Guiding the developmental program of human embryonic stem cells by isolated Wnt factors

Embryonic stem (ES) cells have the remarkable ability to differentiate into all of the cells of the body. During normal development, the differentiation choices of embryonic cells are influenced by extra-cellular factors. The aim of this grant is to understand how these extrinsic signals guide ES cells into differentiation pathways, in particular into endoderm. The endodermal lineage is the precursor tissue for the pancreas and the pancreatic islet cells that make insulin. We are focussing on the ability of Wnt proteins to control how ES cells differentiate, as these are powerful regulators of embryonic development. Our labs have the unique ability to use active Wnt proteins to study how they act directly on ES cells. Over the past year, substantial progress was made. The Nusse lab has shown that the self-renewal of embryonic stem cells, both human and mouse, is dependent on extrinsic Wnt signals. This finding has important consequences for the clinical application and transplantation of ES cells, as it suggests that antagonism of the Wnt pathway will force all pluripotent cells to differentiate, thereby eliminating their tumorigenic potential. Second, these results suggest that Wnts would act to promote the establishment new ES cells. Indeed, we have been able to readily establish various new ES lines from several different mouse strains. Over the next year, we will also attempt to derive new human ES cells based on this technology. The Wong group uses bio-informatic methods to analyze the differentiation of ES cells. They have developed new methods and computer programs to analyze gene expression data from new technologies. The Kim lab has developed a new ES clone, hS17-90 which differentiates into endoderm and characterized the gene expression profile of novel ES cells. These results may lead to a fine-tuned protocol to induce endodermal and pancreatic differentiation of ES cells. All three groups have made inroads into this research area and actively collaborate based on mutual interests and complementing technologies.

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 study 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. We have made progress towards that goal as a new line with hallmarks of bona fide ES cell has been established. 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 native human fetal pancreatic epithelia, 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. 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.

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.