Unknown,Transcriptomics,Genomics,Proteomics

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Early B Cell Factor 2 Regulates Hematopoietic Stem Cell Homeostasis in a Cell-Nonautonomous Manner

ABSTRACT: Hematopoiesis requires the interaction of hematopoietic stem cells (HSCs) with various stromal microenvironments. Here, we examine the role of early B cell factor 2 (Ebf2), a transcription factor expressed in a subset of immature osteoblastic cells. Ebf2-/- mice show decreased frequencies of HSCs and lineage-committed progenitors. This defect is cell nonautonomous, as shown by the fact that transplantation of Ebf2-deficient bone marrow into wild-type hosts results in normal hematopoiesis. In coculture experiments, Ebf2 Ebf2-/-osteoblastic cells have reduced potential to support short-term proliferation of HSCs. Expression profiling of sorted Ebf2-/- osteoblastic cells indicated that several genes implicated in the maintenance of HSCs are downregulated relative to Ebf2+/- cells, whereas genes encoding secreted frizzled-related proteins are upregulated. Moreover, HSCs from Ebf2-/- mice and wild-type HSCs cocultured with Ebf2-/- osteoblastic cells show reduced Wnt responses. Thus, Ebf2 acts as a transcriptional determinant of an osteoblastic niche that regulates the maintenance of hematopoietic progenitors, in part by modulating Wnt signaling. Osteoblastic cells from tibia and femur of Ebf2 heterozygous and knockout mice (n=3 each) were loaded with FDG, FACS sorted and total RNA was isolated. cDNA was synthesised using the One-Cycle cDNA Synthesis Kit (Affymetrix), and labeled cRNA was synthesized by transcription in vitro, using the IVT labelling kit (Affymetrix). The labeled RNA was fragmented and hybridized to GeneChipM-BM-. Mouse genome 430 2.0 arrays (GPL1261), according to the manufacturerM-bM-^@M-^Ys instructions.

ORGANISM(S): Mus musculus

SUBMITTER: Rudolf Grosschedl

PROVIDER: E-GEOD-21700 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Early B cell factor 2 regulates hematopoietic stem cell homeostasis in a cell-nonautonomous manner.

Kieslinger Matthias M Hiechinger Silvia S Dobreva Gergana G Consalez G Giacomo GG Grosschedl Rudolf R

Cell stem cell 20101001 4

Hematopoiesis requires the interaction of hematopoietic stem cells (HSCs) with various stromal microenvironments. Here, we examine the role of early B cell factor 2 (Ebf2), a transcription factor expressed in a subset of immature osteoblastic cells. Ebf2(-/-) mice show decreased frequencies of HSCs and lineage-committed progenitors. This defect is cell nonautonomous, as shown by the fact that transplantation of Ebf2-deficient bone marrow into wild-type hosts results in normal hematopoiesis. In c ...[more]

PMID: 20887955

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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.

Project description:Hamey2017 - Blood stem cell regulatory network This model is described in the article: Reconstructing blood stem cell regulatory network models from single-cell molecular profiles Fiona K. Hamey, Sonia Nestorowa, Sarah J. Kinston, David G. Kent, Nicola K. Wilson, and Berthold Göttgens Proceedings of the National Academy of Sciences of the United States of America Abstract: Adult blood contains a mixture of mature cell types, each with specialized functions. Single hematopoietic stem cells (HSCs) have been functionally shown to generate all mature cell types for the lifetime of the organism. Differentiation of HSCs toward alternative lineages must be balanced at the population level by the fate decisions made by individual cells. Transcription factors play a key role in regulating these decisions and operate within organized regulatory programs that can be modeled as transcriptional regulatory networks. As dysregulation of single HSC fate decisions is linked to fatal malignancies such as leukemia, it is important to understand how these decisions are controlled on a cell-by-cell basis. Here we developed and applied a network inference method, exploiting the ability to infer dynamic information from single-cell snapshot expression data based on expression profiles of 48 genes in 2,167 blood stem and progenitor cells. This approach allowed us to infer transcriptional regulatory network models that recapitulated differentiation of HSCs into progenitor cell types, focusing on trajectories toward megakaryocyte–erythrocyte progenitors and lymphoid-primed multipotent progenitors. By comparing these two models, we identified and subsequently experimentally validated a difference in the regulation of nuclear factor, erythroid 2 (Nfe2) and core-binding factor, runt domain, alpha subunit 2, translocated to, 3 homolog (Cbfa2t3h) by the transcription factor Gata2. Our approach confirms known aspects of hematopoiesis, provides hypotheses about regulation of HSC differentiation, and is widely applicable to other hierarchical biological systems to uncover regulatory relationships. This model is hosted on BioModels Database and identified by: MODEL1610060000. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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Dataset Information

Early B Cell Factor 2 Regulates Hematopoietic Stem Cell Homeostasis in a Cell-Nonautonomous Manner

Publications

Early B cell factor 2 regulates hematopoietic stem cell homeostasis in a cell-nonautonomous manner.

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