Executive Summary Adult mouse spermatogonial stem cells (SSC), which are unipotent adult stem cells, have been shown to reprogram spontaneously in culture, albeit at a very low frequency, into embryonic stem cell (ESC)-like cells termed germ line stem cells (GSC). Since SSCs thus appear to attain pluripotency more easily than somatic cells, the applicant proposes to develop protocols to use human (h)SSCs for reprogramming rather than fibroblasts. The principal investigator (PI) will explore this idea in three steps. First s/he intends to optimize SSC culture conditions, second s/he will perform a molecular analysis of the transition from SSC to GSC, and finally s/he intends to use a full arsenal of molecular tools and screens to improve the rate by which SSCs form GSCs. The general idea of using hSSC to derive new pluripotent hESC-like lines is excellent, highly responsive to the RFA, and should be very feasible. It would be of great advantage if pluripotent stem cells could be generated from hSSCs without the need for genetic manipulation, although one reviewer questioned why one should use SSCs instead of fibroblasts for reprogramming, since SSCs are so much more difficult to obtain. In spite of its potential significance, reviewers agreed that this proposal is overly ambitious. The goal of this project is to render the process of hSSC reprogramming controllable and efficient, and simply expanding with some considerable detail on the first aim would make an excellent proposal. However, the goal as described here to optimize hSSC reprogramming through several ambitious screens lacks direction and makes it very difficult to evaluate the priority or focus of the project. The extraordinarily wide scope and ambition of this multi-screen platform makes it very difficult to assess if hSSC reprogramming will be impacted by the proposed analysis, and there is very little chance that the experiments proposed would be accomplished in the three year time frame of the grant. Concern was also raised about the amount of work to be performed in the mouse system, since the focus of this RFA is the derivation of human cell lines. The PI is an expert in developmental biology but has no background in stem cell biology. S/he has recruited the help of a stem cell expert and a gene therapy expert. Although one reviewer felt that the applicant provided good preliminary data, another pointed out that there is little preliminary data presented regarding the reprogramming process in adult hSSC. Another major drawback of this application is the proposed budget. One reviewer pointed out that it is not possible to carry out the proposed experiments under the proposed budget, since hardly any funds are set aside for experiments, and most of it is allocated for salaries. Taken together, the concerns raised about the feasibility of this proposal led the reviewers to not recommend this application for funding. Reviewer Synopsis This application is based on the observation that spermatogonial stem cells (SSC) transform at a relatively high frequency into multipotent germ line stem cells (mGSC). Thus the idea is to use SSCs for reprogramming rather than fibroblasts since they appear to be much closer to a pluripotent stem cell lineage than somatic cells. They will explore this idea in three steps, first by optimization of SSC culture conditions (mouse). In the second aim they will study the transition from SSC into GSC by expression analysis of SSC, GSC and an intermediate stage, protein profiling, microRNA, histone modifications etc. Finally they want to use a full arsenal of tools like bioinformatics, microRNA manipulation, screening of critical transcription factors, screening for small molecules that increase efficiency or replace some of the factors, an ORFeome screening and improved delivery strategies. Reviewer One Comments Significance: Dr. Izpisua Belmonte proposes to use spermatogonial stem cells, a unipotent adult stem cell, as a starting point for reprogramming to become pluripotent human stem cells. Adult spermatogonial stem cells are closer to pluripotency than adult somatic stem cells and there is some indication they may not need any genetic modification which would be a great advantage. This is innovative and also has the great advantage of being able to derive them from the patient, therefore they would be immunologically compatible. Feasibility: The proposal is to support the PI at 25% effort, three post-docs at 100% effort and salary, plus three additional post-docs at 100% effort and no salary, and a technician at 100% effort and no salary, plus 5 consultants at 2% effort and no salary. I don’t understand the significance of so many staff on the project at 100% effort receiving no salary unless they are supported in another way? If they are being supported another way, this should be stated. Aim 1 will be to improve on preliminary efforts with hSSCs using defined culture conditions. They want to develop methods in parallel in mouse. Aim 2 will be to characterize the event of spontaneous reprogramming of hSSCs. They will use expression and protein profiling. Three phenotypes have been described in the mouse system. Aim 3 will use bioinformatics, microRNAs, transcription factor profiling, ORFeome screening, and small molecule screening (in collaboration) to characterize molecular mechanisms underlying the hSSCs’ reprogramming. This is a very ambitious proposal for three years which is the major weakness. It is likely they will find some methods more fruitful than others and they will run into technical difficulties that will require improvements in the current technology. No doubt, much data can be produced but the analysis and interpretation of it will be key. For example, the small molecule approach will identify hits that may appear to have the activity, but follow up studies will be required to determine if the activity is not due to an artifact of the system and likely chemistry will be required because the molecules will have multiple activities and generally be too weak to use even as a tool. It is important to have someone with chemistry expertise to help with this aspect of the project. The PI has a strong background in development and cell determination in zebrafish but has little or no publications in the area of stem cells. He will have to rely on consultants, collaborators and the expertise of his post-docs for this work. This is a potential weakness. Responsiveness to RFA: This proposal is directly in line with the RFA and falls under the category