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RN1-00536-1: Reprogramming of human somatic cells back to pluripotent embryonic stem cells
Recommendation: Recommended if funds available
Scientific Score: 65
First Year Funds Requested: $588,675
Total Funds Requested: $2,943,375
Public Abstract (provided by applicant)
The ability to dedifferentiate or reverse lineage-committed cells to pluripotent/multipotent cells might overcome many of the obstacles (e.g. cell sources, immunocompatibility and bioethical concerns) associated with using other ES and adult stem cells in clinical applications. With an efficient dedifferentiation process, it is conceivable that healthy, abundant and easily accessible somatic cells could be reprogrammed to generate different types of functional cells for the repair of damaged tissues and organs. However, the cellular processes involved in dedifferentiation remain poorly understood, and methods for the control and study of dedifferentiation to pluripotency in human somatic cells are lacking.
Reprogramming of murine somatic cells in embryonic and adult fibroblast cultures to pluripotent ESC-like cells has recently been achieved by simultaneous viral transduction of four transcription factors together. With such proof-of-principle demonstration, next critical steps would be to “translate” such reprogramming methods into human somatic cells and identify small molecules that would allow temporal reversible treatment to induce/enhance reprogramming without risks of genetic manipulations.
Here we propose to develop homogenous human somatic cell systems to examine reprogramming to pluripotency, and implement a high throughput screen of large and diverse chemical librariesy to identify small molecules that can induce/enhance programming of human somatic cells back to pluripotent hESC-like cells. We will further examine their effects/activities via various in-depth cellular/biochemical assays, and characterize their mechanism of action by integrated chemical and functional genomic approaches. Collectively, the studies described in this proposal will provide novel chemical tools for producing unlimited amount of (autologous) pluripotent cells from differentiated/lineage-restricted cells for various applications as well as studying the underlying molecular mechanisms of pluripotency and epigenetic regulations, and may ultimately facilitate development of small molecule therapeutics to stimulate tissue/organ regeneration in vivo.
Statement of Benefit to California (provided by applicant)
Historically, small molecules have been more useful than genetic approaches in the treatment of human disease. However, much of our ability to control reprogramming of somatic cells to pluripotent cells currently involves either genetic manipulation of these cells or complex mixtures of protein factors. The demonstration that one can systematically identify, optimize and characterize the mechanism of action of small drug-like molecules that selectively control cell fate and reprogramming will: (1) provide important tools to manipulate cell fate in the lab; (2) provide new insights into the complex biology that regulates (stem) cell fate; and (3) provide an important first step which may ultimately lead to drugs that facilitate the clinical application of stem cells.
Review
SYNOPSIS: The Principal Investigator (PI) suggests a screen of a large chemical library to identify small molecules that can induce/enhance the reprogramming of human somatic cells into pluripotent stem cells that resemble human embryonic stem cells (hESC). Previous work from the investigator’s laboratory has resulted in the identification and characterization of small molecules that can reprogram cells, and the PI proposes to develop the lab’s involvement in hES cell biology to identify key biological molecules involved in reprogramming.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The significance of accomplishing this work is at the highest possible level currently for the field. The proposal addresses a fundamental biological issue which could resolve the key drivers for reprogramming and pluripotency. The scientific strength of this proposal is its potential impact on understanding the fundamental cell biology of reprogramming, and the mechanism of stem cell function. Medical advances arising from this work may be some way off, but the applicant’s aim to focus on potential small molecule drugs could yield important therapies in the future. If successful, this would represent a scientific milestone and pave the way for autologous stem cell-based therapies. Novel molecules that can induce reprogramming appear to have been identified; thus, the existing competencies are likely to yield data of value to the stem cell field.
While this proposal appears strong in its scientific aims, it has several important shortcomings. The PI is an excellent chemist, but does not have stem cell expertise and needs a serious collaborator to guide him/her in nuances of hESC biology that cannot be dismissed. It also seems that the PI has made assumptions about the ease of translating methods from mouse ESC to human ESC. One reviewer notes that to date, despite much screening, a compound capable of sustaining hESC stemness has not been identified. The PI describes important experiments in the preliminary results section, including data to establish the proof-of-principle for the proposed approach, but these data are disappointing because the PI does not identify the molecules being used, nor does s/he detail the reproducibility of the experiments, making it difficult to determine the feasibility or assess the quality of the data.
