Right Column

RL1-00652-1: GENERATION OF PLURIPOTENT STEM CELL LINES FROM SPERMATOGONIAL STEM CELLS APPLICABLE TO REGENERATIVE MEDICINE

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

Engineering, economical or biological sciences have undeniably contributed to an increase in the standards and quality of life: we live longer and mostly better. However, medicine has yet to bring cures for many major diseases, such as neuronal degenerative diseases, which might touch every second elderly person above the age of 80, heart diseases, which are the leading cause of death in developed countries, diabetes, cancers, and so many more. Human embryonic stem cells, which are obtained from oocytes of supernumerary embryos, appear as a major contributor of innovative therapies for those diseases, whereby they could replace the sick cells, or deliver necessary factors. But they will not be used as such, mostly due to the ethical issues they raise, and because of the difficulty in obtaining them. A great breakthrough in stem cell research allows for imagining that the therapeutic potential of those cells could be carried out by reprogrammed adult cells in the near future. Cells have indeed been obtained from adult skin cells, using some genetic reprogramming, that can give rise to any kind of cell in the human body. This is only the first of many steps to be taken in order to achieve available cures for the above mentioned diseases. An important issue, which will need to be solved, is the safety of the reprogramming method. As it stands now, cells are permanently genetically modified, and are very likely to give rise to cancers. This, of course, is not viable for therapeutic use. Moreover the extent of similarity or dissimilarity of the generated cells with human embryonic stem cells and with cells that could be used in a clinical setting has yet to be fully characterized, and the mechanism by which reprogrammed cells are able to give rise to any cell type is very poorly understood. This research project elaborates a strategy to obtain, in a safer way, those invaluable cells, and plans to make sure that the obtained cells would be usable for therapeutic applications. In order to do so, we will concentrate in reprogramming human spermatogonial stem cells, which can be obtained by a simple biopsy, and which are not as differentiated as the skin cells used previously, which should make reprogramming easier. We will study the mechanism of reprogramming, which is up to now still a big black box, and hope thereby to improve the efficiency of any type of reprogramming. We will generate pluripotent stem cell lines, which can be later used for both basic and applied research.

Statement of Benefit to California (provided by applicant)

The people and the state of California can gain both scientifically and economically from the work proposed here. Scientifically, California is leading the way in stem cell research and will benefit both from the fruits of our research, and from the added-value to individual scientific efforts conducted in California brought by the establishment of the network of collaborations, both within the State, and around the world, which we will put into place. We conduct research to expand fundamental knowledge of human biology and to bring science close to patients’ bedsides. In this study we would develop an approach that allows the production of unlimited numbers of different pluripotent cell lines, which could later be used for therapeutic purposes, or as disease models by the pharmaceutical industry in order to test new drugs. In the future, those cells may hold the key to replacing cells lost in many devastating diseases such as Parkinson’s and diabetes. Moreover, as the pluripotent stem cell lines developed in our laboratory will be made available to all academic researchers around the world, we are convinced that our technology will have wide-spread use by stem cell researchers and geneticists in California as well as the rest of the world. Economically, the technologies developed or improved throughout our proposed research could lead to economic growth through the creation of new products, technologies, jobs, companies and even new industries and most importantly to advances in health care and improvements in the quality of life.

Review

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.

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:

• Cowan, Chad
• Minger, Stephen