Basic Biology II
$1 712 880
Our lab is known for its discovery of the family of nuclear hormone receptors (NHRs) that use vitamins/hormones to control genes and thereby regulate embryonic development, cell growth, physiology and metabolism. Of 48 known NHRs, we discovered that a unique subset of 38 receptors are expressed in adipose-derived human induced pluripotent stem cells (hiPSCs). The process of converting adult cell types like skin or fat into stem cells literally occurs in the nucleus by a process known as epigenetic reprogramming. A unique property of NHRs that distinguishes them from other classes of receptors is their ability to directly interact with and control the expression of genomic DNA. Consequently, NHRs play key roles in both the etiology and the treatment of disease by controlling genes. Drugs targeting NHRs are among the most widely prescribed in the world. While adipose-derived iPSCs express 38 NHRs, virtually nothing is known about their function in controlling stem cell renewal and differentiation into specific cell types (cell fate). How the extensive family of hormonal ligands can be used to control iPSC generation, maintenance and cell fate has profound implications for regenerative medicine. We wish to take advantage of our lab's expertise to understand, at the molecular and hormonal level, how nuclear receptors can be exploited to accelerate the use of iPSCs in regenerative medicine.
Statement of Benefit to California:
Our lab is known for its discovery of the family of nuclear hormone receptors (NHRs) that use hormones to control genes and thereby regulate embryonic development, cell growth, physiology and metabolism. This work was all done in California and has brought in more than $100,000,000 of private and federal funding to my group over the last 30 years. It has led to my employment of 150+ people and the publication of more than 300 research papers. Three biotech companies were founded from this work that in aggregate raised more than $1B in research and development support. Several FDA approved drugs for cancer, diabetes, osteoporosis and low white blood cells (leukopenia) were developed with this technology. Of 48 known NHRs, we discovered that a unique subset of 38 receptors are expressed in adipose-derived human induced pluripotent stem cells (hiPSCs). Drugs to nuclear receptors are among the most widely prescribed in the world. While adipose-derived iPSCs express 38 NHRs virtually nothing is known about their function in controlling stem cell renewal and differentiation into specific cell types (cell fate). How the extensive family of hormonal ligands can be used to control iPSC generation, maintenance and cell fate has profound implications for regenerative medicine. We wish to take advantage of our lab's expertise to understand, at the molecular and hormonal level, how nuclear receptors can be exploited to accelerate the use of iPSCs in regenerative medicine. Thus, our proposed study should be beneficial to the State of California in several ways: 1) by maintaining a unique training environment for students, postdoctoral fellows and academic and clinical physicians; 2) discovering how to better and more efficiently generate and use human iPSCs; 3) decipher the molecular genetic logic of nuclear reprogramming; 4) determine how a pharmacopeia of hormones and drugs can be brought to play on directing stem cell renewal, differentiation and therapy.
EXECUTIVE SUMMARY This is a proposal to determine how nuclear hormone receptor (NHR) signals regulate the formation, proliferation and differentiation of adipose derived human induced pluripotent stem cells (iPSCs). In Aim 1, the investigator proposes to use sequencing technologies to describe the complete transcriptomes of adipose and keartinocyte iPSCs, in order to define their NHR signatures. In Aim 2, the genomic set of cis-acting targets (cistrome) of six NHRs and the Oct4 cistrome will be evaluated to uncover genomic patterns that correlated with self-renewal or differentiation. In Aim 3, the epigenetic signatures of Oct4 and the NHRs will be probed by mapping key histone acetylation and methylation markers. The investigator proposes that this will reveal genetic and epigenetic codes for iPSC pluripotency and cell fate. Reviewers agreed that this proposal addresses a major unsolved problem in stem cell biology: understanding epigenetic programming in human iPSCs. They commented that the approach is logical and scientifically sound, and praised the mechanistic focus of the research and the innovative approach of examining the role of NHRs in iPSC generation, maintenance, and function. Although the proposal is descriptive and ambitious in focus, the PI’s reputation and experience gave reviewers confidence that the work would yield interesting results. Finally, most reviewers believed this proposal was of high translational value because the study emphasizes new therapies using NHR stimulation and develops feeder-free conditions for hiPSC derivation and maintenance. Overall, the impact and significance of the project for the field of stem cell biology and regenerative medicine was seen as very high. Reviewers praised the experimental design and feasibility. The proposal was well-organized, presented logical specific aims, and included a well-planned three-year timeline. The preliminary data effectively demonstrated the research team’s ability to derive iPSCs and to find NHR expression patterns in their analyses. In particular, reviewers appreciated that the research plan was carefully crafted to give meaningful results regarding NHR downstream effects on iPSCs. In discussing the proposal’s strengths, reviewers commented that the cutting edge genomic technologies and other state of the art approaches proposed to dissect the transcriptional regulatory network would identify new NHRs involved in reprogramming and stem cell functions. Integration of knockdown and overexpression analysis to validate sequencing results was rigorous. Reviewers recognized the robust data analysis component and noted the strengths in bioinformatics analyses contributed by a collaborating lab. Reviewers expressed mild concern over the lack of a discussion of alternate plans or potential pitfalls, the descriptive nature of the proposal, and the lack of focus on a particular NHR. One reviewer felt the proposal over-emphasized the exploration of the NHR expression patterns and the examination of the downstream genetic targets of the adipocyte-derived stem cells and their keratinocyte-derived counterparts, whereas another felt that there was not enough discussion of the analysis and interpretation of this data. However, the reviewers were mostly very positive about the specific experiments proposed and the overall feasibility of the project. The reviewers were overwhelmingly positive about the principal investigator’s (PI’s) expertise, laboratory, and track record. The scientific infrastructure for research on nuclear hormone receptors in the applicant’s laboratory was described as unsurpassed, and the expertise of the collaborating laboratory in bioinformatics was seen as an important strength. Reviewers also commented that the PI’s 20% commitment to this project heightens its chances for success. In summary, this proposal elucidates the role NHRs play in iPSC generation, maintenance, and differentiation. The reviewers commended this proposal for its well-packaged presentation, innovative and logical experimental design, and for addressing a major problem for future iPS cell therapies. The approach was seen as innovative, the results were considered of substantial potential impact, and the PI’s rigor and expertise in the field were described as unsurpassed.