Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Stepwise development of hematopoietic stem cells from embryonic stem cells

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Hideo Ema

PROVIDER: E-GEOD-15053 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Stepwise development of hematopoietic stem cells from embryonic stem cells.

Matsumoto Kenji K Isagawa Takayuki T Nishimura Toshinobu T Ogaeri Takunori T Eto Koji K Miyazaki Satsuki S Miyazaki Jun-ichi J Aburatani Hiroyuki H Nakauchi Hiromitsu H Ema Hideo H

PloS one 20090316 3

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. Bo ...[more]

PMID: 19287487

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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 Affymetrixâ¢ platform. As a control, profiling was also performed with LSK cells expressing unmodified constitutively active HOXB4 (HOXB4const) Â± 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.

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Stepwise development of hematopoietic stem cells from embryonic stem cells

Publications

Stepwise development of hematopoietic stem cells from embryonic stem cells.

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