Although the potential of embryonic stem cell-based therapy to cure disease is tremendous, its progress has been hampered by the biological complexity of stem cells and by limited federal research support. We propose to develop “embryonic stem cell-like” cells, that have been shown in animal models to have pluripotency properties similar to true embryonic stem cells, using adult testis tissue, and not human embryos, as the cell source. This research is based on the recognition that early germ cells (cells later destined to become sperm) in the testis are likely to be pluripotent, unlike other cells in the body. In other words, they have the power to either self-renew or to generate another cell type. As shown in mouse studies, it is possible to extract and culture the critically important testis stem cell (spermatogonial stem cells) in a Petri dish for prolonged periods of time. Under specific and precise culture conditions, it is also possible to “reprogram” these stem cells, or to uncover their pluripotency potential, so that they the gain the properties of true embryonic stem cells. Although these cells are not actually obtained from embryos, these “embryonic stem cell-like” cells perform many activities of true embryonic stem cells, including self-renewal or the ability to develop into other cell types in the body. Work in our laboratory suggests that we also can obtain these early germ (spermatogonial) stem cells from adult men after a testis biopsy. Additionally, in specific culture conditions, we were able to coax these stem cells into cells that looked identical to embryonic stem cells. Despite early success, our first attempts to grow these cells for prolonged periods to verify their identity was not successful. We reasoned that we had not created the optimal culture conditions for these cells to “reprogram” or to uncover their pluripotency. Thus, one aim of this proposal is to study culture conditions, including systems that employ embryonic stem cells, to generate an optimal environment for reprogramming testis stem cells into embryonic stem cell-like cells. Since one goal stem cell therapy is to repair damaged tissues in diverse diseases and individuals, another aim of this study addresses this issue. Because diseases are not limited by age or ethnicity, we will derive embryonic stem cell-like cells from individuals of various ages and ethnicities. We will determine whether the age or ethnicity of the stem cell donor affects our ability to generate embryonic stem cell-like cells. Perhaps it is more difficult to generate stem cells from men who are older compared to younger, or men of certain ethnicities compared to others. This information will have important clinical implications if testis based stem cell therapy is used to treat disease in the future. In summary, through this research we hope to obtain pluripotent stem cells that could potentially be used to treat disease in half the world’s population, without using embryos.
Statement of Benefit to California:
Five decades ago, a fledgling electronics and computer chip industry began to flourish in California. Known now as Silicon Valley, it was the vanguard of one of the world’s greatest industrial revolutions. Three decades ago, biotechnology also found a stronghold in California, and since then has blossomed into an industry of similarly profound size and productivity. Research in stem cell biology, enabled by Proposition 71, is yet another example of a burgeoning revolution that will maintain California’s competitiveness and reputation as the nation’s premier state for biomedical research. As Florence served as a magnet for education and the arts during the Renaissance, California, with its long history of entrepreneurial energy and new interest in funding stem cell research, now has the ability to draw into its fold the best minds in biomedical science with the potential for research funding in one of the most promising fields that biomedical research has ever witnessed. The research, which will use known hESC lines to help create and characterize patient-specific, embryo-free, pluripotent stem cells of potential benefit to half of the world’s population, is uniquely suited for CIRM funding and will benefit the California on several fronts. Through its broad scientific approach and requirement for a highly skilled, broadly trained and multidisciplinary team, this proposal assembles a cohesive group of talented clinicians and scientists, including the hiring of others (post-doctoral fellow), who will work together to improve our ability to recruit and retain premier scientific minds in California. In addition, the mentoring abilities of the lead scientist and the project collaborator are robust, further contributing to the training of future stem cell scientists in California. Lastly, multidisciplinary interactions are essential not only for solving today’s problems in stem cell biology, but they also form a crucible for gestating the next generation of ideas and discoveries. In this way, California’s reputation at the premier state for stem cell research will be maintained, even as other states including Massachusetts, New Jersey, Connecticut, Maryland and Illinois consider similar state-supported, stem cell propositions. In addition to the importance of a broadly trained team to the discovery “process” in the proposed research, the “content” of this proposal will add value to California in the form industry collaborations and patents. Given that a vibrant biotechnology industry coexists with academic centers in California, one can foresee this research in non-embryo-based, pluripotent stem cell technology being carried to its full clinical potential as cell-based therapy through academic-industry collaboration. Finally, by funding this proposal with limited federal funding opportunities, California will maintain its competitiveness as a global leader in high quality, clinically driven, embryonic and non-embryonic stem cell research.
In the mouse it is possible to isolate and differentiate the so-called ESC-like cells from testis spermatogonial stem cell of the adult. The PI of this project has reproduced this observation isolating SSC from adult men after biopsy, and differentiating them into cells that look and behave a lot like ESCs: i.e., they make nice compact colonies and can contribute to derivatives of all germ layers. The problem however is that these cells do not survive the culture conditions after a few passage. The proposal has two specific aims. The first is to determine the potential of human spermatogonial stem cells in vitro. This will begin by improving culture conditions and a thorough examination of their properties and includes tricks like co-culturing SSCs with hESCs to help their “reprogramming”. The second aim will examine whether the derivation of hESC-like cells from the testis is a generalizable and reproducible procedure, and will address their potential to form teratomas. SIGNIFICANCE AND INNOVATION: This is an important and significant project, because if it works it solves two problems at the same time: bypassing the requirement for embryos and the potential of derivation from patient with special genetic conditions. STRENGTHS: The strengths include the previous success the investigators have had in deriving SSCs from human material, their access to human material, and their colloboration with a leading hES stem cell researcher. It’s a well-written grant with good rationale and plan of attack. In addition the investigator has a proven track record of major accomplishments. More importantly, this approach allows the establishment of a collection of different genetic background as the investigator proposes to derive SSC from men of different ages and different genetic backgrounds. The latter is still missing for traditional hESCs. Overall, the research proposal is well written, well planned, and well supported by preliminary data. WEAKNESSES: The major criticism is the definition of what an ESC-like cell is. As the author states, these cells are either being reprogrammed, or they are unveiling their stemness potential. This issue needs to be solved before other strategies are designed. How would the author test the in vivo ability of these cells to contribute to different cell types and germ layers (outside of the traditional teratomas or in vitro differentiation assays)? A second weakness of this proposal is that there is no reason that it could not be funded by the NIH. While they propose to use a non-federally supported hES cell line, it is not clear that this would be necessary or better than federally approved hES cell lines