Public Abstract:
A major goal of regenerative medicine is to understand the regulation of cell fate, and then to use that understanding to develop cell-based and drug-based therapies. Cell fate is determined by the immediate history and the environment of a cell. History and environment are in some senses erased at the formation of the zygote and the restoration of totipotency. This change in developmental potential is associated with genome-wide changes in chromatin, which enable recapitulation of the normal patterns of gene expression that emerge during development. Our methods for purifying and characterizing intact chromatin from genes that regulate self-renewal provide the first opportunity to identify all of the proteins that control these fate determinants. We will focus our study on a gene, Nanog, which is specifically required for ES cell self-renewal and on a second gene, MIR145, which is required for differentiation. In ES cells, Oct4 is an activator of Nanog but a repressor of MIR145. MIR145 encodes the microRNAs, miR143 and miR145; they suppress accumulation of the Oct4, Sox2, and Klf4 proteins. Our goal is to understand how these genes are regulated and, therefore, we must examine them in both their active and inactive states. Accordingly, we will identify all of the proteins that bind these genes in self-renewing hES cells and we will describe the changes in bound proteins that occur when the cells differentiate into extra-embryonic endoderm (XEN). The most promising of these changes will be tested genetically to determine whether they play a causal role in either the maintenance or the repression of stem cell self-renewal. These experiments will provide a deeper understanding of how stem cells maintain and then exit their self-renewing state. The work will also provide a new general method for studying the regulation of gene expression in any stem cell or differentiated cell of interest, accelerating the rate of discovery broadly across the entire field of regenerative medicine.
Statement of Benefit to California:
The benefits of this research will include (1) an acceleration in our understanding of the key determinants of cell fate, (2) new technology to understand gene regulation in stem cells at its most mechanistic level, and (3) the potential to start new companies that exploit this new technology and bring its benefits directly to the public.
Review Summary:
EXECUTIVE SUMMARY
The overall goal of this proposal is to gain novel insights into the molecular mechanisms that control stem cell self-renewal and differentiation in order to enhance the ability to control cell fate towards therapeutic applications. To achieve these ends, the applicant proposes to catalog the entire repertoire of proteins that interact with the genetic loci of Nanog and MIR145, two factors that that play critical roles in the regulation of stem cell fate and behavior. In the first aim, the principal investigator seeks to adapt and refine a novel technology for purifying gene specific chromatin factors from human embryonic stem cells (hESC), the identities of which will be subsequently determined by peptide mass spectrometry. The second Aim will utilize these methods to profile the changes that occur at the Nanog and MIR145 loci as hESC transition towards an extra-embryonic, endodermal fate (XEN).
Reviewers were unanimously impressed by the innovation and creativity of the proposed technology for gene-specific chromatin-immunoprecipitation, which was developed in the applicant’s laboratory. If successfully adapted, the potential to elucidate the molecular mechanisms that underlie cell fate changes could be broadly applied and fill numerous gaps in our current understanding of pluripotency and stem cell behavior. Their enthusiasm was somewhat tempered, however, by the decision to focus on XEN differentiation rather than a lineage whose elucidation might have a more clinically relevant impact.
Although the reviewers appreciated the logical approach, well-conceived milestones and unique expertise of the team, they also expressed a number of concerns that led them to question the feasibility of this effort. Importantly, the majority of the preliminary studies were conducted with HEK293 cells, a transformed kidney cell line whose properties are substantially different from hESCs. Furthermore, there were no data provided to validate the effectiveness of the specific Nanog and MIR145- tools in hES cells, and no alternative approaches were presented to address the possibility that these constructs would not be functional in the embryonic cell system. Additionally, the reviewers identified several serious gaps in the experimental design that were thought to limit its potential for success. For example, some worried that the transgene reporter approach might introduce a bias as the result of chromatin context, a concern that might have been allayed by analyzing several independent lines as opposed to a single hESC source. Others noted that the applicant did not describe markers or reagents that would enable purification of undifferentiated cells from the XEN derivatives to be studied. Finally, reviewers questioned whether the cross-linking and mass spectrometry methodologies would be sufficiently effective at the scale necessary for this effort to have meaningful impact.
The principal investigator is a distinguished, recognized expert in systems biology and a key player in the development of the technological platform that forms the basis of this proposal. However, the reviewers were concerned by his/her limited experience with hESCs, as evidenced by the lack of relevant publications, premature status of the preliminary data, and an underestimation of challenges likely to be encountered. The reviewers also noted a lack of hESC expertise amongst the key personnel, despite an impressive set of qualifications for the methodological aspects of this project. Several suggested that inclusion of a seasoned hESC biologist would have substantially bolstered their confidence in the ability of this talented team to translate their innovations into the field of basic stem cell biology.
In summary, the reviewers appreciated the innovation and utility of this proposal but felt that its impact was limited by problems with feasibility, including the premature status of the preliminary studies and lack of vital expertise within the applicant team.
