Work Package 8

Signal mechanisms required for complete differentiation of ES cells to desired cell lineages

Introduction
All somatic cells in adult mammals develop from a small group of pluripotent cells in the blastocyst. Through a series of differentiation steps, these embryonic cells gradually become lineage restricted, losing their potency as division proceeds. They proliferate as the precursors of particular cell types (lineage restricted stem cells) until receiving new sets of signals that induce them to differentiate further and finally adopt a fully functional, terminally differentiated phenotype. Precursor cells are also present in some adult tissues in small numbers where they remain quiescent until required for tissue repair. Pluripotent and lineage restricted stem cells and the specific signals that control them are thus essential for normal development and tissue repair following injury/disease. In studies to identify relevant control mechanisms, it has emerged that a limited number of signal transduction pathways are used repeatedly in development, in processes as diverse gastrulation, neurogenesis, cardiogenesis and limb development. Notably, the pathways activated by the wnt, Fgf, hedgehog and TGFb ligand superfamilies are used in various combinations in all major morphogenetic events. In addition, embryonic and extraembryonic tissues are sources of yet unidentified signals controlling differentiation. For example, signals from extraembryonic endoderm affect cardiomyocyte differentiation, while the aorta/gonad/mesonephros region of the embryo is a source of signals for haematopoietic stem cell differentiation. Understanding how the order, magnitude and combination of these signals control differentiation in the embryo provides the basis for intelligent translation to stem cells in culture; the perspective being that their differentiation could then be tightly controlled, and lineage precursors or fully differentiated cells with the required phenotype could then be derived and expanded efficiently to yield large cell numbers for research and ultimately transplantation.

Objective
To identify and control the signal mechanisms that result in the earliest differentiation choices of ES cells to the ectoderm, mesoderm or endodermal lineages and subsequent terminal differentiation of partially restricted stem cells that occur throughout the embryo to their terminally differentiated derivatives (eg nerve/skin, muscle/blood and pancreas/liver cells, respectively). We expect to identify sets of signal transduction pathways controlling differentiation, to determine their temporal relationship with differentiation and to develop sets of markers that will allow intermediate states of differentiation to be monitored, expanded and selected.

Approach
Mouse and human ES cells will be induced to differentiate to various lineages using standard protocols of high density growth (“embryoid bodies”) in combination with growth factors, hormones and/or somatic cells as sources of differentiation inducing signals. We will profile gene expression at different stages after induction of differentiation using DNA arrays to determine temporal associations between emergence of various lineages and signal transduction pathways activated. This information will be used to design strategies for enhancing or repressing the cell phenotypes represented in cell cultures, overexpression of signal transduction molecules or transcriptional targets, expressing dominant negative variants or selected RNAis to alter endogenous levels of signalling targets in a dose dependent manner. We will also determine whether homologous recombination as used in mouse ES cells is a viable approach to modifying gene expression in the current generation of human ES cells. In addition to modulating genes specifically associated with the pluripotent state (oct-4, rex-1,UTF-1), we will also modulate the expression of genes known to act in a highly dose dependent manner in differentiation, such as the Smads downstream of the TGFb superfamily and b-catenin, downstream of wnts. Some of these data are already available in mouse ES cells and will be translated to human ES cells following development of effective gene transfer technology. Two existing human ES cell lines will be available to programme at the outset; during the course of the programme, initiatives to derive new human ES cell lines under Dutch law will have been implemented and will yield novel technology available to the programme.

Links with other parts of the programme: Wnt signalling aspects are closely related to work in WP7, genetic programmes in stem cells, whereas RNAi constructs used will derive from work in WP9.

Participants: Mummery, Dzierzak, Embryonic Stem Cells International, PamGene BV