of developing an alternate method for generating human pluripotent stem cells that doesn’t involve IVF embryos. The proposed work also will help understand the mechanism of reprogramming. These are two strengths to the proposal. Reviewer Two Comments Significance: This is a project to derive pluripotent cell lines using adult spermatogonial stem cells (SSC) rather than fibroblasts. Several groups have shown recently that mouse SSC can reprogram spontaneously to ES-like cells (mGSC or MASC). This is a very exciting area of research since it suggests that pluripotent cells can be generated from unipotent adult cells without the need of genetic manipulation. The goal of this project is to make the process controllable and efficient. The PI indicates preliminary data using the chick system, suggesting that the process is evolutionarily conserved. The project unfortunately reads somewhat like an extensive laundry list of everything that one could imagine doing to evaluate SSC and mGSC which makes it difficult to assess the focus or priority. Feasibility: There are three Aims listing an extensive number of approaches to evaluate the efficiency of cell line derivation. There is so far little preliminary data presented regarding this process in human adult SSC. The first Aim is to improve hSSC culture conditions by screening feeder cell lines that might optimize expansion and reprogramming. An attempt will be made to derive reporter lines in mouse SSC using Nanog:cherry or Stra8:gfp which could allow for selection of a more homogeneous population. At some point an attempt will be made to translate this to human cells, although details are lacking. The second Aim is to characterize at the molecular level 3 cell types: the hSSC, a transitional cell type, and the ES-like cells following reprogramming. A variety of potential markers will be sought: cell surface or other antigens, miRNA patterns, histone modifications by ChIP, pluripotency via markers, karyotype, and teratomas. The “transitional cell” is not described nor is it indicated how this will be isolated. The idea is that from this analysis some ideas will come about for what genes might help to facilitate more efficient reprogramming. The third Aim is to use the information from Aim2 to identify reprogramming factors and test them, via bioinformatics, manipulation of miRNAs, testing of known and putative or discovered self-renewal factors, small molecules, random screening of the ORFeome. In addition, it is proposed that novel delivery strategies will be developed such as adeno or protein delivery. The PI is expert in developmental biology and has recruited the help of a stem cell expert (Dr. Gage) and a gene therapy expert (Dr. Verma). However, the extraordinary wide scope and ambition of this multi-screen platform makes it very difficult to assess the likelihood that SSC reprogramming will be impacted by this analysis in a 3 year period. Responsiveness to RFA: The general idea of using hSSC to derive new pluripotent ES-like lines is excellent, highly responsive, and should be very feasible. Simply expanding with some considerable detail on the first Aim would make an excellent proposal. However, the goal as described here to optimize this through 6-7 different ambitious screens lacks direction and it makes it very difficult to evaluate the priority or focus of the project. Reviewer Three Comments Significance: The successful derivation of pluripotent stem cells from hSSCs instead of fibroblasts would be useful if virus/oncogenes were not used. mGSCs cells can be derived from hSSCs spontaneously but this occurs at a very low frequency. If this proposal was successful, hSSCs would be derived at an increased rate from mGSCs. The major drawback of this proposal is there is no money set aside for experiments and that it is unfocused. Feasibility: It is not possible to carry out the proposed experiments under the proposed budget. In year one only 22K will be spent on the proposed experiments, with 271K going towards salaries. This decreases to <$9,000 for experiments for the entire third year of the proposal ($284,579 for salary). On the budget justification page there are no dollar amounts for any of the reagents that are requested for the proposed experiments, most likely because there is no money left after paying all 13 people listed as “key personal” (100% salary requested for at least three people and 25% salary for the PI). It appears that this proposal is being used solely to fund salaries. The proposal is much too ambitious. There is very little chance that the experiments proposed would be accomplished in the three-year time frame of the grant. For example, just Aim 2.5, determining pluripotency of the reprogrammed cells (hSSCs to mGSCs), should be expanded to an entire Aim. If the reprogrammed cells are not pluripotent, alternative experiments will need to be performed. It is unclear why the PI is proposing to investigate miRNAs. The goal of the project is to create useful SSCs to develop pluripotent cell lines. I agree that miRNAs are important regulators of gene regulation, but so are hundreds of other factors. There is no reason to focus on miRNAs when there are much more promising proteins that are already known to influence reprogramming. The above comments are just two examples of the difficulty that the PI will encounter in attempting to complete this proposal. The grant would be much better if the PI focused on a single research question instead of attempting to do too many experiments. By my count there are at least seven different screens/profiling experiments proposed. A more detailed explanation of only one or two of these would have greatly aided this proposal. Responsiveness to RFA: The proposal should generate hES cells and there is an adequate plan to share the cells. Reviewer Four Comments Significance: I do not see why one should use SSCs instead of fibroblasts, they are so much more difficult to obtain. There is some value in understanding the fundamental biology of SSCs or mGSC Feasibility: Good preliminary data, good research plan, certainly overly ambitious and most of the time less hypothesis driven and more of a screening approach. Very ambitious, this is something that the field is doing for many years for ES cells. No clear hypothesis, very screening based approach, outcome uncertain in terms of how this will contribute to our knowledge how reprogramming works. They don’t come back to the original hypothesis and try to reprogram SSCs using any of the identified factors.