Identification of the chemokine CCL28 as a growth and survival factor for human hematopoietic stem- and progenitor cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE45136 | GEO | 2013/06/03
SECONDARY ACCESSION(S): PRJNA192950
REPOSITORIES: GEO
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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.
2013-06-03 | E-GEOD-45136 | biostudies-arrayexpress
Project description:We identified chemokine (C-C motif) ligand 28 (CCL28) as a novel growth factor for HSPCs with the ability to enhance the proliferation potential of fetal, neonatal and adult CD34+ HSPCs after one week of culture in serum-free medium containing limiting stem cell factor (SCF) concentrations. Importantly, CCL28 could increase the progenitor potential of CB- and bone marrow (BM) derived CD34+ cells as measured by colony assays. Furthermore, treatment of CB CD34hiCD38loCD90+CD45RA- cells with CCL28 significantly enhanced the ability of the cells to long-term repopulate immunodeficient mice compared to SCF-treated cultures. Our findings show that CCL28 is a potent growth factor for human HSCs and suggest that it should be further exploited in clinical strategies for ex vivo expansion. Human CD34+CD38-CD45RAlowCD90+ cord blood cells were cultured in vitro for 18 hrs with CCL28 and SCF or SCF alone.
2013-01-01 | E-GEOD-35374 | biostudies-arrayexpress
Project description:We identified chemokine (C-C motif) ligand 28 (CCL28) as a novel growth factor for HSPCs with the ability to enhance the proliferation potential of fetal, neonatal and adult CD34+ HSPCs after one week of culture in serum-free medium containing limiting stem cell factor (SCF) concentrations. Importantly, CCL28 could increase the progenitor potential of CB- and bone marrow (BM) derived CD34+ cells as measured by colony assays. Furthermore, treatment of CB CD34hiCD38loCD90+CD45RA- cells with CCL28 significantly enhanced the ability of the cells to long-term repopulate immunodeficient mice compared to SCF-treated cultures. Our findings show that CCL28 is a potent growth factor for human HSCs and suggest that it should be further exploited in clinical strategies for ex vivo expansion.
2013-01-01 | GSE35374 | GEO
Project description:Hematopoietic stem cells (HSCs) are cells able to self-renew and differentiate into all blood lineages. We previously have performed serum-free single-cell culture for HSC1 (CD201+CD150+CD48-CD41-CD34-LSK) and competitive repopulation with cultured cells. Single highly purified HSC1 were cultured with stem cell factor (SCF), SCF + G-CSF, or SCF + thrombopoietin (TPO) for 7 days. SCF directly acted on HSC1 and maintained their long-term multilineage reconstitution after serial transplantation. Compared with SCF alone, SCF+G-CSF neither enhanced the number of divisions of HSC1 nor enhanced their levels of reconstitution after serial transplantation. SCF+TPO significantly increased the number of divisions of HSC1. The levels of reconstitution of each lineage in SCF + TPO culture were significantly increased at early months after primary transplantation, however, it gradually decreased after secondary transplantation. Therefore, we performed single-cell RNA-Seq to examine the gene expression of cells cultured with different cytokine combinations.
2023-06-28 | GSE153430 | GEO
Project description:Hematopoietic stem cells (HSC) rely on a unique regulatory machinery that facilitates life-long blood production and enables reconstitution of the entire hematopoietic system upon transplantation. However, the biological processes governing human HSC self-renewal and engraftment ability are poorly understood and challenging to recapitulate ex vivo to facilitate robust human HSC expansion. We discovered a novel HSC regulatory protein, MYCT1 (MYCT target 1), that is selectively expressed in endothelial cells (EC) and undifferentiated human HSPCs but becomes drastically downregulated during HSC culture. Lentiviral knockdown of MYCT1 in human foetal liver and cord blood HSPCs revealed a critical role for MYCT1 in governing human HSPC expansion and engraftment ability. Single cell RNAseq of human CB HSPCs after MYCT1 knockdown and overexpression revealed that MYCT1 governs HSC functional competence and modulates cellular properties essential for HSC stemness, such as low mitochondrial metabolic activity. Indeed, restoring the compromised MYCT1 expression in cultured human CB HSPCs improved ex vivo expansion of the most undifferentiated human HSPCs and enhanced their engraftment ability. We found that MYCT1 is localized in the endosomal membrane and interacts with vesicle trafficking regulators and signalling machinery essential for HSC and EC function. Loss of MYCT1 led to excessive endocytosis and hyperactive signalling responses to cytokines, whereas restoring MYCT1 expression in cultured CB HSPCs balanced the abnormal endocytosis associated with prolonged culture and fine-tuned signalling responses. Our work identifies MYCT1-moderated endocytosis and environmental sensing as an essential regulatory mechanism required to preserve human HSC stemness, and pinpoints silencing of MYCT1 as a critical contributor to the dysfunction of cultured human HSCs that needs to be addressed to improve human HSC culture strategies.
