There are thousands of cell types in the animal body, many of which can be derived from embryonic stem cells (ES cells), a pluripotent cell type that thrive in cell culture condition. ES cells differentiate into various cell type in a tissue culture dish in response to different growth factor/cytokine treatment, which can be transplanted back into animals for regenerative medicine application. In recent years, scientists have generated another type of pluripotent stem cell population, designated as induced pluripotent stem cells (iPS cells). These iPS cells are derived from adult cell types, and capable of differentiating into a variety of cell types in a tissue culture dish. Both ES cells and iPS cells not only provide a unique paradigm to study early mammalian development, but also hold great promise for regenerative medicine. Therefore, understanding the molecular network that regulate stem cell maintenance and stem cell differentiation of these cell types are very important for their application in regenerative medicine. In the studies we proposed, we will examine a novel class of gene regulators for their functions to maintain the stem cell population, as well as to trigger their differentiation into specific lineages. The focus of our studies is non-coding RNAs, which are RNA molecules that do not have capacity to encode proteins. Among the best studied non-coding RNAs are the microRNAs, which are small RNA molecules that are potent regulator for gene expression. These small RNAs often have the capacity to each regulate hundred of genes, therefore, regulating diverse developmental processes and physiological processes. If the biogenesis of these small RNAs are removed, stem cells exhibit multiple defects both in the tissue culture dish and in animal development. In this proposal, we aim to investigate the roles of non-coding RNAs in the regulation of stem cell maintenance and differentiation, and to identify novel non-coding RNA regulators that may impact stem cell biology. It is worth noting that RNA therapies, particularly those using synthetic small RNAs or their inhibitors, bypass the need for conventional gene therapy, and provide great promise for clinical application. The methods to delivery small RNAs into the cells and animals have been improved significantly over the past decade, and it is very likely that small RNAs and their inhibitors will be soon utilized for therapeutic applications. Therefore, our proposed research will generate exciting findings to stem cell biology and may lead to the development of novel diagnostic markers and therapeutic approaches for regenerative medicine.
The studies proposed here explore the functions of novel non-coding RNAs in the self-renewal and differentiation in pluripotent stem cells. This is a new area of stem cell research, which, in funded, can benefit the state of the California to develop new diagnostic markers and therapeutic approaches in regenerative medicine. to gain knowledge on the molecular basis for pluripotency, and to train young scientists in stem cell biology. In what follows, the benefit of proposed research to the State of California and its citizens are summarized in details. Pluripotent stem cells not only provide a unique paradigm to study early mammalian development, but also hold great promise for regenerative medicine. RNA therapies are an area of intense investigation, and the in vitro and in vivo delivery methods for RNA molecules have been greatly improved over the past few years. Therefore, it is possible in the near future to use RNA therapy, particularly those involving small RNAs or their inhibitors, for regenerative medicine. RNA therapy is fundamentally different from the “conventional” growth factor or cytokine delivery to modulate stem/progenitor cells. RNA therapy by itself, or in combination with conventional therapy, may provide a novel approach for regenerative medicine. In addition, studies of non-coding RNAs in stem cells may give rise to new markers for stem cells, as well as their differentiated lineages. The proposed research will carefully examine the roles of non-coding RNAs in regulating the pluripotency of stem cells, which is a field in its infancy. Not only the knowledge acquired from this study will enhance our understanding on the molecular basis for stem cell maintenance and differentiation, but the novel research tools developed from this study will also benefit the stem cell research in the state of California and its people. In addition, the proposed research will allow young scientists to be well trained in the stem cell field, and to combine two exciting fields in biology, i.e., non-coding RNA biology and stem cell biology. And such training will be critical for the state of California to have its own research force on stem cell biology in the future.
This proposal is focused on defining the possible roles of non-coding (nc) RNAs, such as microRNAs (miRNAs), in controlling self-renewal and pluripotency of embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells. The principal investigator (PI) proposes to functionally validate pluripotent cell-specific miRNAs by modulating their expression in mouse and human cells (aim 1), and to screen for miRNAs that rescue the differentiation defects of mouse ESC that lack the miRNA biogenesis machinery (aim 2). Lastly, the applicant proposes to identify novel ncRNAs that regulate self-renewal, and specifically focus on identifying RNAs associated with a RNA binding protein known to promote pluripotency (aim 3).
This is an innovative, clearly presented proposal that will identify the role miRNAs play in both ESC and iPS cells. It is based on strong evidence in the field that the miRNA machinery can influence stem cell properties. The proposal addresses an exciting research area, and pursues important tool development. Aims 1 and 2 are solid; reviewers lauded the overall experimental design, but pointed out that the proposed readout for assessing self-renewal/pluripotency may need improvements. Furthermore, the proposal would benefit from expanding the number of human embryonic stem cell (hESC) lines under investigation to allow evaluation of the reproducibility of identified responses to changes in miRNA expression. Finally, reviewers expressed concern that potential variability in the proposed screen, due to varying expression levels within the miRNA overexpression library, and the heterogeneity of ESC cultures, was not sufficiently addressed in the discussion of potential pitfalls. A reviewer questioned the feasibility of aim 3, as it depends on the availability of certain reagents that have not yet been developed. Notwithstanding these specific suggestions and criticisms, reviewers were overall very enthusiastic about the innovation and research design presented in this proposal and about its potential for facilitating progress toward therapeutic approaches using hESC and/or iPS cells. Since preliminary data were considered relatively weak, the proposal was judged to be high risk but potentially highly rewarding. The reviewers expressed confidence that important insights will be gained from the proposed work.
The principal investigator (PI) has only recently been appointed as an assistant professor, and has already attracted some extramural funding. She/he is an exceptional scientist with an outstanding record of publication in the miRNA field, including a number of publications in very high profile journals. She/he was trained by one of the world leaders in the field of miRNA biology, and obtained the expertise necessary to create mouse knockouts as a graduate student. miRNA research is extremely competitive, but the PI appears to be very well trained to be successful in this field of research. The PI has only a limited stem cell background, which is partly addressed by establishing collaborations, and by the fact that the home institution has a strong stem cell center, and a strong commitment to the development of the stem cell biology program. The mentoring plan is solid. Given his/her overall track record it is highly likely the PI will be a future leader in the field.
A strong letter of support from the head of the PI’s division was included. The PI has very little administrative or teaching duties, which will allow him/her to focus on research. A generous start-up package with a full laboratory suite and support for graduate students was provided. Extensive core facilities are readily accessible.