Gene expression data of murine hematopoietic stem cells before and after in vitro culture
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ABSTRACT: Isolation of long-term reconstituting hematopoietic stem cell (HSC) has been possible by utilizing flow cytometry with a combination of multiple antibodies against cell surface markers. However those cell surface phenotypes do not represent functional HSC after in vitro culture. We compared gene expression profiling of phenotypic HSC (CD48-KSL cells) before and after in vitro culture, and discovered that cultured HSC express mast cell-related genes including Cd244. After in vitro culture, phenotypic HSC can be divided into CD244- and CD244+ subpopulations, and only CD244- cells that have low mast cell gene expression and maintain HSC-related genes sustain reconstitution potential. Induction of CD244 may partially be triggered by induction of endoplasmic reticulum (ER) stress, as chemically induced ER stress signal increased CD244+ subpopulation while ER stress suppression using a molecular chaperone, TUDCA, decreased CD244+ population, which were correlated to the output of reconstitution assay. These data suggest CD244 positivity is a potent marker to exclude non-functional HSC after in vitro culture thereby useful to elucidate mechanism of functional decline of HSC during ex vivo treatment.
Project description:Isolation of long-term reconstituting hematopoietic stem cell (HSC) has been possible by utilizing flow cytometry with a combination of multiple antibodies against cell surface markers. However those cell surface phenotypes do not represent functional HSC after in vitro culture. We compared gene expression profiling of phenotypic HSC (CD48-KSL cells) before and after in vitro culture, and discovered that cultured HSC express mast cell-related genes including Cd244. After in vitro culture, phenotypic HSC can be divided into CD244- and CD244+ subpopulations, and only CD244- cells that have low mast cell gene expression and maintain HSC-related genes sustain reconstitution potential. Induction of CD244 may partially be triggered by induction of endoplasmic reticulum (ER) stress, as chemically induced ER stress signal increased CD244+ subpopulation while ER stress suppression using a molecular chaperone, TUDCA, decreased CD244+ population, which were correlated to the output of reconstitution assay. These data suggest CD244 positivity is a potent marker to exclude non-functional HSC after in vitro culture thereby useful to elucidate mechanism of functional decline of HSC during ex vivo treatment. It has been unknown whether HSC respond to acute anemic stress. We compared frequency and differentiation potential of HSC in steady-state vs acute anemic mice, and discovered that HSC rapidly expand upon the anemia induction and change their lineage balance to more erythrocytic.
Project description:Cellular metabolism exceedingly determines hematopoietic stem cells (HSCs) divisional fate and functioning through organelles interaction upon stress-induced response. The outer mitochondrial membrane-localized E3 ubiquitin ligase Mitol/Marchf5 (encoded by Mitol gene) is known to regulate mitochondrial and endoplasmic reticulum (ER) interaction and promote cell survival. To investigate the precise functional involvement of Mitol in HSC maintenance, we analyzed MX1-cre inducible Mitol knockout mice. Mitol deletion in bone marrow (BM) leads to HSCs exhaustion and impairment of BM reconstitution capability. Moreover, Mitol deletion induced prolonged ER stress in HSCs, which triggers cellular apoptosis that is mainly regulated by IRE1a signaling. Inhibition of ER stress by KIRA6 could partially reduce apoptosis of long term-HSC. Our observations indicated that Mitol is principal to maintain hematopoietic homeostasis and protects HSCs from apoptosis mainly through ER function via IRE1a signaling.
Project description:HSC endured stress upon culture, that led to decrease their functional properties (i.e. decreased HSC self-renewal potential). Here we study the impact of antioxidants in the maintenance of HSC cultured 2 days in vitro.
Project description:Cell purification technology combined with whole transcriptome sequencing and small molecule agonist of hematopoietic stem cell self-renewal has allowed us to identify the endothelial protein c receptor protein (EPCR) as a surface maker that defines a rare subpopulation of human cells which is highly enriched for stem cell activity in vivo. EPCR-positive cells exhibit a robust multi-lineage differentiation potential and serial reconstitution in immunocompromised mice. In culture, most if not all of the HSC activity is detected in the EPCR+ subset, arguing for the stability of this marker on the surface of cultured cells, a feature not found with more recently described markers such as CD49f. Functionally EPCR is essential for human HSC activity in vivo. Cells engineered to express low EPCR expression proliferate normally in culture but lack the ability to confer long-term reconstitution. EPCR is thus a stable marker for human HSC. Its exploitation should open new possibilities in our effort to understand the molecular bases behind HSC self-renewal.
