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RS1-00161-1: MicroRNA Regulation of Human Embryonic Stem Cell Self-Renewal and Differentiation

Recommendation: Recommended if funds available
Scientific Score: 86

First Year Funds Requested: $315,063.00
Total Funds Requested: $631,831.00

Public Abstract (provided by applicant)

A major hurdle for regenerative medicine is the safe transplantation of human embryonic stem (ES) cells or their derivatives into patients. While the unlimited growth potential of ES cells is a major asset for their potential in tissue replacement, it is also a major risk for tumorigenesis. Therefore, it is critical to determine what molecules are responsible for silencing the tumorigenic risk of embryonic stem cell derivatives as occurs during the process of normal development. Identification of such molecules should provide both markers for tumorigenic risk as well as potential targets for therapeutic intervention when tumors do develop from transplanted tissue. We now know that most, if not all adult cells can revert to an early stem cell phenotype. This has been proven by a technique called somatic cell nuclear transfer, where adult cell nuclei are transferred into oocytes and allowed to develop as early embryos. These embryos reactivate the embryonic stem cell program within the adult nuclei. Cells derived from these embryos, the embryonic stem cells, have regained the ability to proliferate indefinitely, a property termed self-renewal. Therefore, considering there is in the order of one hundred trillion cells in the adult human, it is amazing that some fraction of cells do not commonly reactivate the stem cell program by chance. This is because human have evolved an amazing and complex network of molecules whose main purpose is to permanently silence critical components of the embryonic stem cell program. Unfortunately, this network is poorly understood. The aim of our laboratory and of this research proposal is to identify these factors and use them to our advantage. We have discovered in the mouse model that small RNA molecules called microRNAs are essential for the silencing of the embryonic stem cell’s capacity to self-renew. In this grant, we propose to confirm a similar role for microRNAs in human ES cell differentiation. Furthermore, we propose to identify the molecular nature of the specific microRNAs responsible for this function. These miRNAs could then be used as markers for both the developmental potential of embryonic stem cells derived by various means as well as a marker for the appropriate silencing of self-renewal in ES cell derived tissues prior to transplant in patients. Furthermore, they could provide targets for therapeutic intervention in the unfortunate scenario of tumor formation from transplanted tissues that had failed to fully silence the stem cell program.

Statement of Benefit to California (provided by applicant)

This grant proposes to identify the molecules that normally silence the potential of human embryonic stem cells to proliferate indefinitely. Identification of such molecules should benefit California in at least two ways. First, understanding the mechanisms our cells have evolved to silence indefinite proliferation would provide novel targets for therapeutic interventions in cancers either resulting spontaneously or from transplanted embryonic stem cell derivatives. Second, expression of these molecules should provide markers and, therefore, tests for analyzing the developmental and tumorigenic potential of embryonic stem cell derivatives pre- and post-transplant of these cells into patients. Such advances would benefit the health as well as the economy of the state of California.

Review

SYNOPSIS: This proposal aims to characterize and probe the function of microRNAs (miRNAs) expressed in human embryonic stem cells (hESC). The miRNA expression profile of multiple hESC lines will be characterized in the undifferentiated state and also during early in vitro differentiation and teratoma formation. The requirement for miRNAs in hESC will be tested by assessing the role of a specific molecular factor, and analyzing the function of individual miRNAs. Loss of pluripotency in hESC also will be determined using a fluorescent reporter.

SIGNIFICANCE AND INNOVATION: The proposed study to evaluate the roles of miRNAs in ESC self-renewal and differentiation represents a very innovative direction. miRNAs represent one of the epigenetic regulatory machineries fundamental to the regulation of self-renewal vs. differentiation of stem cells, and are likely to have profound effects on the behavior and differentiation of hES cells and derivatives. Understanding how miRNAs operate in stem cell behaviors will likely advance our knowledge on the fate decision-making of stem cells, which may in turn enhance our ability to generate target cell population without contamination of stem cells. There are only limited reports on miRNA profiles of human ESCs and no detailed functional studies have been reported. Determining in a comprehensive manner the role of miRNAs could be an important adjunct to other studies of the control of differentiation in hESC. Other labs are investigating similar questions, but this Principal Investigator (PI) appears to have strong preliminary data and hence could make a significant impact in the field.

STRENGTHS: The strength of the proposal includes: 1) the novelty of the proposed study to evaluate epigenetic regulation of stem cell self-renewal vs. differentiation and 2) the necessary experience and technology that the PI has built upon with studies on mouse ESCs. In particular, the preliminary data where loss of a specific molecular factor leads to loss of differentiation is an important finding relevant to the proposed studies, and experiments involving this factor will be extremely useful for the proposed study. In addition, the availability of the fluorescent reporter will significantly simplify the readout of stem cell self-renewal vs. differentiation. The PI’s extensive experience in stem cell biology and his association with hESC investigators is a plus. There is a high likelihood of success in obtaining interpretative data.

WEAKNESSES: Given the large number of miRNAs, particularly following differentiation, determining the function of all as suggested will be a huge undertaking. In addition, too many cell lines are proposed. Two questions that are not addressed are 1) what is the cell identity following the proposed treatments to induce differentiation, and 2) how can the function of specific miRNA be identified in this differentiation system?

DISCUSSION: While this is a huge undertaking, the PI has demonstrated that he is moving in this field with strong preliminary data and parallel studies in the mouse. The PI also has extensive experience in stem cell biology. The weakness is that the differentiation process is not well-defined, therefore looking at miRNA effects may be tough.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• Feit, Marcie
• Lansing, Sherry
• Sheehy, Jeff