Work Package 7

Identification of stem cell versus differentiated cell genetic programs

Introduction
Stem cell fate determination and differentiation, whether in the gut, blood, nervous system, early stage embryo or embryonic stem cells, is a direct result of the genetic program operating within the individual cell together with the specific microenvironment that elicits growth factors. The overall genetic programs of stem cells/differentiated cells, which exist within the complex anatomical structures in the embryo and adult tissue, are difficult to study in the context of the whole organism. However, in vitro growth of undifferentiated ES cells and purification procedures for tissue-specific stem cells and their controlled differentiation, either in vivo or in vitro, provide source material for study of such expression analysis and identification of an overall gene expression program. Of particular interest to the study of cell fate determination and differentiation are signaling pathways employed during embryonic development. Production of the dedicated cDNA screening chip for signaling molecules (see project 1) allows for the signaling program of stem cells and differentiated cell types to be examined and compared. Signaling cascades and interactions and overlaps in signaling pathways can be discerned through such gene expression analyses. Information on the signaling pathways in place within the analysed cell types will allow for further investigation into whether these pathways play a functional role. Current technological advances with RNAi, allows for the knockdown in expression of chosen molecules and hence, once signaling cascades in stem cells/differentiated cells are identified, this innovative molecular approach can be used to block/enhance signaling pathways and thus, provide important information on the control of fate determination and differentiation. This knowledge of the specific genetic programs functioning in stem cells and their differentiated progeny should allow for manipulations that include the maintenance and amplification of such cells for future cell replacement therapeutic applications.

Objectives
To determine the molecular program (signaling pathways) expressed in stem cells and differentiated cells during developmental and/or adult stages and to identify the important signaling molecules that play a role in stem cell fate determination and differentiation. Approach Stem cells (gut, hematopoietic, oligodendrocyte and undifferentiated ES cells) will be isolated and used for expression analysis on the signaling cDNA chips generated by the consortium. Differentiated progeny will also be used for such analyses. Participating consortium laboratories are expert in the isolation, purification, culture and growth of stem cells. To verify gene expression programs, quantitative molecular and immunochemical analyses will be performed on the relevant cells. RNAi for the signaling molecules most likely to be important in stem cell fate determination and differentiation will be generated and used in the relevant cells to test for functionality in the implicated signaling pathways.

Links with other parts of the program and other research: These studies are involved in theme 3, stem cells and differentiation, but rely on information and results from Theme 1. Also, while this project is looking at overall signaling pathway expression and function, Project 2 allows for a more in-depth investigation into the Wnt signaling pathway and will provide corroborative and insightful data for other stem cell/differentiation systems.

Keywords: genetic program, signaling pathways, expression analysis screening, RNAi

Participants: Dzierzak, Mummery, Fodde, Clevers, Meijlink