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)
Project description:Cytotoxic T cells are typically expanded ex vivo for adoptive immunotherapy by culture with IL-2. This culture period leads to a differentiated phenotype and acquisition of effector function, as well as a loss of in vivo proliferative capability and anti-tumor efficacy. Here, we report antigen-specific and polyclonal expansion of cytotoxic T cells in a cocktail of cytokines and small molecules that leads to a memory-like phenotype in mouse and human cells even during extended culture, leading to enhanced in vivo expansion and tumor control. OT-I CD8 T cells were cultured for 14 days in either IL-2 or a cocktail of memory inducing small molecules and cytokines (IL-7, IL-21, 2-deoxyglucose and TWS119). Populations were sorted, IL-2 cells were CD44+CD62L-, cocktail cells were sorted into CD44+CD62L-, CD44+CD62L+ and CD44lowCD62L+ populations, and naive OT-I cells were CD44-CD62L+. Total RNA was extracted from each population and prepared for sequencing as three technical replicates.
Project description:Hematopoietic stem and progenitor cells (HPCs) can be maintained in vitro, but the vast majority of their progeny loses “stemness” during culture. We have analyzed DNA methylation (DNAm) profiles of freshly isolated CD34+ cells and upon expansion on either tissue culture plastic (TCP) or mesenchymal stromal cells (MSCs). Cultured HPCs acquired significant DNA-hypermethylation, particularly in up-stream promoter regions and shore-regions of CpG islands (CGIs). To analyze if these DNAm changes are relevant for differential gene expression we analyzed gene expression profiles of additional samples. As expected highly expressed genes (10% with highest signal intensity in gene expression arrays) were hardly methylated at promoter regions, CGIs and shore-regions. 9 samples were hybridized GeneChip Human Gene 1.0 ST Arrays (Affymetrix)
Project description:Hematopoietic stem and progenitor cells (HPCs) can be maintained in vitro, but the vast majority of their progeny loses “stemness” during culture. In this study, we have analyzed DNA methylation (DNAm) profiles of freshly isolated CD34+ cells and upon expansion on either tissue culture plastic (TCP) or mesenchymal stromal cells (MSCs). DNAm profiles of expanded CD34+ versus CD34- subsets reflected hematopoietic differentiation, whereas culture on TCP or MSCs had little impact. Notably, all cultured HPCs - even those which remained CD34 positive - acquired significant DNA-hypermethylation, particularly in up-stream promoter regions, shore-regions of CpG islands, and binding sides for PU.1 and RUNX1. Our results point to a coordinated epigenetic process which needs to be controlled to enhance self-renewal of HPCs in vitro. 12 samples were hybridised to the Illumina Infinium 450k Human Methylation Beadchip
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 (≥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.
Project description:To discover novel growth factors for hematopoietic stem- and progenitor cells (HSPCs), we have assessed cytokine responses of cord blood (CB)-derived CD34+ cells in a high-content growth factor screen. We identify the immunoregulatory chemokine (C-C motif) ligand 28 (CCL28) as a novel growth factor that directly stimulates proliferation of primitive hematopoietic cells from different ontogenetic origins. Microarrays are used to compare the expression profiles of HSPCs expanded in SCF, TPO and CCL28 respectively. HSPCs were cultured in Serum-Free Expansion Medium with SCF, SCF + TPO, and SCF + CCL28 respectively, and hybridised microarrays in triplicate.
Project description:The occurrence of clonal perturbations and leukemia in patients transplanted with retrovirally-transduced autologous hematopoietic stem and progenitor cells (HSPCs) has stimulated extensive investigation, demonstrating that proviral insertions perturb adjacent proto-oncogene expression. Although enhancer-deleted lentiviruses are less likely to result in insertional oncogenesis, there is evidence that they may perturb transcript splicing, and one patient with a benign clonal expansion of lentivirally-transduced HPSC has been reported. The rhesus macaque model provides an opportunity for informative long-term analysis to ask whether transduction impacts on long-term HSPC properties. We utilized two techniques to examine whether lentivirally-transduced HSPCs from eight rhesus macaques transplanted 1-13.5 years previously are perturbed at a population level, comparing telomere length as a measure of replicative history and gene expression profile of vector positive versus vector negative cells. There were no differences in telomere lengths between sorted GFP+ and GFP- blood cells, suggesting that lentiviral transduction did not globally disrupt replicative patterns. Bone marrow GFP+ and GFP- CD34+ cells showed no differences in gene expression using unsupervised and principal component analysis. These studies did not uncover any global long-term perturbation of proliferation, differentiation, or other important functional parameters of transduced HSPCs in the rhesus macaque model. CD34+ hematopoietic stem and progenitor cells were purified from bone marrow aspirates obtained from 4 rhesus macaques 3-9 years following transplantation with lentivirally-transduced autologous hematopoietic stem and progenitor cells. All lentiviral vectors contained the GFP marker gene. The CD34+ cells from each animal were sorted via flow cytometry into GFP+ and GFP- fractions, and RNA from these cells was used for Affymetrix gene expression analysis as detailed below. There were two samples, GFP+ and GFP-, from each of 4 animals.
