Understanding the role of TGFβ1 and TGFβ3 on differentiation of human embryonic stem cells (ESC) towards haematopoietic lineages
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NESCI would be interested in funding / studentship opportunities for the following proposal.
NESCI are interested in industrial partners who may like to collaborate in one of these fields (for a CASE studentship or other awards), or in anyone who is interested in these areas of research (including other academic groups). For further details, please contact Helen Clamp from NESCI.
Project: Understanding the role of TGFβ1 and TGFβ3 on differentiation of human embryonic stem cells (ESC) towards haematopoietic lineages
Lead: Dr Majlinda Lako
The transforming growth factor beta (TGFβ) superfamily contains highly pleiotropic members that encompass diverse functions during embryogenesis and adult tissue homestasis. Mammalian members of the TGFβ superfamily include TGFβ1, TGFβ2, TGFβ3, Activins, Inhibins, BMPs (bone morphogenetic proteins), GDFs (growth differentiation factors), GDNFs (glial derived neurotrophic factors), Nodal, Lefty etc. There are two branches of TGFβ signalling: the TGFβ/Nodal/Activin branch and the BMP signalling branch. The TGFβ/Nodal/Activin branch of TGFβ signalling involves the activation of intracellular Smad2/3, which becomes phosphorylated and complexes with co-Smad4 before being translocated to the nucleus. The other branch, the canonical BMP signalling pathway is initiated by the binding of BMP to heterodimers of BMPR1α and BMPRII. This leads to activation of Smad1/5/8, which forms a heteromeric complex with Smad4 prior to translocation to the nucleus. Suppressors of cytokine signalling (SOCS) and inhibitory Smads such as Smad7 provide negative feedback regulatory mechanisms.
In vertebrates, strong evidence exists to support a crucial role for both branches of this family in fate specification within the primitive streak, mesoderm development, emergence of haematopoietic cells from mesoderm and proliferation of haematopoietic cells. Our own work has shown that two members of this family, TGFβ1 and TGFβ3 act as positive enhancers of human ESC haematopoietic differentiation (Ledran et al. 2008). Together these data suggest an important role for several members of the TGFβ family during early haematopoietic development. Notwithstanding this, the precise action and target cell types in which TGFβs act during early human embryonic development is not known. This is the key thrust of this proposal outlined herein which will aim to focus on three important lines of investigation as follows:
• Define the expression of TGFβ superfamily members (in particular, the TGFβ/Nodal/Activin branch) during differentiation of human ESC to mesoderm, specification of haemangioblast and further differentiation to haematopoietic and lineages. We will make use of our efficient differentiation protocol already reported in Ledran et al. 2008, MIXL1-GFP marked human ESC line obtained on collaboration basis from Profs. Elfanty and Stanley (Monash, Australia) and the ability to FACS purify selected cell populations of interest on the basis of cell surface markers such as PDGFRα+MIXL1+ (mesoderm), CD31+KDR+ (haemangioblast) and CD34+CD45+ (haematopoietic progenitors).
• Define the role of members of the TGFβ/Nodal/Activin branch during each stage of haematopoietic differentiation of human ESC (specification of mesoderm, emergence of haemangioblast and commitment to haematopoietic cells) using both inhibition and overexpression based studies (reported in Ledran et al. 2008) and the cell culture tools described in the above section. Once the cell type (medoderm vs haemangioblast or hematopoietic precursor) is identified, microarray expression profiling will be carried out to investigate molecular targets of this pathway.
• Investigate the role of TGFβ/Nodal/Activin branch members on the engraftment ability of human ESC derived haematopoietic progenitors in NOD/LtSz-Scid IL2Rγnull immunocompromised recipients. Our own work has shown that co-culture of human ESC with the stromal cell line derived from the aorta region of aorta-gonad-mesonephros region results in an efficient engraftment of hESC derived blood cells and this co-culture is associated with high levels of TGFβ1 and TGFβ3 mRNA levels. In view of this, we will stimulate and inhibit TGFβ1 and TGFβ3 pathway and assess the impacts on engraftment in the blood system of these animal models.
Reference:
Ledran MH, Krassowska A, Armstrong L, Dimmick I, Renstrom J, Lang R, Yung S, Santibanez-Coref M, Dzierzak E, Stojkovic M, Oosterndorp RAJ, Forrester L and Lako M (2008) Efficient haematopoietic differentiation of human embryonic stem cell on stromal cells derived from hematopoietic niches. Cell Stem Cells 3:85-98.
