Stepwise development of hematopoietic stem cells from embryonic stem cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE15053 | GEO | 2009/03/18
SECONDARY ACCESSION(S): PRJNA114823
REPOSITORIES: GEO
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Project description:The cellular ontogeny of hematopoietic stem cells (HSCs) remains poorly understood because their isolation from and their identification in early developing small embryos are difficult. We attempted to dissect early developmental stages of HSCs using an in vitro mouse embryonic stem cell (ESC) differentiation system combined with inducible HOXB4 expression. Here we report the identification of pre-HSCs and an embryonic type of HSCs (embryonic HSCs) as intermediate cells between ESCs and HSCs. Both pre-HSCs and embryonic HSCs were isolated by their c-Kit+CD41+CD45- phenotype. Pre-HSCs did not engraft in irradiated adult mice. After co-culture with OP9 stromal cells and conditional expression of HOXB4, pre-HSCs gave rise to embryonic HSCs capable of engraftment and long-term reconstitution in irradiated adult mice. Blast colony assays revealed that most hemangioblast activity was detected apart from the pre-HSC population, implying the early divergence of pre-HSCs from hemangioblasts. Gene expression profiling suggests that a particular set of transcripts closely associated with adult HSCs is involved in the transition of pre-HSC to embryonic HSCs. Experimental strategy consisted of EB formation, co-culture with OP9 cells, and functional assays. iHOXB4 ESCs were allowed to differentiate spontaneously into EBs for 6 days without HOXB4 expression. We decided to fractionate EB6 cells mainly because by day 6 of culture the number of multipotent progenitors reaches a plateau and several surface markers become detectable. CD41 is known as a marker for the initiation of definitive hematopoiesis. CD41 appeared in a significant proportion of EB cells on day 6 of culture. Induced HOXB4 expression during EB formation did not affect the generation of colony forming cells and repopulating cells in the OP9 and iHOXB4 system or the appearance of surface markers in EB cells. EB6 cells were analyzed and sorted by flow cytometry. Sorted EB6 cells were co-cultured with OP9 cells for various days under HOXB4-on or -off conditions. The minimal requirement of the co-culture period appeared to be only 4 days, which is much shorter than previously thought. After a second analysis and fractionation by flow cytometry, cells were subjected to in vivo repopulating assays under HOXB4-on or -off conditions.
2009-03-18 | E-GEOD-15053 | biostudies-arrayexpress
Project description:HOXB4 mediates expansion of adult and embryo-derived hematopoietic stem cells (HSCs) when expressed ectopically. To define the underlying molecular mechanisms, we performed gene expression profiling in combination with subsequent functional analysis using enriched adult HSCs expressing inducible HOXB4. A substantial number of the identified HOXB4 target genes are involved in signaling pathways important for controlling self-renewal, maintenance and differentiation of stem cells. Functional assays performed on selected pathways confirmed the biological coherence of the array results. HOXB4 activity protected adult HSCs from the detrimental effects mediated by the proinflammatory cytokine TNF-alpha. Furthermore, we demonstrate that HOXB4 activity and FGF-signaling are intertwined. HOXB4-mediated expansion of adult HSCs was enhanced by specific and complete inhibition of FGF-receptors. Based on our results we propose that HOXB4 governs pivotal cell-intrinsic pathways involved in the regulation of cell cycle, differentiation and apoptosis. Our results strongly suggest that HOXB4 modulates the response of HSCs to multiple extrinsic signals in a concerted manner, thereby shifting the balance towards stem cell self-renewal. Experiment Overall Design: To understand the mechanisms of HOXB4 activity, we wished to identify target genes of HOXB4 in adult hematopoietic stem and progenitor cells (HSC/HPCs). We thus transduced murine HSC/HPCs with a retroviral vector that co-expresses EGFP and a tamoxifen-inducible form of HOXB4 (HOXB4-ER). Upon addition of 4-hydroxytamoxifen (TMX), the HOXB4-ER fusion protein translocates from the cytoplasm to the nucleus, consequently being capable of modulating gene expression. Transduced cell populations were expanded for 14 days in the presence of TMX. Thereafter, HOXB4-ER+LSK (GFP+ , lineage negative, Sca1+, ckit+) cells were flow cytometrically isolated and cultivated either with or without TMX for 1 or 4 hours. Inactivation of HOXB4 activity by TMX withdrawal was intended to mimic the naturally occurring down-regulation of HOXB4 in differentiating stem cells. RNA was prepared after the aforementioned times and the transcriptional profiles of HOXB4-ER+LSK +/- TMX analyzed using the Affymetrix⢠platform. As a control, profiling was also performed with LSK cells expressing unmodified constitutively active HOXB4 (HOXB4const) ± TMX, to exclude changes in gene expression due to unknown effects of tamoxifen itself. RNAs from adult LSK cells were processed for use on Affymetrix GeneChips Mouse Genome 430 2.0. All quality parameters for the arrays were confirmed to be in the recommended range.
