Basic Biology II
The most prominent feature of the stem cell is its pluripotent capacity to differentiate into various types of cells. The importance of the orchestrated interplay between molecular regulators has been demonstrated in the maintenance of self-renewing pluripotent property or the initiation of differentiation. Advance in the generation of the induced pluripotent stem cells (iPSCs) have been benefitted by our knowledge on the molecular regulation in stem cell renewal/differentiation. Furthermore, the practical use of stem cells for regenerative medicine will be possible through our understanding on the mechanism underlying distinct differentiation process. Recent progress in stem cell biology has unveiled some important features of molecular and cellular regulations in stem cell pluripotency and differentiation, but it remains largely elusive. The proposed study is based on our recent published findings that demonstrate the significance of the cell cycle regulatory molecule in embryonic stem cell self-renewal and differentiation. Our published data strongly supports that CDK2AP1 (CDK2 associating protein 1) is a competency factor in mouse embryonic stem cell (mESC) differentiation. Even though the difference in molecular regulation between mouse and human has been documented, it is also accepted that they share common molecular mechanism in the maintenance of self-renewal/differentiation. Especially the importance of the role of OCT3/4 in stem cell maintenance and pluripotency has been well documented in both models. This study is focused on the molecular and cellular mechanism and is an innovative research in a sense that the proposed study will unveil a novel mechanism in stem cell regulation. Specific Aims proposed in this application will significantly advance our understanding in hESC biology towards (1) the epigenetic control of OCT3/4 and its functional contribution to the stem cell pluripotency, (2) the role of cell cycle regulatory mechanism in stem cell self-renewal/differentiation decision, and (3) the potential utilization of molecular regulatory mechanism for future regenerative therapeutics.
Statement of Benefit to California:
The medicine today is facing two equally pressing issues in the treatment of patients- providing a life-saving quality treatment in the mean time at an affordable cost to every patient. California has the highest healthcare costs of any state in the nation – more than $110 billion per year. In that sense, the regenerative medicine offers an initiative for the future of medicine. The regenerative medicine is the ultimate goal of the future medicine in treatment of patients suffering from both genetic and non-genetic disorder. The benefit of stem cell research is almost unlimited in developing breakthrough cures and treatment for debilitating diseases and injuries, including diabetes, cancer, heart disease, Alzheimer’s, Multiple Sclerosis, HIV/AIDS, Parkinson’s, ALS, osteoporosis and spinal cord injuries. Unfortunately, our current knowledge on stem cell biology is far behind what we need to know in order to make the stem cell therapy available to the patients in the clinics. Only way to improve our practical knowledge and move the field forward is to devote our efforts and resources toward better understanding of the biology and mechanism, which will ultimately lead to the practical translation of our basic knowledge from the laboratory to the treatment of patients in the clinics. In the long run, stem cell therapies may cut California’s skyrocketing healthcare expenditures by reducing the need for expensive, long-term supportive care, which will be unavoidably pressing issues to the citizens of California in the very near future.
EXECUTIVE SUMMARY This proposal focuses on the regulation of stem cell proliferation and differentiation. The project investigates the requirement for CDK2-associating protein 1 (CDK2AP1) as an essential factor for differentiation of human embryonic stem cells (hESCs) and explores the action of this protein in epigenetic regulation of the OCT3/4 gene and modulation of the phosphorylation of the retinoblastoma protein (pRB). These studies are based on the applicant’s previous research using mouse embryonic stem cells. In the first Aim, the applicant proposes to investigate the relationship between CDK2AP1 expression and hESC self-renewal/differentiation. Next, a systems biology approach will be used to indentify key molecular pathways that underlie CDK2AP1-mediated human ESC regulation. For the final aim, the applicant proposes small molecule screens to identify inducers of OCT3/4 expression that are either dependent or independent of CDK2AP1 function. Reviewers agreed that this proposal could lead to an improved understanding of the molecular mechanisms that regulate the function, fate and behavior of stem cells. As the CDK2/pRB pathway has not been well studied in hESCs, the reviewers were optimistic that this work would contribute novel insights to the field. Furthermore, reviewers praised the innovation and creativity of the combined systems biology approach. Reviewers expressed some concern as to whether the mouse data would be translatable to the hESCs and whether the study would yield data of sufficient quality. Nonetheless, the intriguing premise and rational approach heightened reviewer enthusiasm for the project’s potential to illuminate novel mechanisms regulating stem cell differentiation. Reviewers acknowledged the logical rationale and sound approach of the research plan. They were particularly impressed by the high quality preliminary data including significant methylation and RB analyses. Additionally, reviewers appreciated the sophisticated molecular analysis and use of a systems biology approach to elucidate key molecular components. Reviewers noted some weaknesses in the experimental design including a lack of adequate discussion of potential pitfalls and alternative approaches, insufficient accounting of expected endpoints and timelines, and the absence of preliminary data addressing the role of the CDK2/pRB pathway in hESC. Nonetheless, reviewers found the proposal compelling and were positive about the project’s feasibility The reviewers described the principal investigator (PI) as a highly trained expert with relevant expertise in the proposed area of research. While some questioned his/her experience with systems biology, most agreed that the steady stream of recent publications, together with the recruitment of a strong collaborative team with complementary expertise, supported the overall potential for the project’s success. In summary, this application proposes an investigation of an understudied but potentially significant hES cell cycle regulatory pathway. Despite some minor reservations about achievability, the reviewers remained enthusiastic about the project’s potential to advance the field. Significant strengths included the creative approach, compelling preliminary data, and the highly relevant expertise of the principal investigator.
- Ali Brivanlou