Funding opportunities

Replenishing Dysfunctional Testes with Stem Cells

Funding Type: 
New Faculty I
Grant Number: 
RN1-00552
Funds requested: 
$1 785 540
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Men’s health and quality of life are largely dependent on fully functional testes. Testes have two main functions: they produce sperm and the male hormone testosterone. Evidence from clinical and population based surveys suggests an increasing incidence of male reproductive problems. About 15% of couples are infertile. Male infertility and low testosterone levels due to abnormal function of the testis are caused either by defects in the germ cells, cells that support maturation of sperm or cells that made testosterone. Stem cell therapy provides new opportunities to replace and replenish damaged and dysfunctional testicular cells. Studies have demonstrated that transplantation of stem cells of testis origin into dysfunctional recipient testes can restore the capability to regenerate sperm and produce more testosterone. Because of the unavailability of stem cells from testes and ethical challenges of genetic paternity for future clinical applications, our proposed studies will use bone marrow stem cells as a more feasible source for replacement of dysfunctional testicular cells. Previous studies including a study from our lab have demonstrated that bone marrow stem cells have potential capabilities to replenish germ cells, supporting cells and testosterone producing cells in dysfunctional testes. In the proposed studies, we will first isolate bone marrow stem cells from donor animals and increase the population of stem cells in culture before transplanting these enriched stem cells into recipient testes of various animal models for male infertility and men with low testosterone levels. In addition, we will test the effect of different testis environments, hormones, growth factors on stem cell plasticity. This study is important because: 1) we can use this model to study the cellular and molecular mechanisms controlling stem cell function and plasticity within the microenvironment of the testis. This is crucial to the future use of stem cells in regenerative medicine, sperm production, male aging, and testicular tumor formation; 2) we can explore the potential of novel stem cell therapy for male Infertility and low testosterone. With diminished ethical issue by harvesting the patient’s own bone marrow stem cell and transplanting into their own testes for treatment of male testicular failure, we can more easily translate our findings into clinical studies; 3)This method could also be important for re-establishing genetic paternity, with or without assisted fertilization technology, in the setting of patients with cancer (not involving the bone marrow) following chemotherapy; 4) we can develop stem cell therapy to replace the need of life-long testosterone supplementation in testicle failure males and aging population. Thus, our study will have major consequences for the understanding of reproductive biology, pathology and clinical implications for novel future therapies in men with congenital and/or acquired primary testicular failure.
Statement of Benefit to California: 
Infertility affects 15% of couples in attempting their first pregnancy and of these 30% is due to male factor infertility. 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.
Review Summary: 
SYNOPSIS: In this proposal, Dr. Lue outlines a strategy to identify cells in bone marrow that are capable of regenerating somatic and germ lineage cells after transplantation into the testis in rodents. These studies follow on the applicant's prior finding that unfractionated bone marrow cells generate these cell types after transplant. Experiments will focus on two bone-marrow derived populations - MSCs and MAPCs. The investigator will take advantage of specific testis toxins and engineered mouse strains that create cell-deficient niches to be filled by bone marrow stem cells. In Aim 1, MSCs will be marked with GFP and their differentiation status after transplant into ethylene treated rat testes will be assessed by measuring testosterone secretion. MSC differentiation potential for Leydig cells will be tested in an LH -receptor knockout mouse, LuRKO, that is hypogonadal secondary to lack of testosterone production by Leydig cells. If mouse MSCs can differentiate into Leydig cells in the testicular environment, the animals will no longer be hypogonadal. Almost 200 mice will be used for these studies, including bone marrow cell donors. Aims 2 and 3 will examine the ability of MAPCs injected directly into seminiferous tubules to differentiate into Sertoli and germ cells, and whether or not cell-to-cell fusion occurs. Almost 200 mice will be used for these aims. In Aim 2, MAPC differentiation into will be tested by transplant into busulfan-treated and kit mutant mice and read out by detection of GFP+ sperm and by fertility experiments. Finally, in Aim 3, potential contributions via cell-cell fusion will be assessed by tranplant of GFP+ MSCs or MAPCs into ROSA26-beta-galactosidase-marked recipients and analysis by microscopy and FACS. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: This project proposes to identify novel sources of replacement cells for somatic and germ lineages in the testis. The proposal addreses an important scientific and medical issue, using both in vitro and in vivo models in seeking to regenerate reproductive organ function in dysfunctional murine models. The applicant relates his proposed studies almost entirely to his interest in male reproduction, infertility, and contraception, but the results from these studies could have far-reaching consequences to stem cell research with respect to differentiation in situ in specific niches, if his hypotheses are correct. While not a requirement of this RFA, human embryonic stem cells are not involved and all the studies could be funded by federal sources. The rationale provided by the applicant for the use of bone marrow cells as a source of replacement cells for the testicular lineage is problematic. The applicant states that the use of bone marrow cells is preferable because it enables autologous transplantation; however, autologous transplant would not be effective in cases of testicular dysfunction due to cell-autonomous genetic mutations. In this regard, the applicant proposes that autologous transplantation of bone marrow cells in conjunction with in vitro genetic manipulation could be used to correct genetic defects; however, such an approach has obvious ethical complications that are not considered or addressed by the applicant. Also, the proposed use of cell transplantation to treat testiclular failure in aging individuals could be less than effective if cell dysfunction arises from microenvironmental or systemic defects. Likewise, the hypothesis, stated in the specific