Testicles have two main functions: they produce sperm and testosterone. Evidence from clinical and population based surveys suggests an increasing incidence of male reproductive damages. Our long term goal is to study how to let stem cells become testosterone producing cells (Leydig cell), sperm production supporting cells (Sertoli cell) in the testis. In this proposal, we will focus on how to turn stem cells into Leydig cells in the testis. Testis is not closely monitored by immune system. We hypothesize that 1) a proper cells coming from human embryonic stem cells (hESCs) in culture once transplanted into the interstitial space of recipient testes can become Leydig cells in a proper testicular environment; 2) certain hormones promote and stimulate transplanted stem cells turning into Leydig cells in testes. This study has high significance because: 1) we can establish an experimental model to test the plasticity of stem cells (adult or embryonic) in the testis. Utilizing this model we can demonstrate the cellular and molecular mechanisms controlling stem cell function and plasticity in testes. This is crucial to the future use of stem cells in regenerative medicine, as well as in understanding aging, tumor formation, testosterone and sperm production; 2) we can translate the results to clinical studies relevant to the novel treatment of male infertility and testosterone deficiency. The cause of male infertility is either due to the germ cell defect or supporting cells’ dysfunction. In many cases, germ cells are present. The presence of donor-derived healthy cells replacing defective nurturing cells is critical since both Leydig and Sertoli cells support sperm production. Defects in these cells have been believed to contribute to abnormal sperm production. The possibility of beneficial hormonal effects of Leydig cell transplantation independent of their support of sperm production also exits. Since Leydig cells are responsible for testosterone production, stem cell transplantations may replace the need of life-long testosterone supplementation in testicle failure males and aging population. The findings from these studies will have a major impact in the understanding reproductive physiology and recovery from testicular pathology, and also may have the potential for novel future therapies in patients with testicular failure.
Statement of Benefit to California:
California is the biggest state in population in the United States. Infertility affects 15% of couples and evidence from clinical and population based surveys suggests an increasing incidence of male reproductive problems. In addition to the mentally and physically suffering in men, testicle failure sometimes contributes to social instability of the family. Our long term goal is to develop regenerative strategies to maintain, improve and rescue testicular functions through the stem cell research. In addition, understanding the cellular and molecular mechanisms controlling stem cell function and plasticity is crucial to the future use of stem cells in regenerative medicine, as well as in understanding aging, tumor formation, testosterone secretion and sperm production. Techniques generated from this study will contribute to biotech industrial development in California.
SYNOPSIS: The PI is interested in generating Leydig cells from hESCs. Leydig cells are responsible for testosterone production. In order to accomplish this the PI proposes to first differentiate hESCs into mesenchymal stem cells (MSCs) in vitro, transplant these cells into the testis of adult rat testis and look for Leydig cell differentiation. This proposal has two specific aims. This first is to examine if MSCs transplanted into interstitium of testes are able to differentiate into Leydig cells. The second specific aim is to determine the role of gonadotropin in stimulating the differentiation of MSCs derived from hESCs into Leydig cells in testes. INNOVATION AND SIGNIFICANCE: This proposal is significant and innovative, as it combines both in vitro and in vivo differentiation toward the generation of the Leydig cells. If this system works, it will provide the paradigm to dissect the molecular aspects as well as practical clinical application for testosterone therapies. STRENGTHS: The experiments are well-defined and ordered, and certainly appropriate for the period of funding. Specific aim 1 is the most important aspect of the work and the authors have considerable experience in this area. WEAKNESSES: This proposal has a number of critical weaknesses. First,if the goal is to develop human models of testicular progenitors that can be used to study fertility and testicular cancer, the authors should focus on the derivation of testicular progenitors, with properties similar to the physiological progenitors of Leydig cells, rather than a bone marrow population that likely plays no physiological role in the generation of germ cells or testicular cancers. Secondly, even if one concluded that mesenchymal stem cells were desirable for the generation of Leydig cells, there is little rationale for deriving these cells from ES cells: MSCs are readily available by culturing bone marrow or other cells from affected patients. Obtaining them from hES cells would be much more difficult, and would introduce safety and histocompatibility issues. Third, the authors have not devoted sufficient attention to the histocompatibility issues that will arise from the transplantation of human cells into the rodent testis. Even though the testis is 'immunopriviledged' the human cells are likely to induce a signficant xenogeneic immune response. This will be a major confounding factor. Fourth, one reviewer is concerned that the preliminary data supporting the potential for bone marrow cells to form Leydig cells and sperm were not carefully done. In general, data related to bone marrow transdifferentiation into other cell types has been notoriously difficult to reproduce and therefore it is appropriate to insist upon a high standard of proof for such claims. While strong preliminary data are not requested in these applications, I believe the history of work in this area merits skepticism. Fifth, magnetic beads are not a sufficiently robust approach for purifying MSCs from ES cell cultures. The authors are likely to end up with significant contamination by ES cells and other cells. Finally, Specific Aim 2 hurts this project because it relies entirely on the successful accomplishment of the first aim. If the first aim fails, the second aim becomes irrelevant. DISCUSSION: Reviewers questioned the rational for deriving testicular progenitors from MSC and furthermore, deriving MSC from hESCs rather than obtaining MSC from readily available bone marrow. Reviewers also expressed concern that the applicant did not address histocompatibly issues likely to arise upon transplantation of human cells into rodent testis.