Embryonic Stem Cell Differentiation Laboratory

Laboratory Heads: Andrew Elefanty and Ed Stanley

Background

The embryonic stem cell (ESC) differentiation laboratory focuses on the directed differentiation of ESCs to cells types with a potential use in cell transplantation therapies. The two main interests of the laboratory are the generation of haematopoietic cells (for the treatment blood disorders and the provision of transfusible blood products) and pancreatic beta cells (for the treatment of type 1 diabetes). The underlying strategy is to guide ESC differentiation along the same developmental pathways traversed by cells during the ontogeny of these cell types or organs during embryogenesis. Indeed, gene-profiling experiments have shown that ESCs move sequentially from the undifferentiated state through stages corresponding to epiblast, primitive streak and mesoderm and endoderm (Hirst et al, 2006 in press). To assist in the analysis of this process, we have used homologous recombination to insert DNA sequences encoding reporters such as green fluorescent protein (GFP) into genes whose expression marks obligate intermediates in the genesis of the end cell types of interest. The first example of such a gene is Mixl1, which is expressed in the primitive streak, a structure from which the precursors of mesoderm (that gives rise to blood) and endoderm (that gives rise to pancreas) emerge during gastrulation. Using Mixl1^GFP/w ESCs we have defined the growth factors required for efficient induction of gastrulation stage differentiation in a serum free defined media and shown that Mixl1 is required for proper haematopoietic development (Ng et al, Dev. Biol., 2005 (pdf, 899kb) ). A similar strategy in which GFP sequences were used to tag the endogenous Pancreatic and duodenal homeobox gene 1 (Pdx1) has been used to monitor the emergence of endodermal cells that are committed to forming pancreatic precursors during ESC differentiation (Micallef et al, Diabetes, 2005 (pdf, 352kb) ). These experiments with mouse ESCs established a conceptual framework for a similar systematic approach to the directed differentiation of human ESCs (HESCs). To this end, our laboratory is now establishing technologies that will enable genetic manipulation and directed differentiation of HESCs in a serum free culture media. Our Laboratories first foray into stable transfection of HESCs resulted in the isolation of a cell line, Envy, which constitutively expresses robust levels of GFP in all differentiated lineages (Costa et al, Nat. Meth., 2005 (pdf, 391kb)) Envy cells are likely to prove useful in transplantation experiments or other settings where easy identification of input cells is desirable. We have also devised a differentiation protocol for HESCs that leads to uniform and predictable outcomes (Ng et al, Blood, 2005 (pdf, 248kb) ). Using this method in conjunction with a recently developed anti-Mixl1 monoclonal antibody, we were able to show that like mouse ESCs, differentiating HESCs pass through a gastrulation stage identified by co-expression of Mixl1 and Oct4 (Mossman et al, Stem Cells Dev., 2005 (pdf, 170kb) ).

Current work is focussed on isolating HESCs carrying genetic tags similar to those that have proved useful in the mouse ESC system and the finer definition of growth factor requirements for directed differentiation of cells towards blood and pancreatic cell types.