Project description:The objective of the study is to identify the genes regulated by STAT5 in very primitive human hematopoietic stem progenitor cells (HSPCs). We used Affymetrix Human Genome U133 Plus 2.0 microarrays to investigate gene expression in human CD34+CD38- HSPC upon expression of STAT5 or control luciferase shRNA-encoding vectors.
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:The comparative characterization of hematopoietic stem cells from healthy stem cell donors and patients with acute myeloid leukemia on a proteome level has the potential to reveal differentially regulated proteins which might be candidates for specific immunotherapy target molecules. Exemplarily, we analyzed the proteome of the cytosolic and the membrane fraction of CD34 and CD123 co-expressing FACS-sorted leukemic progenitors from five patients with acute myeloid leukemia employing mass spectrometry. As a reference, CD34+CD123+ normal hematopoietic progenitor cells from five healthy stem cell donors were analyzed. In this TMT 10-plex labeling based approach 2068 proteins were identified with 256 proteins differentially regulated in one or both cellular compartments. This study demonstrates the feasibility of a mass spectrometry based proteomic approach to detect differentially expressed proteins in two compartment fractions of leukemic stem cells as compared to their healthy stem cell counterparts.
Project description:RNA-seq analysis was performed to compare differentially expressed genes in freshly isolated and ex-vivo cultured human cord blood CD34+ cells. Mitochondrion related genes are upregulated in CD34+ hematopoietic stem and progenitor cells upon ex vivo culture. In vivo transplantation experiments demonstrate that stemness of CD34+ cells is significantly decreased due to oxidative stress induced by ex vivo culture.
Project description:To facilitate comparative genomic analyses of human fetal and adult cells undergoing erythropoiesis, we employed a serum-free two-phase liquid culture system to expand and differentiate primary human CD34+ hematopoietic stem/progenitor cells (HSPCs) ex vivo. In this experimental context, highly enriched populations of stage-matched, differentiating, primary proerythroblasts (ProEs) were generated. We selected four time points (day 0, CD34+ HSPCs; day 3, 5, and 7, differentiating ProEs) that represented similar stages differentiation for gene expression profiling using microarrays. Primary maturing fetal or adult erythroblasts were generated ex vivo from CD34+ hematopoietic stem/progenitor cells (HSPCs) using a serum-free two-phase liquid culture system. Total RNA from primary fetal and adult HSPCs (day 0) and differentiating proerythroblasts (ProEs; day 3, 5, and 7) were extracted and used to hybridize to Affymetrix expression arrays using the HG-U133 Plus 2.0 platform.
Project description:To facilitate comparative genomic analyses of human fetal and adult cells undergoing erythropoiesis, we employed a serum-free two-phase liquid culture system to expand and differentiate primary human CD34+ hematopoietic stem/progenitor cells (HSPCs) ex vivo. In this experimental context, highly enriched populations of stage-matched, differentiating, primary proerythroblasts (ProEs) were generated. We selected four time points (day 0, CD34+ HSPCs; day 3, 5, and 7, differentiating ProEs) that represented similar stages differentiation for gene expression profiling using microarrays.
Project description:To better understand the early events regulating lineage-specific hematopoietic differentiation, we analyzed the transcriptional profiles of CD34+ human hematopoietic stem and progenitor cells (HSPCs) subjected to differentiation stimulus. CD34+ cells were cultured for 12 and 40 hours in liquid cultures with supplemented media favoring myeloid or erythroid commitment. Serial analysis of gene expression (SAGE) was employed to generate four independent libraries. CD34+ Hematopoietic Stem Progenitor Cells with no differentiation stimulus were used as a control library.