Another potential complication for the PI to consider in transitioning from mouse to human ESCs is in culture scale up to allow for screening, which may not be as easy as expected and for which some timeline adjustment may be required. Preliminary data on the establishment of long term cultures seems to be valid, but there is no support for the capacity to scale up readily. Also, one of the key scientists for performing the studies does not appear to have recent key relevant publications, and there is no information on the other core staff to be funded.
The experimental plan is extremely ambitious particularly with regard to the large number of somatic cell types that may be used in the reprogramming experiments. Preliminary data with one of these somatic cell types would strengthen the proposal. Experiments to analyze the molecular pathways targeted by the active small molecules are diffuse in nature and are likely to be beyond the scope of the current proposal.
One final criticism is that no figure legends are included in the proposal.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The investigator is a very talented and accomplished chemical biologist who has been recently promoted to Associate Professor. The PI has made many important contributions to this field, which includes publishing at a good rate and in relevant areas, such as at the interface of chemical biology, stem cells and regenerative medicine. The PI has performed very well in his/her current position, which has resulted in a recent promotion, and appears to have a great deal of drive and commitment to good fundamental science, which here is being expanded from basic chemistry to more biological issues. The PI has the requisite expertise and resources to execute the technically challenging high-throughput chemical screens. There is good career development potential, and strong and committed mentoring in hES cell work, although this is not local and there is no apparent intention to have regular formal interaction with these mentors. The PI and institution have funds from more than one source and both are engaging in the stem cell field. Reviewers have no hesitation affirming that this PI and team are perfectly qualified to perform all the specific aims of this grant, although one reviewer questioned the level of hESC expertise represented in this proposal.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The applicant institution provides an exceptional environment for pursuing the proposed research, encompassing all of the necessary resources and centers to successfully execute the proposed specific aims. Substantial funds are already supporting the PI’s development into the hES cell area and the applicant institution has made good lab space available. The PI has been provided with good facilities and support in genomics and chemical screening to support the proposal. There also is good local support on modeling and physical/chemical analysis and with other helpful technical platforms. There is a stem cell activity within the screening program for bioactive molecules and there are strong funded links to workers elsewhere.
DISCUSSION: The proposed work from this strong PI could have a wide impact on therapy. The PI has a good track record, good support, and strong mentoring, although one reviewer would liked to have seen more mentoring. A panelist asked about the stem cell expertise at the institution, and a reviewer replied that there was plenty of local talent. This person is a good candidate for the stem cell field who plans to move into new areas based on existing strengths, and bring a new technology into the field.
The aims are strong, and while the timeline might need adjustment, the plan is good, but the technical assumption that protocols for mouse ESC are transferrable to human ESC weakens the proposal. Another problem is that there is no support for scale up of lines. Another reviewer emphasized that this is an inventive chem-bio program from people who are leading the field, and the PI has shown feasibility for this kind of work in the mouse, therefore this reviewer is confident that human cell work is achievable. However, one reviewer disagrees, saying that the PI has no expertise in ESC in humans or mouse, and needs to recruit collaborators to help determine whether cells meant to be differentiated are actually differentiated. The proposal ignores substantial differences between mouse and human models – e.g., a chemical compound that sustains stemness in mouse ESC has not been shown to have the same effect in human ESC despite years of research. This reviewer also was disappointed by the preliminary results, where the PI states that functional compounds have been identified, but does not show their structure; thus, this reviewer felt that it is not possible to evaluate the preliminary data. One panelist asked about the readout methods for the screens. A reviewer answered that they are using tagged hESC line from a collaborator and doing side-by-side comparisons with mouse cells, which is appropriate.
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:
- Wagers, Professor Amy
- Orkin, Stuart