Module 1: Haematopoietic Differentiation and Expansion of Human Pluripotent Stem Cells

Module Leaders	Professor Andrew Elefanty
Host Organisations	Monash Immunology and Stem Cell Laboratories (MISCL), Monash University

Module description

Pluripotent embryonic stem cells (ESCs) have the capacity to develop into all cell types in the adult. This Module’s first hypothesis proposes that it will be possible to direct the in vitro differentiation of hESCs to haematopoietic tissues, specifically to generate transplantable haematopoietic stem cells and expandable precursors of definitive erythroid and granulocytic lineages.

It is widely accepted that the in vitro differentiation of ESCs recapitulates aspects of embryonic development. Therefore, a second hypothesis argues that growth factors important in embryonic blood cell development will be required to promote haematopoietic differentiation of hESCs. Specifically, the module hypothesises that definitive haematopoietic precursors will derive from a distinct mesendodermal population formed under the influence of critical combinations and concentrations of inducing factors.

We believe that differentiating ESCs will express specific milestone genes (such as MIXL1 and RUNX1c) as they transit through stages corresponding to those that embryonic cells pass through during haematopoietic ontogeny. Consequently, our third hypothesis states that the expression of these genes can be used to monitor haematopoietic differentiation and isolate haematopoietic cells and their progenitors.

The image to the right is showing red fluorescent protein expression in haemoglobinised erythroid cells and green fluorescent protein in a central core of endothelial cells.

Aims

Formation of haematopoietic mesoderm: Analysis of growth factors involved in primitive streak/mesodermal patterning and subsequent haematopoietic lineage allocation.

Generation and characterisation of haematopoietic stem cells: Establishment of protocols for the development and analysis of definitive haematopoietic lineages.

Culture and expansion of definitive erythroid and granulocyte progenitor cells: Establishment of protocols for generation, expansion and maturation of erythroid and granulocytic lineage cells.

Module Leader biographies

Professor Andrew Elefanty is the Joint Laboratory Head of the Embryonic Stem Cell Differentiation Laboratory of the Monash Immunology and Stem Cell Laboratories.

The laboratory has focused on human embryonic stem cell differentiation along mesodermal (blood, endothelium and heart) and endodermal (pancreas) lineages. The group has made significant contributions to the field in the culture of human embryonic stem cells, and they have developed a robust system for the efficient differentiation of human embryonic stem cells, complemented by the development of a safe animal product free medium in which human embryonic stem cell differentiation can be reproducibly directed to different lineages by the inclusion of specific growth factors. A major goal of their work is to regulate human embryonic stem cell differentiation in order to understand human development, to generate tools for drug discovery, and eventually to provide a source of cells for therapy.

Contact details

Professor Andrew Elefanty         

E-mail	andrew.elefanty@med.monash.edu.au
Phone	+61 3 9905 0650
Web	www.med.monash.edu.au/miscl

Selected publications

1. Ng ES, Azzola L, Sourris K, Robb L, Stanley EG, Elefanty AG. The primitive streak gene, Mixl1, is required for efficient haematopoiesis and BMP4-induced ventral mesoderm patterning in differentiating ES cells. Development, 132, 873-884, 2005.
2. Costa M, Dottori M, Ng ES, Hawes SM, Sourris K, Jamshidi P, Pera MF, Elefanty AG and Stanley EG. The hESC line Envy expresses high levels of GFP in all differentiated progeny. Nat Methods, 2, 259-260, 2005.
3. Ng ES, Davis RP, Azzola L, Stanley EG and Elefanty AG. Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation. Blood, 106, 1601-3, 2005.
4. Peng JC, Hyde C, Pai S, O’Sullivan BJ, Nielsen LK, Thomas R (2006) Monocyte-derived DC primed with TLR agonists secrete IL-12p70 in a CD40-dependent manner under hyperthermic conditions. Journal of Immunotherapy 29:606-615.
5. Pick M, Azzola L, Mossman A, Stanley EG and Elefanty AG. Differentiation of human embryonic stem cells in serum free medium reveals distinct roles for BMP4, VEGF, SCF and FGF2 in hematopoiesis. Stem Cells, 25, 2206-2014, 2007
6. Quek L-E, Nielsen LK (2008) On the reconstruction of the Mus musculus genome-scale metabolic network model. Genome Informatics 21: 89-100.
7. Hines M, Nielsen LK, Cooper-White J (2008) The hematopoietic stem cell niche: what are we trying to replicate? Journal of Chemical Technology and Biotechnology 83:421-443.
8. Davis RP, Ng ES, Costa M, Mossman AK, Sourris K, Elefanty AG and Stanley EG. Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak-like cells and enables isolation of primitive hematopoietic precursors. Blood, 111, 1876-1884, 2008
9. Ng ES, Davis R, Stanley EG and Elefanty AG. A protocol describing the use of a recombinant protein-based, animal product free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies. Nat Protocols, 3, 768-776, 2008.
10. Hatzistavrou T, Micallef SJ, Ng ES, Vadolas J, Stanley EG and Elefanty AG. ErythRED, a hESC line enabling easy identification of erythroid cells. Nat Methods, in press, 2009.