Basic Biology II
This research proposal is aimed at gaining a better understanding of the factors that control the differentiation of human embryonic stem cells into one or another cell type. We propose a research program that focuses on the roles of a particular class of soluble, extracellular differentiation factors, i.e. the TGF-beta family, and the signaling molecules that they activate in cells. This class of differentiation factors is known to function as key regulators of many types of cell differentiation, and to play key roles in the generation of many tissues. Remarkably, these soluble factors have the ability to redirect differentiation from one cell type to another, raising the distinct possibility that changes in signaling by these factors may redirect the differentiation potential of embryonic stem cells, allowing them to give rise to cell types with different properties. Yet, very little is known about their effects in human embryonic stem cells, and how changes in signaling by TGF-beta family proteins affect embryonic stem cell differentiation. We are exceptionally well-positioned to study the role of these factors and their signaling molecules, as this lab has been at the basis of much of the current knowledge on what roles these factors play in cell dfferentiation. In the proposed research program, we will tinker with the embryonic stem cells to introduce changes in the signaling networks that are activated by TGF-beta family proteins, and then ask how these changes affect the potential of these cells to become different cell types. Furthermore, we will study the effects of such changes on the potential of cells, derived from embryonic stem cells, to become pluripotent again and to give rise to the full spectrum of cells. Finally, we will address whether changes in the cells that form the niche for embryonic stem cells affect the ability of embryonic stem cells to become many different cell types. Taken together, these studies should provide a basis for targeted manipulations of embryonic stem cells that will then result in directed changes in the cell types that can be generated from embryonic stem cells.
Statement of Benefit to California:
This research proposal addresses basic research questions and therefore does not directly address the potential use of human embryonic stem cells for therapeutic purposes or toward a particular disease. Nevertheless, we believe that this proposal may have important implications for future uses of embryonic stem cells, in particular for our desire to instruct the cells at will to differentiate into a particular cell type. Through our research on other cell types, e.g. fat cells, muscle cells and bone cells, we have found that we can enhance the potential of precursor cells to differentiate into one or another cell type by modifying the signaling pathways of TGF-beta family proteins. Thus, we have been able to enhance bone cell differentiation and muscle cell differentiation using such approaches. In addition, we are even able to redirect differentiation by modifying the signaling by TGF-beta proteins. For example, using this approach, we have been able to redirect pre-fat cells to become bona fide bone cells or muscle cells, thus providing a possible basis for the use of pre-fat cells, obtained through liposuction, for bone and muscle regenerative therapy. We now propose to study the role of these same TGF-beta family signaling pathways in embryonic stem cells, and to evaluate whether changes in these signaling pathways will change the capacity of the embryonic cells to differentiate into one or another cell type. If successful, this approach may have substantial implications for the derivation of various cell types from embryonic stem cells.
EXECUTIVE SUMMARY This proposal focuses on the role of the TGF-β signaling pathway in human embryonic stem cell (hESC) self-renewal and pluripotency. The proposal is based heavily on an observation, published in 2007 in Nature by another group, that some cultured hESCs differentiate into niche cells (hDFs) that support and regulate the undifferentiated hESCs in the culture. The applicant hypothesizes that TGF-β signaling is important in interactions between hDFs and hESCs. In Aim 1, the applicant proposes to study the roles of TGF-β and its downstream signaling effectors in hESC pluripotency and differentiation into hDFs using a variety of molecular techniques. In Aim 2, the applicant will use similar techniques to examine the roles of these molecules in hDF reversion and reprogramming to pluripotency. Finally, in Aim 3, the applicant proposes to manipulate TGF-β signaling pathway components in hDFs and study the effects on hESC pluripotency and differentiation potential. Reviewers commented that the principal investigator submitting this application is a leader in the TGF- β signaling field, but they raised doubts about the significance of this particular proposal for human stem cell biology and regenerative medicine. They noted that TGF-β signaling is already known to play important roles in hESC pluripotency and differentiation, and questioned the novelty of experiments proposed in Aim 1. While they appreciated the importance of studying signaling pathways in hESCs, they noted that the hypothesis of an in vitro niche, which forms the basis of the proposal, remains controversial. Reviewers felt that there was insufficient data supplied to support the idea that hDFs are critical for maintaining undifferentiated hESC cultures, and thereby questioned the rationale for the proposal. The reviewers raised several concerns about the research plan and preliminary data. Overall, the preliminary data to support the role of TGF- β and Smad signaling by these so-called niche cells are indirect and very weak. Reviewers would have appreciated further characterization of the hDFs and additional data supporting their role in hESC colony growth. One reviewer wondered whether hDFs might be early differentiated hESC progeny, such as mesendoderm, and suggested additional gene expression analysis to better define these cells. This reviewer noted heterogeneous staining patterns in the preliminary data that suggest suboptimal maintenance of self-renewal in the cultures. Reviewers did appreciate the use and development of novel tools and reagents to study TGF-β signaling in hESCs, and they found the cell assembly technology interesting. However, they were unconvinced that hESCs are a good first target for these tools, given the lack of characterization of the system. Reviewers described the PI on this proposal as an exceptional investigator who has published extensively and is an expert in TGF-β signaling biology. They did note that hESC expertise is lacking in the research team and that a collaborator in this field would have enhanced the application. Overall, reviewers raised a number of concerns about the novelty and significance of this proposal. They appreciated that the project would generate useful reagents and tools, but were not convinced it would make a major impact on the field.