Expression data from CD34+/CD71- cells following knockdown of HSPA9
ABSTRACT: Knockdown of HSPA9 causes a dose-dependent decrease in erythroid maturation of CD34+ cells differentiated in culture. Due to differences in the degree of differentiation, a more homogeneous population was selected for using FACS and the gene expression profile of these cells was compared. We used a lentiviral vector (pLKO.1) expressing short hairpin RNAs targeting either luciferase (control shLUC) or HSPA9 (shHSPA9-433) to knock down expression of HSPA9. We isolated CD34+ cells from human cord blood (Day 0), transduced cells with a lentiviral vector (Day 1), selected for transduced cells with puromycin and differentiated them in erythroid culture media before FACS isolation of the CD34+/CD71- population (Day 5). Four independent CD34+ populations were isolated, differentiated and sorted for biologic replicates.
Project description:Activated T cells inhibit neurogenesis in adult animal brain and cultured human fetal neural stem cells (NSC). However, the role of inhibition of neurogenesis in human neuroinflammatory diseases is still uncertain because of the difficulty in obtaining adult NSC from patients. Recent developments in cell reprogramming suggest that NSC may be derived directly from adult fibroblasts. We generated NSC from adult human peripheral CD34+ cells by transfecting the cells with Sendai virus constructs containing Sox-2, Oct3/4, C-MyC and Klf-4. The derived NSC could be differentiated to astroglia and action potential firing neurons. Co-culturing NSC with activated autologous T cells or treatment with recombinant granzyme B caused inhibition of neurogenesis as indicated by decreased NSC proliferation and neuronal differentiation. Thus, we have established a unique autologous in vitro model to study the pathophysiology of neuroinflammatory diseases that has potential for usage in personalized medicine. 11 Human samples from 7 sources representing 4 different cell types: 2 CD34 (CD34+ cells purified from adult peripheral blood), 3 iNS (induced Neural Stem Cells derived directly from CD34+ cells), 2 iNS derived from iPSC (Neural Stem cells differentiated from induced Pluripotent Stem Cells from CD34+ cells), 4 NPC (human primary cultured neural progenitor cells)
Project description:Gene expression profiling of primary cord blood hematopoietic stem cell (day 0, CD34+ cells), enriched control (untreated), Scriptaid and Valproic acid expanded CD34+ cells after a week in culture Individal cord blood CD34+ cells were processed individually and equal number of reisolated CD34+ cells from 3-4 samples were pooled after expansion to avoid the sample variations. Gene expression profiles of primary human cord blood CD34+ cells (day0), primary cells (PC) expanded in the presence or absence of histone deacetylase inhibitors (HDACIs) in serum containing cultures supplemented with a combination of cytokines (SCF, FLT3, IL3 and TPO) for 7 days.
Project description:Gene expression analysis from erythroid progenitors (CD34+/CD71(high)/CD45- mononuclear cells from the bone marrow) of patients with Diamond-Blackfan anemia (due to RPS19 mutations) and control individuals. Case-control microarray gene expression analysis
Project description:Hematopoietic stem and progenitor cells are a rare, self-renewing bone marrow resident population capable of giving rise to all circulating hematopoietic cells. They can be used therapuetically for reconstituting defective or ablated hematopoietic systems following chemotherapy, and for inducing tolerance toward allografts of the same haplotype as the HSC donor. There are several sources for HSCs, such as the adult bone marrow, or umblical cord blood, which is more replete with such HSCs. However, HSCs obtained from such sources may be immunogenic, especially if isolated from adult bone marrow. To overcome this issue, our lab has establsihed human induced pluripotent stem cell-derived HPCs with the hope of creating a nonimmunogenic, readily available and unlimited source of HSCs to use for therapy. The goal of this study was to compare the gene expression profiles of naturally found HSCs (UCB-CD34+ HSCs) and HPCs differentiated from 4 different human iPS cell lines (iPS-HPCs), so as to determine the variation between the four iPS-HPCs and whether there were any differences between these HPCs and naturally found HSCs. We utilized 4 iPS cells for this study (detailed descriptions are provided below). iPS cells were differentiated into hematopoietic progenitor cells by coculture on OP9 stromal cells, followed by enrichment of CD34+ cells through immunomagnetic bead separation. The UCB-CD34+ cells were isolated from frozen cord samples through immunomagnetic bead separation. Total RNA was isolated and human gene Affymetrix ST 1.0 arrays performed at the University of Iowa DNA core facility. Data was analyzed, normalized and plotted on BRB Array Tools.