Project description:Estrogens are potential regulators of the hematopoietic stem cell (HSC) niche and have effects on mature hematopoietic cells; however, whether estrogen signaling directly regulates normal and malignant HSC remains unclear. We demonstrate differential expression and specific roles of estrogen receptors (ER) in hematopoietic progenitors. ERa activation in short-term HSC and multipotent progenitors induced apoptosis. In contrast, the selective ER modulator (SERM) tamoxifen induced proliferation of quiescent long-term HSC, altered their self-renewal signature and compromised hematopoietic reconstitution following myelotoxic stress. Treatment with tamoxifen alone abolished hematopoietic progenitor expansion induced by JAK2V617F by restoring normal levels of apoptosis, blocked JAK2V617F-induced myeloproliferative neoplasm in vivo, and sensitized MLL-AF9+ leukemias to chemotherapy. Tamoxifen showed selective effects on mutant cells compared to normal ones, and had only a minor impact on steady-state hematopoiesis in disease-free animals. These results uncover specific regulation of hematopoietic progenitors by estrogens and potential anti-leukemic properties of SERM LT-HSCs, ST-HSCs and MPPs sorted from the bone marrow of mice treated with tamoxifen or vehicle (3 biological replicates per group)
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.
Project description:Cell purification technology combined with whole transcriptome sequencing and small molecule agonist of hematopoietic stem cell self-renewal has allowed us to identify ITGA3 as a surface maker that defines a rare subpopulation of human cells which is highly enriched for stem cell activity in vivo. ITGA3-positive cells (within the EPCR+CD90+CD133+CD34+CD45RA- fraction) exhibit a robust multi-lineage differentiation potential and serial reconstitution in immunocompromised mice. In culture, most if not all of the HSC activity is detected in the ITGA3+ subset, arguing for the stability of this marker on the surface of cultured cells, a feature not found with more recently described markers such as CD49f. Functionally ITGA3 is essential for human HSC activity in vivo. Its exploitation should open new possibilities in our effort to understand the molecular bases behind HSC self-renewal.
Project description:Estrogens are potential regulators of the hematopoietic stem cell (HSC) niche and have effects on mature hematopoietic cells; however, whether estrogen signaling directly regulates normal and malignant HSC remains unclear. We demonstrate differential expression and specific roles of estrogen receptors (ER) in hematopoietic progenitors. ERa activation in short-term HSC and multipotent progenitors induced apoptosis. In contrast, the selective ER modulator (SERM) tamoxifen induced proliferation of quiescent long-term HSC, altered their self-renewal signature and compromised hematopoietic reconstitution following myelotoxic stress. Treatment with tamoxifen alone abolished hematopoietic progenitor expansion induced by JAK2V617F by restoring normal levels of apoptosis, blocked JAK2V617F-induced myeloproliferative neoplasm in vivo, and sensitized MLL-AF9+ leukemias to chemotherapy. Tamoxifen showed selective effects on mutant cells compared to normal ones, and had only a minor impact on steady-state hematopoiesis in disease-free animals. These results uncover specific regulation of hematopoietic progenitors by estrogens and potential anti-leukemic properties of SERM
Project description:Hematopoietic stem cells (HSCs) play a crucial role in lifelong hematopoiesis by generating all blood lineages. The long-term maintenance of HSCs relies on the regulation of endoplasmic reticulum (ER) stress at a low level. However, the precise control of ER stress in HSCs remains largely unknown. In this study, we demonstrate that suppression of ER stress enhances the in vitro maintenance and in vivo repopulation capacity of HSCs, while also protecting HSCs against radiation-induced injury. Integrated multi-omics analysis has revealed that epithelial membrane protein 1 (Emp1) functions as an ER stress sensor in HSCs. Loss of Emp1 leads to increased protein aggregates and elevated ER stress, ultimately resulting in impaired HSC maintenance and self-renewal. Mechanistically, EMP1 is located within the ER and interacts with ceramide synthase 2 (CERS2) to limit the production of dihydroceramides (dhCers), which are a class of sphingolipids. DhCers accumulate in Emp1-deficient HSCs and directly induce protein aggregation. Inhibition of CERS2 significantly restores the maintenance of Emp1-deficient HSCs. Furthermore, Emp1 deficiency renders HSCs more susceptible to radiation, while overexpression of Emp1 or inhibition of CERS2 protects HSCs against radiation-induced injury. These findings highlight the critical role played by the EMP1-CERS2-dhCers axis in constraining ER stress and preserving HSC potential, uncovering a new link between sphingolipid metabolism and HSC homeostasis, and have clinical implications.