Basic Biology II
Stem cell therapies have the potential to result in entirely new treatments for a vast variety of currently incurable diseases. The goal of these therapies is to regenerate lost cells or tissues through the use of established human embryonic stem cell lines or even from the patient's own cells. One major limitation of current stem cell therapy approaches is that they may give rise to cancer. Embryonic stem cells have a limitless potential to reproduce and if they remain in an undifferentiated state could give rise to tumors. This proposal seeks to make stem cell therapies safer by using two novel targets that can selectively kill undifferentiated stem cells while sparing differentiated cells or normal cells. We propose to use small molecule inhibitors of the cell cycle and inhibitors of specific small RNAs to kill embryonic stem cells that have remained in an undifferentiated state. If validated, these therapeutic approaches could be developed to make stem cell therapies safer.
Statement of Benefit to California:
The State of California is committed to developing new approaches to treat currently incurable diseases through the application of stem cell biology technologies. While there is much excitement about the prospects for these new treatments, there is also potential concern, that current strategies may also increase the risk of developing cancer. This proposal seeks to use new strategies to block the growth of embryonic stem cells that pose the risk of developing into cancer. It is hoped that such approaches will make stem cell-based therapies safer for us all.
EXECUTIVE SUMMARY The goal of this proposal is to develop methods that would improve the safety of pluripotent stem cell derived therapies. Undifferentiated human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have the potential to form tumors following transplantation. The applicant proposes to study small molecules that may selectively eliminate undifferentiated hESCs or iPSCs from populations of differentiated cells. In Aim 1 the applicant will test small molecule cell cycle inhibitors for this selective killing capability and determine what cellular properties are required for the effect. In Aim 2 the applicant proposes to perform similar experiments with a microRNA (miRNA) inhibitor and test this inhibitor in vivo against hESC- and iPSC-derived tumors in a xenografted animal model. Reviewers agreed that this proposal addresses a significant issue in the field of regenerative medicine. The development of methodologies that could specifically deplete undifferentiated cells that have the potential to form teratomas could have a major impact on the advancement of stem cell derived therapeutics. Reviewers did question the scientific rationale for two key aspects of the proposal however. In particular, previous studies have shown that mouse ESCs lacking all miRNAs (Dicer and DGCR8 mutants) are viable, and therefore, it seems improbable that knocking down a single miRNA could lead to selective killing of undifferentiated hESCs or iPSCs. Reviewers also found little compelling rationale for targeting the miRNA to eliminate undifferentiated cells in vivo rather than using this strategy to deplete these cells ex-vivo prior to transplantation. Reviewers expressed serious concerns about the project’s experimental design and feasibility. This concern included problems with the relevance and interpretation of the preliminary data. For example, the inhibitor proposed in Aim 1 is a known anti-proliferative agent and its actions described in the preliminary data are most likely due to its general effects on proliferating cells rather than any special selectivity for stem cells. Given the inhibitor’s general effects, a reviewer suggested that bone marrow progenitor cells would constitute a better control than the differentiated fibroblasts presented in Figure 1. Another reviewer noted that most of the preliminary data for Aim 2 are limited to mouse cells, and this is problematic because the relevant sites on the proposed gene target, particularly the seed complementary region required for miRNA activity, are poorly conserved between mouse and human. The proposal would have been strengthened by evidence of miRNA repression of the target gene in human cells. Reviewers also criticized some of the experiments addressing the potential mechanisms of inhibitor action. For example, experiments in which gene knockdowns cause cell death are unlikely to be useful in identification of specific cellular targets. In Aim 2, knockdown of the proposed miRNA gene target is proposed as a method of determining if the miRNA inhibitor is operating through that gene, but this approach ignores potential non-specific, anti-apoptotic effects. Reviewers felt that these and other weaknesses in experimental design undermined the potential of experiments to adequately address the project’s goals. The reviewers described the PI as a talented young investigator with a good publication record. They noted that while the PI has yet to produce a paper as senior author, two well-established collaborators add strength to the proposal. In general the reviewers found the research team to be well qualified to carry out the proposed research. In summary, although the reviewers appreciated the significance of the problem addressed by this proposal, their overall enthusiasm was significantly dampened by serious deficiencies in the scientific rationale, experimental design and project feasibility.