Statement of Benefit to California:
SYNOPSIS: This project will look at roles for the RB gene in hESC growth and differentiation. In particular, RB and its family members p107 and p130 will be studied with regard to mechanisms of cell cycle progression in hESCs by way of: (1) Controlling the expression of RB family genes in hESCs; (2) Investigating how RB family members control cell cycle progression in hESCs; and (3) Determining how changes in RB family members affect the differentiation of hESCs. SIGNIFICANCE AND INNOVATION: This proposal is both innovative and significant because it will use different hESC lines for the first time to study a well known tumor suppressor gene (RB) that could lead to the development of better therapies for human cancers known to have lost RB, and to potentially gain insights into the control of the inherent tumorigenicity of hESCs which is a major problem in transplantation paradigms. It is also innovative and significant given the state of the field regarding the cell cycle and pluripotency of hES cells. STRENGTHS: As pointed out by the PI, “…Nothing is known about the role of RB family proteins in early human development or in hESCs…” Since RB normally acts as a transcriptional regulator and its loss can result in many cellular defects including hyperproliferation, lack of differentiation, and genomic instability (hallmarks of oncogenic transformation), its relevance toward an understanding of developmental biology and tumor biology, in ES cell studies as proposed here, is tremendous. Cell cycle progression in hESCs is understudied, and the present model is extremely well-suited for gleaning important normal and abnormal cell cycle events, related to RB expression, in different hES cell lines. Previous studies of hES cell cycle mechanisms have focused on only two lines that could skew our understanding of hES cell cycle regulation; the present proposal will examine several lines. Not only will this proposal examine consequences of altering RB family function in hES cells by way of looking at cell cycle control (e.g. G2/M transition), but also self renewal abilities (Oct 3/4 and Nanog expression) AND fate choice and differentiation by way of looking at ectoderm, mesoderm, and endoderm markers and potential altered differentiation into retinal cells (Reh collaboration). The application, from a gifted young PI, is extremely well-written, planned, and justified. The investigators have extensive experience in understanding Rb/pocket proteins in a number of other cellular contexts. Moreover, the proposal appears highly collaborative bringing together groups with distinct, but complementary strengths. The rational for CIRM support is strong, e.g. use of several independent hESC lines (including non-NIH approved lines), and the need to develop novel tools before going on to study something not known to be involved in hES cell development (therefore risky). WEAKNESSES: The PI has no experience at all on working with hESCs; however, a letter of support from Dr. Baker, who has apparently derived novel hESC lines (although all publications listed are on Xenopus), does help the cause, but the PI will be relying heavily on the collaboration with Dr. Baker. The mouse feeder layer could add problems in interpretation amongst other things to these hESC studies. The PI acknowledges this and rather boldly proposed to employ new feeder-free technologies (without details or experience). One other concern was the fact that there was no mention of the phenotype of the triple Rb knockout mouse ESCs (previously published) and whether the investigators expect a similar or different phenotype in hESCs. DISCUSSION: This proposal aims to study Rb in hESCs, specifically to look at a role for Rb in hESC growth and differentiation, in cell cycle progression control, and to assess changes in Rb and resultant differentiation effects in hESCs with the goal of finding better therapies for cancers and to provide insight on tumorigenicity. Studying Rb function in hESCs has significant potential to impact better therapies for types of cancer that have lost Rb and so the project is extremely innovative. The PI proposes a well-planned and unique cell cycle examination in several hESC lines. One strength is that nobody knows anything about the role of Rb in hESCs or a role for Rb during development. In addition, the cell cycle progression in hESCs is understudied. Weakness: the PI has no experience with hESCs; mouse feeders could significantly complicate this project. The secondary reviewer felt that understanding cell cycle in hESCs is a hot topic in the field. Because the Rb/pocket proteins are at the heart of the G2/G0 transition, they may play an important role in tumorigenicity. This study may shed light on how the Rb proteins contribute to tumorigenicity. The applicant is a leader in the Rb field and the lack of experience may not be overly problematic. A minor criticism - the applicant neglected to mention a study of the Rb protein in mESC. There were only minor technical issues highlighted during discussion. The PI has no experience with hESCs, and despite the collaboration letter from Dr. Baker, one reviewer recommended that he take a good hESC course.