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Direct Conversion from Mouse Fibroblasts Informs the Identification of Hemogenic Precursor Cells In Vivo

ABSTRACT: Definitive hematopoiesis emerges via an endothelial-to-hematopoietic transition in the aorta-gonad-mesonephros (AGM) region and placenta. We have recently demonstrated the induction of hematopoietic stem/progenitors (HSPCs) from mouse fibroblasts with a combination of transcription factors progressing through endothelial-like precursors. Here, guided by our in vitro programming experiments we analyzed mouse placentas for the presence of the precursor phenotype. We identified a small population of CD34+ Sca1+Prom1+ (34PS) cells in mid-gestation placentas that do not express the pan-hematopoietic marker CD45. After isolation and culture 34PS cells acquire CD45 and generate large hematopoietic as well as cobblestone colonies. Prom1+ cells localize to the placental vascular labyrinth where HSPCs emerge. 34PS cells express markers associated with the hemogenic endothelium (CD31, Tie2, VE-Cadherin, Sox17, Runx1, Scl) and also markers identified by direct induction (Itga6/CD49f). This population is heterogeneous for the early hematopoietic marker CD41 and expresses the programming transcription factors. Remarkably, global gene expression profiles of placental 34PS cells correlate with AGM-derived hemogenic endothelium and fibroblast-derived precursors. Finally, when co-cultured with stroma placental 34PS cells give rise to B/T lymphoid cells as well as mixed colonies containing erythroid, myeloid and megakaryocytic cell lineages. In summary, we show that direct in vitro conversion provided a cell surface phenotype for the isolation of hemogenic precursors in vivo. Our findings provide insights into the specification of definitive hemogenesis in the placenta, in depth characterization of hemogenic precursor populations and the first evidence that direct in vitro conversion approaches can be used as a valuable tool to address basic developmental questions in vivo.

ORGANISM(S): Mus musculus

PROVIDER: GSE54574 | GEO | 2016/03/07

SECONDARY ACCESSION(S): PRJNA237041

REPOSITORIES: GEO

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Project description:Transcriptome analysis of adult hematopoietic stem cells (HSC) and their progeny has informed our understanding of blood differentiation and leukemogenesis, but a similarly transformative analysis of the embryonic origins of hematopoiesis is lacking. To address this issue, we acquired gene expression profiles of developing HSC purified from over 2500 dissected murine embryos and adult mice, and applied a network biology-based analysis to reconstruct the gene regulatory networks of sequential stages of HSC development. We found that embryonic hematopoietic elements clustered into three distinct transcriptional states characteristic of the definitive yolk sac, HSCs emerging from hemogenic endothelium, and definitive HSCs. We functionally validated several candidate transcriptional regulators of HSC ontogeny by morpholino-mediated knock-down in zebrafish embryos, confirming changes in the expression of HSC markers runx1 and c-myb in the aorta-gonads-mesonephros (AGM), the site of definitive HSC specification. Moreover, we found that HSCs derived from differentiating embryonic stem cells in vitro (ESC-HSC) most closely resemble definitive HSC, yet lack a signature indicative of specification by Notch signaling, which likely accounts for their deficient lymphoid development. Our analysis and accompanying web resource will accelerate the characterization of regulators of HSC ontogeny, facilitate efforts to direct hematopoietic differentiation and cell fate conversion, and serve as a model to study the origins of other adult stem cells. Hematopoietic stem cells and their precursors were isolated from mouse embryos and distinct stages and sites during development, purified by fluorescence activated cell sorting, and gene expression profiled on Affymetrix arrays. We also profiled HSCs and their precurors derived by in vitro differentitation of embryonic stem cells [expression data from GSE16925 and GSE14012 was used for mESCs, available at http://hsc.hms.harvard.edu/].

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Direct Conversion from Mouse Fibroblasts Informs the Identification of Hemogenic Precursor Cells In Vivo

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