Model of the Human Testis for Reproductive Toxicology

描述(由申请人提供)：在过去的50年里，已经开发和分发了超过87,000种化学品。其中绝大多数还没有在人类或动物身上进行潜在毒性影响的测试。大多数系统的毒理学研究都是在动物身上进行的，而且非常昂贵。虽然动物研究提供了重要的信息，但它们可能不太接近接触或接触对人类的影响。对模拟和评估人类暴露及其生殖影响的成本效益生物测定系统的需求尚未得到满足，以便对潜在有毒物质进行系统的、前瞻性的调查。这项研究的总体目标是开发一种基于人类细胞的体外精子发生的功能性模型，作为生殖毒理学的临床相关产品。第一代产品的开发是因为我们能够分离和繁殖成人人类捐赠者(HSertCs)的原代Sertoli细胞，并在体外操纵原始生殖细胞(PGCs)、人类胚胎干细胞(HESCs)和诱导的多能干细胞(IPSCs)产生单倍体细胞。自2009年5月以来，通过与Lonza(马里兰州沃克斯维尔)的合作，hSERTC已向世界各地的研究人员提供，并已被用于在Transwell钢板涂层嵌入物中创建血-睾丸屏障(BTB)的模型。通过沉默和过度表达编码生殖细胞特异的细胞质RNA结合蛋白的基因，我们已经能够调控人类生殖细胞的形成和发育，促进减数分裂的后期和单倍体配子的发育。在第一阶段，我们的目标是利用原代人类支持细胞、间质细胞和肾小管周围肌样细胞建立模型，在模拟生理剪应力条件的封闭中空纤维系统中形成功能性(BTB)，并对这些细胞进行分析和毒理学研究。第一阶段有三个具体目标：1.建立基于支持细胞的体外睾丸三维模型。2.开发检测和量化睾丸模型内细胞的方法。3.证实生殖毒素对人睾丸细胞活力的影响。在第二阶段，我们打算推动诱导多能干细胞(IPSCs)和精原干细胞(SSCs)在模型中向单倍体分化，并测试毒素对这一过程的影响。通过这项努力，我们希望为生殖毒物测试确定可减少的、可靠的和相关的终点，然后建立结果标志和分析，以便FDA批准该模型，并与所有新药申请所需的其他毒理学测试一起进行。对更相关和更好的生殖毒理学测试的需要是一个重要的商业机会，特别是在制药行业。生物检测产品最初将以按服务收费的方式提供给行业。这一努力可能导致在体外复制正常的人类精子发生，并为包括癌症幸存者在内的严重不育、无精子症男性产生从头开始成熟的精子。 与公共健康相关：在过去的50年里，开发和分发了超过8.7万种被丢弃的化学品。其中绝大多数还没有在人类或动物身上进行潜在毒性影响的测试。对更具成本效益的生物测定系统的需求尚未得到满足，该系统将模拟和评估人类暴露及其生殖影响，以便对潜在有毒物质进行系统的、前瞻性的调查。这项研究的总体目标是开发一种基于人类细胞的体外精子发生的功能性模型，作为生殖毒理学的临床相关产品。更好地识别生殖毒素可以帮助保护发育中的胎儿免受不必要的有毒暴露，降低男性不育的发生率，潜在地降低睾丸癌的发生率，并为市场创造更安全的药物。此外，我们开发睾丸模型的部分兴趣是开发在体外复制正常人类精子发生的能力，以潜在地为包括癌症幸存者在内的严重不育、无精子症男性产生从头开始成熟的精子。

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.