2008-06-16 | E-GEOD-9010 | biostudies-arrayexpress
Project description:Overexpression of HOXB4 in hematopoietic stem cells (HSCs) leads to increased self-renewal without causing hematopoietic malignancies in transplanted mice. The molecular basis of HOXB4-mediated benign HSC expansion in vivo is not well understood. To gain further insight into the molecular events underlying HOXB4-mediated HSC expansion, we analyzed gene expression changes at multiple time points in Lin-Sca1+c-kit+ (LSK) cells from mice transplanted with bone marrow (BM) cells transduced with a MSCV-HOXB4-ires-YFP vector. A distinct HOXB4 transcriptional program was reproducibly induced and stabilized by 12 weeks after transplant. Dynamic expression changes were observed in genes critical for HSC self-renewal as well as genes involved in myeloid and B cell differentiation. Prdm16, a transcription factor associated with human acute myeloid leukemia (AML), was markedly repressed by HOXB4 but upregulated by HOXA9 and HOXA10, suggesting that Prdm16 downregulation was involved in preventing leukemia in HOXB4 transplanted mice. Functional evidence to support this mechanism was obtained by enforcing co-expression of sPrdm16 and HOXB4, which led to enhanced self-renewal, myeloid expansion, and leukemia. Altogether, these studies define the transcriptional pathways involved in HOXB4 HSC expansion in vivo and identify repression of Prdm16 transcription as a mechanism by which expanding HSCs avoid leukemic transformation. 5-FU treated bone marrow cells from female C57Bl/6L mice were transduced with concentrated supernatant from GPE+86-derived producer cells containing MSCV-HOXB4-ires-YFP or MSCV-ires-GFP retroviral vectors. Transduced cells were transplanted into lethally irradiated C57Bl/6L mice. HOXB4-YFP or GFP expressing LSK cells were sorted from transplanted mice at 6, 12 and 24 weeks post transplantation, of which RNA was extracted for microarray.
2014-10-01 | E-GEOD-59804 | biostudies-arrayexpress
Project description:Efficient in vitro generation of hematopoietic stem cells (HSCs) from embryonic stem cells (ESCs) holds great promise for cell-based therapies of hematological diseases. To date, HoxB4 remains to be the most effective transcription factor (TF) whose over-expression in ESCs confers long-term repopulating ability to ESC-derived HSCs. Despite its importance, the components and dynamics of the HoxB4 transcriptional regulatory network is poorly understood, hindering efforts to develop a more efficient protocol for in vitro derivation of HSCs. Towards this goal, we performed global gene expression profiling and chromatin immunoprecipitation coupled with deep sequencing (ChIP-Seq) at four stages of the HoxB4-mediated HSC development. Joint analyses of ChIP-Seq and gene expression profiles unveil a number of global features of the HoxB4 regulatory network.
2012-03-23 | GSE34013 | GEO
Project description:Efficient in vitro generation of hematopoietic stem cells (HSCs) from embryonic stem cells (ESCs) holds great promise for cell-based therapies of hematological diseases. To date, HoxB4 remains to be the most effective transcription factor (TF) whose over-expression in ESCs confers long-term repopulating ability to ESC-derived HSCs. Despite its importance, the components and dynamics of the HoxB4 transcriptional regulatory network is poorly understood, hindering efforts to develop a more efficient protocol for in vitro derivation of HSCs. Towards this goal, we performed global gene expression profiling and chromatin immunoprecipitation coupled with deep sequencing (ChIP-Seq) at four stages of the HoxB4-mediated HSC development. Joint analyses of ChIP-Seq and gene expression profiles unveil a number of global features of the HoxB4 regulatory network.
2012-03-23 | GSE33953 | GEO
Project description:Precise study of HSCs during regeneration has been impeded by the rarity of the HSC population and depletion of phenotypic HSCs early following genotoxic stresses, such as total body irradiation (TBI). We isolated bone marrow (BM) ckit+sca-1+lin- (KSL) cells, which are enriched for HSCs, from adult C57Bl6 mice before and at several time points following TBI, as a means to map the dynamic molecular response of HSC regeneration. We isolated BM KSL cells and myeloid progenitor cells (c-kit+sca-1-lin- cells) at day +14 after irradiation and compared the gene expression profile of regenerating HSCs versus steady state HSCs (non-irradiated) and committed progenitor cells.
