Year 4

A central question in Human Embryonic Stem Cell biology is how the state of differentiation the cells can be controlled. Solving this problem will require numerous issues to be addressed. Among these are to define conditions and factors that promote the differentiation of human ES cells into particular lineages; and to identify intermediate stages of fate commitment. We have hypothesized that the best candidate factors are those that regulate cell fate decisions in normal embryos and in this grant, we test this hypothesis directly. We address the function of these developmental signaling molecules in the decisions that embryonic stem cells make during differentiation. The signals are called Wnts, molecules that are known to have multiple roles during embryogenesis. In one line of research, we are interested in generating new human ES cells using Wnt proteins. Among the cell fate choices we focus on are those that result in the generation of endoderm, the precursor tissue for pancreatic insulin-producing beta-cells. During this work, we have been able to directly isolate endodermal cells using novel cell surface markers. We also identify cell culture conditions that permit FACS-isolated cells to resume differentiation towards cells expressing markers of liver, and intestinal epithelium. These conditions include exposure of cells at early stages to purified Wnt proteins. To our knowledge, this re-establishment of development by isolated endoderm-like hESC progeny has not been previously described. We also showed that expression of a subset of surface proteins identified from our hESC studies was maintained in human pancreatic tissue, demonstrating the usefulness of our system for modeling human organ development. An unexpected new finding is that the formation of heart precursor cells from ES cells is also under the control of Wnt signals, allowing us to design a new protocol to generate these important cells in culture. In a complementary line of work, based on the realization that advanced genomics and computational methods are essential to understand the differentiation of human ES cells, we have developed new tools to analyze the transcriptional activity in human ES cells. In parallel work, we found that Wnt protein can maintain the developmental potential of mouse ES cells, serving as a model for human ES cells.
Hence, we have made significant progress in achieving our initial aims and are now able, based on the use of Wnt growth factors, to direct human ES cells along several important developmental pathways. Among the differentiation endpoints are two, cardiac mesoderm and endoderm, that may become relevant in the use of human ES cells for clinical purposes.