Model of the Human Testis for Reproductive Toxicology

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DESCRIPTION (provided by applicant): More than 87,000 chemicals have been developed and distributed over the past 50 years. The vast majority of these have not been tested for potential toxic effects in humans or animals. Most systematic toxicology research is conducted in animals and is very expensive. Although animal studies contribute important information, they may not closely approximate the exposure or the exposure effects in humans. There is an unmet need for cost-effective bioassay systems that mimic and assess human exposures and their reproductive effects for systematic, prospective investigation of potentially toxic substances. The overall goal of the research is to develop a functional in vitro human cell-based testis model of spermatogenesis as a clinically relevant product for reproductive toxicology. Development of the first generation product is enabled by our capability to isolate and propagate primary Sertoli cells from adult human donors (hSertCs) and to manipulate primordial germ cells (PGCs), human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPSCs) in vitro to produce haploid cells. The hSertCs have been available to researchers worldwide through a partnership with Lonza (Walkersville, MD) since May 2009, and have been used to create a model of the blood-testis barrier (BTB) in coated inserts of transwell plates. By silencing and overexpressing genes that encode germ-cell-specific cytoplasmic RNA-binding proteins, we have been able to modulate human germ-cell formation and developmental progression, to promote later stages of meiosis and development of haploid gametes. In Phase I, we aim to establish a model using primary human Sertoli, Leydig, and peritubular myoid cells to form a functional (BTB) in a closed hollow fiber system mimicking physiologic shear stress conditions, and to perform analytical and toxicological studies of the cells. There are three Specific Aims for Phase I. 1. Develop an in vitro human Sertoli cell-based, 3-dimensional (3-D) model of the testis. 2. Develop methods to detect and quantify the cells within the testis model. 3. Demonstrate the effect of reproductive toxins on the viability of human testicular cells. In Phase II, we intend to drive the differentiation of induced pluripotent stem cells (iPSCs) and spermatogonial stem cells (SSCs) to the haploid state within the model, and test the effect of toxins on this process. From this effort, we expect to identify reducible, reliable, and relevant endpoints for reproductive toxicant testing, and then to establish outcome markers and assays enabling for F.D.A. approval of the model in conjunction with other toxicology testing required for all New Drug Applications. The need for more relevant and improved reproductive toxicology testing represents a significant commercial opportunity, especially in the pharmaceutical industry. The bioassay product would be provided to industry on a fee-for-service basis initially. This effort potentially could lead to the capability to replicate normal human spermatogenesis in vitro and generate de novo mature sperm for severely infertile, azoospermic men, including cancer survivors. PUBLIC HEALTH RELEVANCE: More than 87,000 chemicals have been developed and distributed discarded over the past 50 years. The vast majority of these have not been tested for potential toxic effects in humans or animals. There is an unmet need for more cost-effective bioassay systems that will mimic and assess human exposures and their reproductive effects for systematic, prospective investigation into potentially toxic substances. The overall goal of the research is to develop a functional in vitro human cell-based testis model of spermatogenesis as a clinically relevant product for reproductive toxicology. Better identification of reproductive toxins could help protect developing fetuses from unwanted toxic exposures, reduce the incidence of male infertility, potentially lower rates of testis cancer, and create safer drugs for the marketplace. Also, part of our interest in developing a testis model is develop the capability to replicate normal human spermatogenesis in vitro to potentially generate de novo mature sperm for severely infertile, azoospermic men, including cancer survivors.