Basic Biology II
Embryonic stem cells have the potential to generate all tissue types that could be used for regenerative medicine, such as replacement of damaged neurons, replenish of insulin secreting beta cells, or generation of blood cells. The discovery of in vitro reprogramming of somatic cells (normal cells in our body) into induced pluripotent stem cells (iPS, which has the potential to differentiate into many different cell types) offers an exciting reality that patient specific pluripotent stem cells could be obtained. Cells derived from patient specific iPS cells would less likely to cause immune rejection when transplanted back into the patient. The rapid progresses in stem cell research make regenerative medicine from scientific fiction close to medical reality. However, many key issues, such as the efficiency of iPS induction and efficiency of stem cell differentiation in vitro (outside of our body), remain to be resolved before stem cell therapy becomes a routine medical practice. YAP is a transcription co-activator, which can help certain transcription factors to stimulate gene expression. Previous studies have shown that elevated YAP activity makes organ bigger. For example, YAP overexpression in mouse livers increases liver size by 4-5 fold. High YAP activity has also been observed in some human caners. We have found that YAP has an important role in mouse embryonic stem cells. Decreased YAP activity forces stem cell to differentiate while high YAP activity maintains stem cell properties even under differentiation conditions. We also found that in mouse embryonic stem cells YAP stimulates expression of many genes known to be important for stem cell pluripotency. In this project, we will study the function of YAP in human embryonic stem cells. We hope to understand how YAP promotes the stem cell properties. In addition, we would like to know how YAP itself is regulated in the human embryonic stem cells, by specifically determining YAP protein levels, localization in the cell, and degradation. Finally, we will isolate YAP activators and inhibitors. YAP activators may help us more efficiently generating iPS cells from somatic human cells. Conversely, YAP inhibitors may facilitate in vitro differentiation of human embryonic stem cells, therefore reduce the risk of teratoma formation caused by residual undifferentiated ESC in stem cell therapy. Completion of this project will help us to understand the basic biology of stem cells and may provide candidates for future drug development.
Statement of Benefit to California:
Stem cell therapy has the potential to revolutionize the treatment of many common diseases that afflict residents of the State of California. Alzheimer’s disease, diabetes, heart failure, anemia and arthritis are just a few of the illnesses that could potentially be treated. The benefits of the proposed research to the State of California and to its residents will be multiple. It will accelerate the pace of stem cell research, by discovering the novel function of YAP in human embryonic stem cells. The function of YAP in embryonic stem cells has not been reported while our preliminary study strongly indicates a success of this project. Our study will provide scientific knowledge of ESC stemness maintenance and research tools to more efficiently reprogramming iPS from normal somatic human cells. This would facilitate generation of patient-derived iPS cells. Moreover, as we hypothesized, inhibition of YAP activity will deplete human embryonic stem cells in in vitro differentiation; therefore decrease the risk of tumor formation in receipt patients. This project will generate knowledge beneficial to stem cell therapy and provide drug candidates for regenerative medicine in the state of California.
EXECUTIVE SUMMARY This proposal focuses on the role of the protein YAP in the regulation of human embryonic stem cell (hESC) pluripotency as well as the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs). YAP is a transcriptional co-activator known to be critical for organ size control during development. The applicant presents preliminary data that YAP promotes self-renewal and opposes differentiation in mouse ESCs. In Aim 1, the applicant proposes to determine if YAP plays a similar role in human ESCs and during iPSC generation. In Aim 2, the applicant proposes to identify key YAP-regulated genes and to assess their function. In Aim 3, the applicant will examine the mechanism that causes down regulation of YAP activity during hESC differentiation, and increased YAP activity during iPSC reprogramming. Finally, in Aim 4, the applicant proposes to develop an arsenal of YAP inhibitors and activators and to test their effects on hESC pluripotency, differentiation and iPSC reprogramming. Reviewers found this proposal to be highly significant, as YAP and its signaling pathway have not been examined in detail in hESCs. Successful completion of the project would provide a deeper understanding of human stem cell pluripotency and reprogramming. Aim 4 is particularly compelling, as it could result in the discovery of small molecules that sustain self-renewal of hESCs or alternatively, promote cell cycle exit and differentiation. With the exception of Aim 4, reviewers did not find the proposal to be especially creative or innovative, as much of the proposed work has already been done in mouse cells. The reviewers found the research plan to be logical, comprehensive and feasible. They appreciated the substantial preliminary data that form the basis of sound hypotheses. Reviewers did note that much of the preliminary data were obtained using mouse cells and felt the proposal would be stronger if it included convincing evidence that YAP is important in hESCs, especially since Aims 2 through 4 entirely depend on that outcome. However, reviewers were relatively confident that the applicant would confirm conservation of YAP’s functional roles from mouse to man. Reviewers presented two minor criticisms of the experimental design. They cautioned that the functional analysis of YAP target genes identified in Aim 2 might be difficult, as phenotypes following knockdown of individual genes could be quite nuanced. In addition, in Aim 4, the peptide inhibitor approach was judged to be technically problematic, and reviewers felt that the small molecule screen was the more promising strategy in this Aim. Reviewers especially appreciated that the screen had already yielded several preliminary hits. Reviewers described the applicant as an authority on signal transduction mechanisms that regulate cell growth and differentiation. They noted that s/he has consistently published in top-tier journals and has outstanding qualifications to carry out the proposed research. Reviewers also noted that two collaborators with extensive human stem cell expertise have equally outstanding credentials. Overall, reviewers were enthusiastic about this proposal, which features an impressive research team investigating a significant cell signaling pathway that has not been studied in detail in human stem cells. They raised concerns about the dependence of Aims 2-4 on Aim 1, but they were ultimately convinced the research plan is likely to be successful and have a major impact on the field.
- Ali Brivanlou