2024-04-15 | PXD042257 | Pride
Project description: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)
2015-08-07 | E-GEOD-58461 | biostudies-arrayexpress
Project description: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.
2015-08-07 | GSE58461 | GEO
Project description:Thrombopoietin (TPO) is a critical cytokine regulating hematopoietic stem cell maintenance and differentiation into the megakaryocytic lineage. However, the transcriptional and chromatin dynamics elicited by TPO signaling are poorly understood. Here, we study the immediate early transcriptional and cis-regulatory responses to TPO in hematopoietic stem/progenitor cells (HSPCs) and use this paradigm of cytokine signaling to chromatin to dissect the relation between cis-regulatory activity and chromatin architecture. We show that TPO profoundly alters the transcriptome of HSPCs, with key hematopoietic regulators being transcriptionally repressed within 30 minutes of TPO. By examining cis-regulatory dynamics and chromatin architectures, we demonstrate that these changes are accompanied by rapid and extensive epigenome remodeling of cis-regulatory landscapes that is spatially coordinated within topologically associating domains (TADs). Moreover, homotypic TPO-responsive enhancers are spatially clustered and engage in preferential intra- and inter-TAD interactions that are only moderately perturbed by TPO signaling. By further examining the link between cis-regulatory dynamics and chromatin looping, we show that rapid modulation of cis-regulatory activity is largely independent of chromatin looping dynamics. Finally, we show that activated and repressed cis-regulatory elements share a remarkably similar DNA sequence composition and that in vivo transcription factor binding patterns accurately predict rapid cis-regulatory responses to TPO.
2018-01-25 | GSE100835 | GEO
Project description:MIXL1-GFP reporter lines (HESC3MIXL1-GFP/w and MEL1MIXL1-GFP/w, referred to as HES3 and MEL1 in this submission) were differentiated as spin embryonic bodies (Ebs) supplemented with mesodermal inducing growth factors (BMP4,SCF, VEGF based). Methylcellulose hematopoietic colony forming assays were performed by culturing dissociated day 4 EBs in the presence of hematopoietic growth factors (VEGF, SCF, IL3, IL6, TPO, EPO and FLT3L) for 7-9 days. Addition of WNT3a to the methylcellulose lead to the formation of compact, mesodermal colonies, which we term 'mesosphere' (Meso-balls). We demonstrated that the inclusion of WNT3a in the methylcellulose could be replaced with BIO, a GSK3 inhibitor, acting as a canonical WNT signaling agonist. Mesospheres formed in the culture supplemented with BIO molecules were termed 'BIO-balls'. Expression profiling between 'meso-balls' and 'BIO-balls' were compared and analyzed for markers assisiting in defining their phenotypes.
2012-12-01 | E-MEXP-3738 | biostudies-arrayexpress
Project description:CB CD34+ cells were isolated by Miltenyi miniMACS column. Cells were prestimulated in HPGM with 100 ng/ml KITL, FLT#L and TPO for 48 hrs. Cells were transduced with control MiNR1 or STAT5A-ER in three rounds over 48 hrs. Hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and megakaryocyte/erythroid progenitors (MEPs) were sorted (for details see Blood 2011, Fatrai et al). cells were stimulated with 100 ng/ml 4OHT for 24 hrs after which RNA was isolated for Illumina beadhchiop arrays HT12 v3 CB CD34+ cells were isolated by Miltenyi miniMACS column. Cells were prestimulated in HPGM with 100 ng/ml KITL, FLT#L and TPO for 48 hrs. Cells were transduced with control MiNR1 or STAT5A-ER in three rounds over 48 hrs. Hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and megakaryocyte/erythroid progenitors (MEPs) were sorted (for details see Blood 2011, Fatrai et al). cells were stimulated with 100 ng/ml 4OHT for 24 hrs after which RNA was isolated for Illumina beadhchiop arrays HT12 v3
2011-01-22 | E-GEOD-26775 | biostudies-arrayexpress