aims, that MSCs are bone marrow precursors for Leydig cells while MAPCs are bone marrow precursors for Sertoli and germ cells is not clearly supported by prior data. There is no rationale given for the decision to focus on these 2 cell types exclusively. The experiments proposed are technically feasible if sufficient quantities of MSCs and MAPCs are obtained by the procedures described, although details are lacking for how MSC and MAPCs will be obtained, how these cells differ, the purity of the preparations, or what quality control measures will be in place to ensure reproducible isolation of equivalent populations. The "MAPC" cultures shown in the preliminary data do not resemble previous reports of MAPC from the Verfaillie lab, which indicated a strict requirement for culture at very low density. Functional tests for MAPC identity should be included. There is a noteworthy lack of reference to a class of macrophages, F4/80 positive in the mouse, known to be important in testis development and testosterone production by the Leydig cells. The existence of this cell could confound the interpretation of transplanted bone marrow stem cells, so it needs to be controlled for. It is not clear why some experiments will use rats, while others use mice. The research design focuses intensely on methodology, but doesn't give a rationale for the experimental design or the choice of assay system. An enormous amount of tissue examination work is described because of the numbers of animals proposed for each group, but Dr. Lue and his team have had experience with the techniques, and resources needed are available at LA BioMed. However, pitfalls and alternatives are not discussed to account for the possibility that MSC and MAPC may not in fact be the populations that are responsible for their prior observation of BM transdifferentiation in the testis. If these cells are not involved, then the experiments will not be informative. Aim 1 will determine if MSCs transplanted into interstitium of testes are able to differentiate into Leydig cells. The description of the MSC preparation is lacking in details of determining the purity of the preparation. Aim 1a proposes experiments for rats, whose Leydig cells will be ablated by a single intraperitoneal injection of ethylene dimethane sulfonate; regeneration occurs approximately 2 weeks later. At one week, 2 million MSCs recovered from bone marrow of GFP-transgenic rats and expanded in culture will be micro-injected into testis interstitium. Almost 200 rats will be used for this aim: 44 bone marrow stem cell donors, 48 non-manipulated controls, 48 EDS-only injected controls and 48 EDS-injected, MSC-transplanted experimental animals, to be examined at 2, 4, 6 and 8 weeks after transplantation. Statistical justification of this many rats is lacking. QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Dr. Lue is a mid-career scientist, originally trained as a Chinese urologist. His enthusiasm for his research and teaching activities are clear from his application and his Goals and Milestones, #7 of which is to “Attain Professorship in 3 years from award of the grant.” His record to date, his enthusiasm for his research, and his many collaborators provide strong support that this work will be carried out. Dr. Lue received his MD in 1993 from Shandong University School of Medicine, and did fellowship work at UCLA. He was an Assistant Professor at Shandong from 1993-1995, and has been a Research Assistant Professor at UCLA and Harbor UCLA Medical Center, LA Biomedical Research Institute, from 2001-2004 and was promoted to Associate Professor in 2004. He lists 17 publications since 1999, mainly in reproduction and endocrinology journals. The applicant lists funding support only from a seed grant, and a pending R03 application. The applicant outlines a career development plan focused on expanding research interests in stem cell biology particularly as it relates to male reproduction. The PI has clearly stated career goals and the proposal contains clear and measurable objectives for career development with a formal review process .He will seek out additional training in stem cell biology and collaboration with other stem cell biologists, participate in stem cell seminars and train students in his lab and through a course in stem cell biology which he hopes to develop. His career is monitored by twice yearly evaluation by anonymous faculty, fellows and students. He also meets quarterly with senior faculty (Dr. Ronald Swerdloff and Christina Wang, who are his former mentors when he was a research fellow) to discuss progress and problems. His progress is also monitored yearly by the institutional Scientific Advisory Committee (including external and internal reveiwers). INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The institution has committed a very small lab (250 sq. ft.), although the PI is supported by core facilities (tissue culture, morphometric analysis, flow cytometry, confocal microscopy, mass spec., and hormone assay cores are available). It is not clear that the personnel requested could be accomodated in this space. The PI has support from a very a solid and well-qualified team of mentors, and has strong letters of support from collaborators at UCLA and institutional officials at LA BioMed, with statements of a strong commitment to stem cell research. LA Biomedical Research Institute is an independent, not-for-profit center. LA Biomed recently decided to increase its efforts in stem cell research by forming a Stem Cell Seed Grant Program and a Stem Cell Research Advisory Committee. It is recruiting additional stem cell biologists as well, though it has not had a strong presence in this area in the past. The level of interaction of scientists at LA Biomed with other academic institutions in the LA area is unclear. DISCUSSION: This project is unique in that it uses testes as a model system. The PIs career development plan is good, and he has strong support from his institute, although it has committed very little space. The experiments seem feasible. The reviewers felt that the proposal had very significant weaknesses: the preliminary data is not convincing and scientific descriptions are somewhat naïve. For instance, new literature suggesting that a macrophage is required for Leydig cell differentiation is not discussed. Also, the rationale for the proposal is not well-supported. Using bone marrow stem cells to treat infertility would be great, since one then could use autologous cells, but: 1) if infertility is due to a genetic defect, using autologous cells is not going to work; and 2) if the defect is environmental, placing stem cells cells back into bad environment is not going to work. Finally, there are ethical issues with manipulating the testes genetically that the applicant does not discuss.
Conflicts: 

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