Project description:Background: Developmental stage-specific globin expression is a complex phenomenon that involves both trans- and cis-acting elements. While functional analyses ensuing recent genome-wide association studies have highlighted the important roles of trans-factors in regulating hemoglobin expression, these factors can not exert their functions without permissive chromatin domains. By transferring thoroughly profiled beta globin locus of undifferentiated human embryonic stem cells (hESCs) or hESC-derived erythroid cells into an adult erythroid transcriptional environment, we studied the influences of histone modifications on the globin expression decision within a fixed transcriptional environment. Shortly after the locus transfer, embryonic epsilon globin was not expressed regardless of original chromatin states, whereas fetal gamma globin was either expressed or not activated depending on original chromatin configurations, and the originally silent adult beta globin either remained silent or became activated depending on the expression status of gamma globin. These data suggest the interplay between transcriptional environment and the chromatin modifications determine the outcome of globin expression. As the ultimate silencing of gamma globin from hESC-derived erythroid cells in the adult transcriptional environment occurred after months-long cell proliferation, our work also has implications on attempts to generate beta globin expressing erythroid cells from hESCs or induced pluripotent stem cells. hESC line H1 (NIH code WA01, WiCell, Madison, WI) and adeno-associated virus (AAV)-targeted lines, were maintained and differentiated as previously described.12 (Details can be found in the supplemental information.) The expression of stem cell markers including SSEA-3, SSEA-4, TRA-1-60, and TRA-1-61 was detected by flow cytometry. To determine whether AAV-targeted lines retained their hematopoietic differentiation potential, confluent hESCs were harvested off the feeder layers and transferred to ultra-low attachment plates to allow for the formation of embryoid bodies (EBs). Day-14 EBs were dissociated into single cells and the expression of surface markers including CD34, CD71, CD45, CD31, CD41 and glycophorin-A was determined by flow cytometry. To induce erythroid differentiation, day-7 EBs were made into single cell suspension and cultured in erythroid-inducing medium for 14 days. Primers for real time PCR analysis of beta locus globin mRNA expression are provided in the supplemental information.
Project description:Gene expression analyses of hematopoietic stem cells (HSCs), progenitor cells (HPCs), and differentiated cell. Gene expressions of long-term HSCs (CD34-ckit+Sca1+Lineage-), short term HSCs (CD34+ckit+Sca1+Lineage-), Progenitor cells (ckit+Sca1- Lineage-), and differentiated cels (Lineage+) were examined by microarray. Results provide insight into the mechanism of hematopoietic cell differentiation. Long-term HSCs (CD34-ckit+Sca1+Lineage-), Short term-HSCs (CD34+ckit+Sca1+Lineage-), Progenitor cells (ckit+Sca1- Lineage-, and Lineage+), and differentiated cell (Lineage+) were sorted from mouse bone marraw and were examined by microarray. Results provide insight into the mechanism of hematopoietic cell differentiation.
Project description:We applied a novel approach of parallel transcriptional analysis of multiple, highly fractionated stem and progenitor populations in a genetically defined subset of AML (AML with monosomy 7). We isolated phenotypic long-term HSC (LT-HSC), short-term HSC (ST-HSC), and committed granulocyte-monocyte progenitors (GMP) from individual patients with AML, and measured gene expression profiles of each population, and in comparison to their phenotypic counterparts from age-matched healthy controls. Bone marrow samples from AML patients bearing monosomy 7 and age-matched healthy controls were used in this study. Hematopoietic stem and progenitor compartments were purified by multiparameter-high speed fluorescence-activated cell sorting (FACS) from CD34+ enriched bone marrow to isolate LT-HSC (Lin-/CD34+/CD38-/CD90+), ST-HSC (Lin-/CD34+/CD38-/CD90-), and GMP (Lin-/CD34+/CD38+/CD123+/CD45R+).
Project description:We applied a novel approach of parallel transcriptional analysis of multiple, highly fractionated stem and progenitor populations from patients with acute myeloid leukemia (AML) and a normal karyotype. We isolated phenotypic long-term HSC (LT-HSC), short-term HSC (ST-HSC), and committed granulocyte-monocyte progenitors (GMP) from individual patients, and measured gene expression profiles of each population, and in comparison to their phenotypic counterparts from age-matched healthy controls. Bone marrow samples from AML patients with normal karyotype and age-matched healthy controls were used in this study. Hematopoietic stem and progenitor compartments were purified by multiparameter-high speed fluorescence-activated cell sorting (FACS) from CD34+ enriched bone marrow to isolate LT-HSC (Lin-/CD34+/CD38-/CD90+), ST-HSC (Lin-/CD34+/CD38-/CD90-), and GMP (Lin-/CD34+/CD38+/CD123+/CD45R+).
Project description:Identification of cell-type specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Enhancers are typically marked by the co-transcriptional activator protein p300 or by groups of cell-expressed transcription factors. We hypothesized that a unique set of enhancers regulates gene expression in human erythroid cells, a highly specialized cell type evolved to provide adequate amounts of oxygen throughout the body. Using chromatin immunoprecipitation followed by massively parallel sequencing, genome-wide maps of candidate enhancers were constructed for p300 and four transcription factors, GATA1, NF-E2, KLF1, and SCL, using primary human erythroid cells. These data were combined with gene expression analyses and candidate enhancers identified. Consistent with their predicted function as candidate enhancers, there was statistically significant enrichment of p300 and combinations of co-localizing erythroid transcription factors within 1-50 kb of the TSS of genes highly expressed in erythroid cells. Candidate enhancers were also enriched near genes with known erythroid cell function or erythroid cell phenotypes. Candidate enhancers exhibited only moderate conservation with mouse and minimal conservation with nonplacental vertebrates. Candidate enhancers were mapped to a data set of erythroid-associated, biologically relevant, SNPs from the GWAS catalog of the NHGRI. Fourteen candidate enhancers, representing 10 genetic loci, mapped to sites associated with biologically relevant erythroid traits. Fragments from these loci directed statistically significant expression in reporter gene assays. Identification of enhancers in human erythroid cells will allow a better understanding of erythroid cell development, differentiation, structure, and function, and provide insights into inherited and acquired hematologic disease. CD34+-selected stem and progenitor cells were expanded for three days in the absence of EPO, and total RNA was isolated from a portion of the cells. The cells were further cultured in the presence of EPO, and RNA was isolated after cells differentiated into R3/R4 nucleated erythroid cells. There were 3 replicates of CD34 cells and 9 replicates of R3/R4 erythroid cells.