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Hematopoietic specification inside the embryo

Research unit

UMR_S 949 - Biologie et pharmacologie des plaquettes sanguines : hémostase, thrombose, transfusion (BPPS)
10, rue Spielmann - BP 36 67065 Strasbourg Cedex

Group

Name: Biologie et pharmacologie des plaquettes sanguines : hémostase, thrombose, transfusion

Group leader: LANZA Francois - francois.lanza@efs-alsace.fr

Group leader's phone: 03 88 21 25 25

Website: Visit website

Group organization:
- Chercheurs: 1
- ITA: 1
- Doctorants: 1
- Post-Docs: 1
- Autres: 1

Publications of the team linked to the topic (3 last years):
1) (1) Travnickova J, Tran Chau V, Julien E, Mateos-Langerak J, Gonzalez C1, Lelièvre E, Lutfalla G, Tavian M, Kissa K. Primitive macrophages control HSPC mobilization and definitive haematopoiesis. Nat Commun. 2015 Feb 17; 6:6227.


2)
3)

About PhD

PhD Director: TAVIAN Manuela - manuela.tavian@inserm.fr

Phone: 03 88 213 844

Junior advisor: non

Co-tutely: non

Co-Director: non

About PhD topic :

Title: Hematopoietic specification inside the embryo

Project: The continuous generation of blood cells throughout life relies on the existence of a small cohort of hematopoietic stem cells (HSC) generated during embryogenesis. These cells are characterized by the dual ability to self-renew, thereby maintaining their numbers, and to differentiate into all the lineages of the blood. HSC reside in the bone marrow of the adult and are clinically used to treat patients with inducted and congenital blood disorders, but the availability of immune-compatible donors remains a problem. The advent of induced pluripotent stem cell (iPS) technology has raised the possibility of making HSCs from a patient’s own non-hematopoietic tissues, but it is not possible so far to convert pluripotent cells to HSC capable of long-term self-renewal and generation of the complete set of mature blood cell lineages. This suggests that key specification requirements are unknown. We previously demonstrated that during ontogeny HSC are first generated in the aorta, but an elusive blood forming potential is present earlier in the embryo, into the splanchnopleura (P-Sp. In a recent study, we have shown that the angiotensin-converting enzyme (ACE) identifies inside the human P-Sp rare undifferentiated cells, which are responsible for the hematopoietic activity detected in the early embryo (Sinka, 2012). At later stages of development, ACE is expressed at the surface of HSC emerging associated with the ventral side of the aorta as well as of the subjacent endothelium. This double specificity of the expression of ACE - endothelial and hematopoietic - suggests the existence of a common ancestral precursor to these lineages, which is identified by ACE and would correspond to the haemangioblast.
Dissecting successive steps of the emergence and maturation of HSCs in vivo and the identification of novel factors promoting HSC induction/expansion in vitro is the ultimate objective of the proposal. To ascertain the haemangioblastic nature of the ACE+ precursors inside the human embryo, we intend to examine their differentiation abilities. The identification of the haemangioblast will allow the analysis of its expression profile improving our knowledge in the field of stem cells.
(1) Embryonic ACE+ cells will be sorted from the early embryo by flow cytometry and their potential analyzed by in vitro approaches. The fate and behavior of ACE+ cells will also be analyzed in vivo by transplant into the zebrafish embryo. The rationale of this approach is that, being transparent the zebrafish embryo allows direct visualization of hematopoietic cells into live animals (coll. K. Kissa, Univ. Montpellier). Sorted ACE+ cells will be engineered by lentiviral transduction to constitutively express fluorescent protein markers. (
2) The transcriptome profile of ACE+ cells within the human embryo will be analyzed using the RNAseq technology that is now applicable for very limited size samples (Microarray and Sequencing Platform, IGBMC). In addition we will perform quantitative expression analysis of known master-genes involved in hematopoietic commitment and/or in endothelial differentiation. The goal of this analysis is to identify human embryonic specific genes that may be responsible for inducing and activating a hematopoietic program.
(3) Differentially expressed genes identified in aim 2 could be potentially involved in the activation of a hematopoietic pathway. This hypothesis will be verified by modulating the expression of these genes by shRNA-knockdown techniques in ACE+ cells or by exogenous overexpression in the ACEcells of the splanchnopleura. Both approaches will rely on the use of lentiviral vectors In addition functional analyses will be assessed in zebrafish model by knocking down their expression in embryos injected with morpholinos. The planned experiments are well defined and the methods and technologies required for the success of the project are used daily in our laboratory.

Wished skills: Basic knowledge in Molecular and Cell Biology – Developmental Biology

Expertises which will be acquired during the training: The project will allow acquiring a large panel of expertise namely: - Analysis of hematopoietic stem cells – enzymatic and mechanic dissociation of tissues, culture of progenitors, organotypic culture – basic techniques in molecular biology (production of vectors, cell transfection, RT-PCR, q-PCR) – cell transplantation in animal models – flow cytometry (analysis and cell sorting) – Histology (immunohistochemistry and immunofluorescence) – Analysis in confocal microscopy –