Unknown,Transcriptomics,Genomics,Proteomics

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PRC2 inhibition counteracts the culture-associated loss of engraftment potential of human cord blood-derived hematopoietic stem and progenitor cells

ABSTRACT: Cord blood hematopoietic stem cells (CB-HSCs) are an outstanding source for transplantation approaches. However, the amount of cells per donor is limited and culture expansion of CB-HSCs is accompanied by a loss of engraftment potential. In order to analyze the molecular mechanisms leading to this impaired potential we profiled global and local epigenotypes during the expansion of human CB hematopoietic stem and progenitor cells (HPSCs). Human CB-derived CD34+ cells were cultured in serum-free medium together with SCF, TPO, FGF, with or without Igfbp2 and Angptl5 (STF/STFIA cocktails). As compared to the STF cocktail, the STFIA cocktail maintains in vivo repopulation capacity of cultured CD34+ cells. Upon expansion, CD34+ cells genome-wide remodel their epigenotype and depending on the cytokine cocktail, cells show different H3K4me3 and H3K27me3 levels. Expanding cells without Igfbp2 and Angptl5 leads to higher global H3K27me3 levels. ChIPseq analyses reveal a cytokine cocktail-dependent redistribution of H3K27me3 profiles. Inhibition of the PRC2 component EZH2 counteracts the culture-associated loss of NOD scid gamma (NSG) engraftment potential. Collectively, our data reveal chromatin dynamics that underlie the culture-associated loss of engraftment potential. We identify PRC2 component EZH2 as being involved in the loss of engraftment potential during the in vitro expansion of HPSCs. 6 samples were hybridized GeneChip Human Gene 1.0 ST Arrays (Affymetrix)

ORGANISM(S): Homo sapiens

SUBMITTER: Martin Zenke

PROVIDER: E-GEOD-58461 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Mononuclear cells were isolated from umbilical cord blood (UCB) using Lymphoprep sucrose-gradient centrifugation (1.077 g/ml, Nycomed Pharma, Oslo, Norway). Immunomagnetic cell separation, using magnetic beads coated with CD34 antibodies (Miltenyi Biotec, Gladbach, Germany), was performed to isolate CD34-positive hematopoietic progenitor cells (CB-CD34+ cells). To generate retrovirus, bicistronic retroviral DNA constructs were used expressing the ETV6-RUNX1 or AML1-ETO fusion gene and enhanced Green Fluorescent Protein (eGFP). As a control, a construct expressing only eGFP was used. Both vectors consisted of a pMSCV promoter region, an internal ribosomal entry site and an ampicillin resistance cassette. HEK293T cells were co-transfected with these constructs and second-generation retroviral packaging vectors using XtremeGENE 9 tranfection reagents (Roche, Basel, Switzerland). Viral particles were collected in IMDM 48 hours after transfection. CD34+ haematopoietic progenitors, pre-cultured overnight as described above, upon which cells were divided in two fractions. One fraction was transduced with ETV6-RUNX1-IRES-eGFP or AML1-ETO-IRES-eGFP, while the other fraction was transduced with control EV-IRES-eGFP. Transductions were performed with fresh retrovirus in retronectin (Takara, Otsu, Japan) coated wells. After sorting, DAPI- CD34+ GFP+ CB-CD34+ cells were lysed and RNA was extracted using Nucleospin RNA XS extraction columns according to manufacturer’s protocol (Macherey-Nagel, Düren, Germany). Quality of RNA was determined by on-chip-electrophoresis using a RNA Pico Chip according to manufacturer’s protocol (Agilent Technologies, Santa Clara, CA, USA). RNA Integrity scores (RIN) were higher than 8 for all samples. RNA was subsequently linearly amplified using the Nugen WT-Amplification™ pico system (Nugen, San Carlos, CA, USA). This system is based on RNA-dependent DNA polymerase activity and was previously reported to be most suitable for amplification and gene expression of picograms of input RNA.

Project description:The supply of red blood cells (RBCs) is not sufficient in many developing countries or in developed countries for patients who need chronic transfusion from best-matched donors. Ex vivo expansion and maturation of human erythroid precursor cells (erythroblasts) could represent a potential solution. Proliferating erythroblasts can be expanded from human umbilical cord blood mononuclear cells (CB MNCs) ex vivo for 10^6-10^7 fold (in ~50 days) before undergoing senescence. Here, we report that ectopic expression of three to four genetic factors that have been used for iPS cell derivation enables CB-derived erythroblasts to undergo extended ex vivo expansion (M-bM-^IM-%10^51 fold in ~9 months) in a defined suspension culture condition without change of cell identity or function. These vastly expanding erythroblasts maintain homogeneously immature erythroblast phenotypes, a normal diploid karyotype and dependence on specific combination of cytokines and hormone for survival and proliferation throughout the continuous expansion period. When switched to a culture condition for terminal maturation, these immortalized erythroblasts gradually exit cell cycle, decrease cell size, accumulate hemoglobin, condense nuclei and eventually give rise to enucleated hemoglobin-containing erythrocytes. Our result may ultimately lead to the development of unlimited sources of cultured RBCs for optimally-matched or personalized transfusion medicine. We compared the global gene expression profiles of different human cell types: iE: immortalized erythroblasts generated by genetic reprogramming from pCBE; pCBE: primary cord blood-derived erythroblasts; CD34+: CD34+ purified hematopoietic stem/progenitor cells from adult blood or fetal liver; TF-1: a human erythroleukemia cell line; ESC: human embryonic stem cells; iPSCs: human induced pluripotent stem cells. We want to see the relationship among these cell types. We included multiple samples (biological replicates) for most cell types.

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PRC2 inhibition counteracts the culture-associated loss of engraftment potential of human cord blood-derived hematopoietic stem and progenitor cells

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