Embryonic stem (ES) cells possess a remarkable property of pluripotency to give rise to all cells of the organism; therefore holding great promise for regenerative medicine. ES cells are envisioned as potential sources for use in cell replacement therapies. However, as with any allogeneic material, ES cells generated from fertilized embryos, and derivatives of such cells, inevitably face risks of immunorejection when transplanted into a host. A solution to the problem of rejection is to derive ES cells from embryos cloned from a host patient’s own cells, because any replacement cells would be genetically identical to the host. ES-like cell lines have been generated from somatic cells via somatic cell nuclear transfer (SCNT) technology. By this technique the developmental fate of a somatic cell nucleus can be reprogrammed following transfer into an enucleated oocyte, resulting in a cloned embryo that can be used to derive novel ES cells. The nuclear transferred ES cell lines have been shown to possess the same characteristics for self-renewal and unlimited differentiation capacity as those of conventional ES cell lines derived from normal embryos produced by fertilization. Although these cloning experiments are direct evidence that a terminally differentiated cell can be fully reset to a pluripotent ES cell state, the fidelity of reprogrammed nuclei is very inefficient as evidenced by the extremely low frequency (<5%) of producing healthy offspring in animals. Thus, the current available SCNT technique makes ES cell related therapies only an academic possibility at this time. Alternative approaches in deriving stem cells are based on genome-wide analysis on genes that are involved in the molecular control of pluripotency. Takahashi and Yamanaka (2006) have recently reported the induction of pluripotent stem cells from mouse embryonic and adult fibroblast cells by introducing four factors (Oct3/4, Sox2, c-Myc and Klf4) under ES cell culture conditions. All four factors play a crucial role in maintaining the undifferentiated state and proliferation of ES cells in culture. The resultant induced pluripotent stem (iPS) cells are similar but not identical to bona fide ES. The iPS cells are not fully reprogrammed and have a limited capacity to stably integrate into normal tissues in vivo. Thus, this approach is unlikely to sufficiently reprogram somatic cells to a state that will be optimal for human therapeutic cloning. The proposed research is to develop a new high-efficiency nuclear transfer (HENT) approach to obtain high fidelity ES cells. The approach is based on the current existing SCNT method in combination with transcription factors that are known to be required for ES cell renewal and pluripotency. The long-term goal of the proposed research is to apply HENT to derive high fidelity human ES cells and subsequently for cell replacement therapies.
Statement of Benefit to California:
Efficient derivation of host patient-specific embryonic stem (ES) cells is a fundamental step, currently a bottleneck, for cell replacement therapies and regenerative medicine. The proposed research aims to increase the efficiency of ES cell derivation from somatic cells through a modified nuclear transplantation technique. The result of such work will potentially benefit the state of California and its citizens in many aspects including health, life, costs, and leadership in biomedicine. ∑ About half of California families have a child or adult who suffers or will suffer from devastating medical conditions that could potentially be treated or cured with stem cell therapies. ∑ Development of ES cell therapies that treat diseases and injuries with the ultimate goal to cure them will improve the California health care system and reduce the long-term health care cost burden on California. ∑ Intellectual property resulted from the research will provide an opportunity for the state to benefit from royalties, patents, and licensing fees. ∑ The research will contribute to the advancement of the biomedical research in California to world leadership.
SYNOPSIS: The PI wishes to improve the efficiency of SCNT reprogramming of somatic cells, principally by conditionally overexpressing c-myc in the donor cells, and also by examining the roles of other transcription factors in the process of reprogramming. Conditional overexpression will be engineered by the presence or absence of doxycycline. The four factors identified by Yamanaka as reprogramming components in mouse cells will be examined. SIGNIFICANCE AND INNOVATION: The goals to improve SCNT for reprogramming and for development of individual-specific ES cell lines are important goals in the field. The major novelty lies with the proposed conditional overexpression system. Given the attention that the Yamanaka work has already received, there will be many similar attempts to apply the experimental approach to the human system. Consequently, the proposal is not extremely innovative as it builds on previous work which suggests that overexpression of key pluripotency genes in somatic cells can reprogram them back to ES cells. This proposal merely asks whether the overexpression of these same genes might also improve reprogramming via SCNT. That said, the significance of the proposal is high in that real improvements in the efficiency of SCNT might directly translate into an increased chance at creating disease and patient-specific hES cells by SCNT. STRENGTHS: The proposal is a relatively straightforward approach to examine whether manipulation of the concentration of specific components will affect the efficiency of reprogramming. A key strength of the proposal is the quality and track record of the researchers, who have significant experience and expertise in SCNT and mES cell derivation. The proposal is very collaborative, bringing together two groups with complementary skills and strengths. In addition, the recent progress in the field in the mouse system supports the rationale for the proposed work, though one may question whether reprogramming by SCNT is the same process as the forced expression of components as in the Yamanaka experiments which show that 4 factors can achieve substantial reprogramming in mouse somatic cells. WEAKNESSES: The proposal has some real scientific weaknesses. For instance, if we assume that the overexpression of these genes in somatic cells helps reprogram them, the best possible outcome is that they are almost ES-like in nature. A number of experiments have shown that ES cells are still very inefficiently reprogrammed by SCNT. In fact, even nuclei from 2, 4, and 8-cell embryos are not extremely good donors for SCNT. Thus it is unlikely that merely making the somatic cells less differentiated will in fact significantly improve their reprogrammability by SCNT. Differences between mouse and human ES cells with respect to their properties might also suggest that a different set of factors (other than the Yamanaka 4) might be required. Thus, reliance on the 4 factors already described might be a limitation. An additional minor concern is the fact that the proposal uses a CMV-based promoter to drive expression of the pluripotency associated genes. CMV promotors are often silenced in pre-implantation embryos, and always in ES cells, which could pose a problem for the proposed experiments. Also, the justification for focusing on c-myc alone for a start is not strong. The PI might consider additional approaches for the selection of factors to be tested as the reliance on c-myc or even the 4 Yamanaka factors might be shortsighted. DISCUSSION: There was no discussion further following the reviewers' comments.