Project description:The debilitating autoimmune disease Systemic Lupus Erythematosus (SLE) is closely associated with Toll-like receptor (TLR) 7 and type I interferon (IFN) activity in humans and in murine SLE-like disease. Two central manifestations of SLE affect the myeloid lineage of the immune system, myeloid expansion and anemia. Yet, whether these symptoms are linked and the role of TLR7 and/or type I IFN in these processes is unclear. Here we show that TLR7 signaling promotes cell-autonomous, phosphoinositide 3-kinase (PI3K)- and mammalian target of rapamycin (mTOR)-dependent macrophage development from the common myeloid progenitor (CMP). Strikingly, this TLR7-driven macrophage development requires and is enhanced by type I IFN. Genome-wide transcriptional profiling and functional studies demonstrated that TLR7 promoted the expression of Spic, the master regulator of splenic red pulp macrophages (RPM) and preferential development of hemophagocytic RPM-like cells from CMP in vitro. We found increased incidence of RPM-like cells in vivo in a mouse model of SLE caused by TLR7 overexpression, which correlated with decreased red blood cell (RBC) count and anemia. These findings demonstrate a mechanism by which TLR7 signaling promotes anemia that is of clinical significance in SLE, other rheumatological diseases and chronic viral infections. This work also identifies a previously unknown molecular pathway by which TLR signaling and type I IFN synergize to promote myeloid development from hematopoietic progenitors. CMP were sorted from the bone marrow of wild-type C57BL/6 mice, cultured with SCF+R848 or SCFr+MCSF, and CD11b+F4/80+ macrophages sorted after 5 days, n=3 per group
Project description:PPARγ antagonist GW9662 treatment could enhance ex vivo expansion of human cord blood hematopoietic stem and progenitor cells (HSCs/HPCs). To gain mechanistical insights into how antagonizing PPARγ promotes expansion of HSCs/HPCs, we performed RNA sequencing (RNA seq) analysis to identify genes involved in this process. Loss of function of PPARγ in CB CD34+ cells resulted in downregulation of a number of differentiation associated genes, including CD38, CD1d, HIC1, FAM20C, DUSP4, DHRS3 and ALDH1A2, suggesting that PPARγ antagonist may maintain stemness of CB CD34+ cells, at least in part by preventing differentiation. We also observed that FBP1, encoding fructose 1, 6-bisphosphatase, a negative regulator of glycolytic flux, was significantly downregulated by treating CB CD34+ cells with GW9662. Our study demonstrates that antagonizing PPARγ signaling drives ex vivo expansion of human CB HSCs/HPCs by switching on FBP1 repressed glycolysis and preventing differentiation. Overall design: Total RNA was extracted from Vehicle or GW9662 treated cord blood CD34+ cells, and then RNA sequencing analysis was performed.
Project description:Signaling through the AKT and ERK pathways controls cell proliferation. However, the integrated regulation of this multistep process, involving signal processing, cell growth and cell-cycle progression, is poorly understood. Here we study different murine hematopoietic cell types, in which AKT and ERK signaling is triggered by erythropoietin (Epo). Although these cell types share the molecular network topology for pro-proliferative Epo signaling, they exhibit distinct proliferative responses. Iterating quantitative experiments and mathematical modeling, we identify two molecular sources for cell-type-specific proliferation. First, cell-type-specific protein abundance patterns cause differential signal flow along the AKT and ERK pathways. Second, downstream regulators of both pathways have differential effects on proliferation, suggesting that protein synthesis is rate-limiting for faster-cycling cells while slower cell-cycles are controlled at the G1-S progression. The integrated mathematical model of Epo-driven proliferation explains cell-type-specific effects of targeted AKT and ERK inhibitors and faithfully predicts based on the protein abundance anti-proliferative effects of inhibitors in primary human erythroid progenitor cells. Our findings suggest that the effectiveness of targeted cancer therapy might become predictable from protein abundance patterns.