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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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE58461 | GEO | 2015/08/07

SECONDARY ACCESSION(S): PRJNA252679

REPOSITORIES: GEO

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

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

MYCT1 Controls Environmental Sensing in human Hematopoietic Stem Cells [scRNA-seq]

Project description:We identified a novel HSC regulatory gene, MYCT1 (MYC target 1), that is selectively expressed in undifferentiated human HSPC and is critical for human HSC expansion and engraftment. MYCT1 knockdown (KD) in human fetal liver and cord blood (CB) HSPCs impaired expansion ex vivo and engraftment after transplantation, whereas restoring the compromised MYCT1 expression via lentiviral overexpression (OE) in cultured human CB HSPCs improved ex vivo expansion of the most undifferentiated human HSPCs (CD34+CD38-CD90+CD45RA-EPCR+ITGA3+) and enhanced their engraftment ability. We performed single cell RNAseq of uncultured human CB HSPCs, as well as 72 hours after MYCT1 KD and OE to identify MYCT1-regulated programs in HSC and understand the role of MYCT1 in governing human HSPC expansion and engraftment ability. Functional enrichment analysis linked the loss of MYCT1 in HLF+ HSCs to a profound dysregulation of multiple cellular programs essential for HSC stemness, such as oxidative phosphorylation or proteostasis, while MYCT1 OE had the opposite effect and showed closer similarity to uncultured HLF+ HSCs. These data indicate that the loss or gain of MYCT1 expression elicits major effects on programs regulating human HSC functional competence.

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MYCT1 Controls Environmental Sensing in human Hematopoietic Stem Cells [scRNA-seq Dextran]

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RNA sequencing analyisis to identify genes regulated by GW9662 in human cord blood hematopoietic stem and progenitor cells

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.

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Gene expression analysis of CCL28 signaling in ishematopoietic stem and progenitor cells (HSPC) in culture

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

MYCT1 CONTROLS ENVIRONMENTAL SENSING IN HUMAN HEMATOPOIETIC STEM CELLS

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

Targeting JNK Pathway Expands Human Hematopoietic Stem Cells

Project description:The limited number of human hematopoietic stem cells (HSCs) has restrained their widespread clinical application. Despite great efforts in recent years, the in vitro expansion of HSCs remains a challenge due to incomplete understanding of the signaling networks underlying HSC self-renewal. Here we show that culturing human cord blood (CB) CD34+ cells with JNK-IN-8, an inhibitor of the JNK signaling pathway, can enhance the self-renewal of HSCs with a 6-fold increase in number. These expanded CD34+ cells repopulated recipient mice for 20 weeks and can form secondary engraftment that lasted for more than 21 weeks. Knockdown of c-Jun, a major downstream target in the JNK pathway, significantly promoted the expansion of hematopoietic stem and progenitor cells (HSPCs). Our findings demonstrate a critical role of JNK pathway in regulating HSC expansion, provide new insights into HSC self-renewal mechanism, and may lead to improved clinical application of HSCs.

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Gene expression analysis of CCL28 signaling in ishematopoietic stem and progenitor cells (HSPC) in culture

2013-01-01 | GSE35374 | GEO

RNA-seq of Lin-CD34+ cord-blood cells overexpressing PLAG1-S or firefly Luciferase [OE]

Project description:To characterize transcriptional changes associated with gain of the transcription factor PLAG1-S that could contribute to transient ex vivo expansion of human cord-blood (CB) derived HSC we performed RNA-seq of 2 pools of Lin-CD34+ CB cells 72 hours following lentiviral introduction of ectopic PLAG1-S or firefly Luciferase (control).

2022-12-01 | GSE181976 | GEO

In Vivo Chemical Screen Nominates Valproic Acid as Pharmacologic Modulator of Hematopoietic Stem and Progenitor Cell Activity

Project description:The identification of small molecules which either increase the number and/or enhance the activity of CD34+ hematopoietic stem and progenitor cells (HSPCs) during ex-vivo expansion has remained challenging. Applying an unbiased in vivo chemical screen in a transgenic (c-myb:EGFP) zebrafish embryo model, histone deacetylase inhibitors (HDACI) (valproic acid, resminostat and entinostat) were shown to significantly amplify the number of phenotypic hematopoietic precursors. The identified HDACIs were confirmed to significantly enhance also the expansion of human HSPCs during ex vivo treatment. Long-term functionality of ex vivo expanded human HSPCs was verified in a xenotransplantation model using NSG mice. However, the HDACI induced proliferation of HSPCs was associated with short-term functional changes. One of the identified hits, valproic acid (VPA), increased the adhesion capacity of CD34+ cells on primary mesenchymal stromal cells and reduced their chemokine-mediated migration capacity in vitro. In line with the reduced migratory potential in vitro, homing as well as early engraftment of VPA treated human CD34+ cells was significantly impaired in the xenotransplantation model. Our data confirms that HDACI treatment leads to a net expansion of HSPCs cells with long-term engraftment potential across different species. However impaired homing and short-term-engraftment has to be kept in mind when designing clinical transplantation protocols. In addition, our gene expression analysis (RNA-Seq) revealed expression of several genes that were altered in CD34+ cells by VPA treatment including cell adhesion molecules and Notch and wnt genes which has been shown to be involved in preservation of stem cell properties.

2017-11-01 | GSE90552 | GEO

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