The continuous generation of blood cells throughout life is based on the existence of a small cohort of hematopoietic stem cells (HSCs) generated during embryogenesis. These cells are characterized by the capacity for self-renewal and multipotency (ability to differentiate in all blood lines). HSCs reside in the bone marrow of adults and are clinically used to treat patients with hematological disorders, but the availability of immunocompatible donors remains a problem.
The recent discovery of induced pluripotency stem cells (iPS) has raised the possibility of making HSCs from non-hematopoietic cells of the patient. However, until now it has not yet been possible to convert iPS into actual CSH, but only committed progenitors could be generated. This would indicate that the key steps for induction and hematopoietic specification are still poorly understood.
Understanding how HSCs are produced during embryonic life is therefore crucial in attempting to generate HSCs in vitro. We have found that during ontogeny in the human embryo, HSC is generated in the aorta, but a hematopoietic potential is present earlier in the para-aortic splanchnopleura (P-Sp). Our recent results show that angiotensin-converting enzyme (ACE) labels the rare embryo cells in the embryonic P-Sp, which are responsible for hematopoietic activity (Sinka, 2012). In later stages of development, ACE is expressed by the CSHs associated with the aorta as well as by the underlying endothelium. This double specificity of ACE, hematopoietic and endothelial, suggests the existence of an ancestral precursor common to both lineages – the hemangioblast ?? who would be identified by ACE.
The study of the stages of emergence and maturation of the CSH as well as the identification of new factors favoring their induction / expansion in vitro is the objective of this project.
To determine the hemangioblastic nature of embryonic ACE + precursors, we will examine their differentiating abilities. The identification of the hemangioblast will allow us to analyze its expression profile.

1. The ACE + embryonic cells will be sorted by cytometry and their potential for differentiation evaluated by in vitro approaches. The fate of ACE + cells will also be analyzed in vivo by grafting in the zebrafish embryo (K. Kissa, Univ., Montpellier). The zebrafish embryos, being transparent, allow direct visualization of injected cells in live animals. In this regard, the cells to be injected will be fluoresced by lentiviral transduction.
2. Because of the low number of ACE + cells (about 100 per embryo), the study of their transcriptome will be conducted using RNAseq technology (Bio-Chips IGBMC Platform). The purpose of this analysis is to identify specific embryonic genes that may be responsible for the induction and activation of a hematopoietic program. In parallel, we will perform a qPCR quantitative analysis of the expression of master genes, hematopoietic and / or endothelial commitment.
3- In order to test whether differentially expressed genes are involved in the activation of a hematopoietic pathway, expression modulation approaches will be developed, either by blocking their expression by shRNA in ACE + cells or by exogenous overexpression in the cells. cells of the splanchnopleure. A functional approach will also be developed in fish by blocking their expression by the injection of Morfolino into the embryo. The experiments envisaged are well defined and the various techniques necessary for the success of this project are commonly applied in our laboratory.

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