2016-04-05 | E-GEOD-79744 | biostudies-arrayexpress
Project description:Efficient in vitro generation of hematopoietic stem cells (HSCs) from embryonic stem cells (ESCs) holds great promise for cell-based therapies of hematological diseases. To date, HoxB4 remains to be the most effective transcription factor (TF) whose over-expression in ESCs confers long-term repopulating ability to ESC-derived HSCs. Despite its importance, the components and dynamics of the HoxB4 transcriptional regulatory network is poorly understood, hindering efforts to develop a more efficient protocol for in vitro derivation of HSCs. Towards this goal, we performed global gene expression profiling and chromatin immunoprecipitation coupled with deep sequencing (ChIP-Seq) at four stages of the HoxB4-mediated HSC development. Joint analyses of ChIP-Seq and gene expression profiles unveil a number of global features of the HoxB4 regulatory network. Cells from three time points of the developmental processes were collected, including days 6, 16, and 26. Deep sequencing were done for both IP and input DNA from cells from each time point.
2012-03-23 | E-GEOD-34013 | biostudies-arrayexpress
Project description:Efficient in vitro generation of hematopoietic stem cells (HSCs) from embryonic stem cells (ESCs) holds great promise for cell-based therapies of hematological diseases. To date, HoxB4 remains to be the most effective transcription factor (TF) whose over-expression in ESCs confers long-term repopulating ability to ESC-derived HSCs. Despite its importance, the components and dynamics of the HoxB4 transcriptional regulatory network is poorly understood, hindering efforts to develop a more efficient protocol for in vitro derivation of HSCs. Towards this goal, we performed global gene expression profiling and chromatin immunoprecipitation coupled with deep sequencing (ChIP-Seq) at four stages of the HoxB4-mediated HSC development. Joint analyses of ChIP-Seq and gene expression profiles unveil a number of global features of the HoxB4 regulatory network. Cells from four time points of the developmental processes were collected, including days 0, 6, 16, and 26. Triplicate microarray hybridizations were done for cells from each time point.
2012-03-23 | E-GEOD-33953 | biostudies-arrayexpress
Project description:Adult and fetal hematopoietic stem cells (HSCs) display a glycolytic phenotype, which is required for maintenance of stemness; however, whether mitochondrial respiration is required to maintain HSC function is not known. Here we report that loss of the mitochondrial complex III subunit Rieske iron sulfur protein (RISP) in fetal mouse HSCs allows them to proliferate but impairs their differentiation, resulting in anemia and prenatal death. RISP null fetal HSCs displayed impaired respiration resulting in a decreased NAD+/NADH ratio. RISP null fetal HSCs and progenitors exhibited an increase in both DNA and histone methylation concomitant with increases in 2-hydroxyglutarate (2-HG), a metabolite known to inhibit DNA and histone demethylases. RISP inactivation in adult HSCs also impaired respiration resulting in loss of quiescence resulting in severe pancytopenia and lethality. Thus, respiration is dispensable for adult or fetal HSC proliferation, but essential for fetal HSC differentiation and maintenance of adult HSC quiescence.
2017-09-13 | PXD006054 | Pride
Project description:HOXB4 mediates expansion of adult and embryo-derived hematopoietic stem cells (HSCs) when expressed ectopically. To define the underlying molecular mechanisms, we performed gene expression profiling in combination with subsequent functional analysis using enriched adult HSCs expressing inducible HOXB4. A substantial number of the identified HOXB4 target genes are involved in signaling pathways important for controlling self-renewal, maintenance and differentiation of stem cells. Functional assays performed on selected pathways confirmed the biological coherence of the array results. HOXB4 activity protected adult HSCs from the detrimental effects mediated by the proinflammatory cytokine TNF-alpha. Furthermore, we demonstrate that HOXB4 activity and FGF-signaling are intertwined. HOXB4-mediated expansion of adult HSCs was enhanced by specific and complete inhibition of FGF-receptors. Based on our results we propose that HOXB4 governs pivotal cell-intrinsic pathways involved in the regulation of cell cycle, differentiation and apoptosis. Our results strongly suggest that HOXB4 modulates the response of HSCs to multiple extrinsic signals in a concerted manner, thereby shifting the balance towards stem cell self-renewal. Keywords: plus/minus induction of HOXB4 activity by treatment with 4-hydroxytamoxifen (TMX)
2007-09-11 | GSE9010 | GEO