/mL. Control cord blood samples that were CD34 purified were expanded for 5-8 weeks in the same culture media as used for AML1-ETO cells. All samples were magnetically selected for the CD34+ population, returned to culture, and one week later again selected for CD34+ cells and then lysed for RNA isolation.
Project description:Transcription profiling analysis was performed on purified CD34+ cell lines (Cord Blood CD34+) treated with ExtracellularVescicles (EVs) isolated from bone marrow mesenchymal stem cells (BM-MSC).
Project description:We report on the characterization of JMML hematopoietic stem and progenitor cells (HSPCs). In order to characterize the single cell heterogeneity in comparison to normal cord blood we preformed droplet based single cell RNA-seq of FACS sorted CD34+ cells using the 10x genomics platform. We identified distinct clustering of unique JMML HSPC populations in relation to cord blood control.
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:ADGRG1/GPR56 was knocked down by two different shRNAs in cord blood CD34+ cells via lentiviral transduction. GFP was used as marker indicating successful transduction.