Model of the Human Testis for Reproductive Toxicology

用于生殖毒理学的人类睾丸模型

• 批准号: 8201518
• 负责人: Constance M John
• 金额: $ 25万
• 依托单位: MANDALMED, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-18 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8201518
• 关键词: 3-Dimensional; Adult; Animals; Aromatic Polycyclic Hydrocarbons; Asbestos; Asthma; Automobile Driving; BAX gene; Biological Assay; Blood-Testis Barrier; Cadmium; Cancer Survivor; Cells; Chemical Exposure; Chemicals; Clinical Trials; Data; Development; Drug Industry; Environment; Environmental Exposure; Fee-for-Service Plans; Fertility; Fertility Study; Fetus; Fiber; General Population; Generations; Genes; Germ Cells; Goals; Growth; Haploid Cells; Haploidy; Human; Human Experimentation; In Vitro; Incidence; Industry; Infertility; Investigation; Lead; Male Infertility; Malignant Neoplasms; Malignant neoplasm of testis; Marketing; Mediating; Meiosis; Mercury; Methods; Microfluidics; Modeling; Outcome; Overdose; Pathway interactions; Pesticides; Pharmaceutical Preparations; Phase; Physiological; Poison; Process; RNA-Binding Proteins; Reference Standards; Reporting; Reproduction; Research; Research Personnel; Seminal fluid; Spermatogenesis; Staging; Stem cells; Structure of primordial sex cell; System; Temperature; Testing; Testis; Toxic effect; Toxicant exposure; Toxicity Tests; Toxicology; Toxin; Vinyl Chloride; aromatic hydrocarbon receptor; base; bisphenol A; cell type; clinically relevant; cost; cost effective; developmental disease; drug development; exposed human population; human disease; human embryonic stem cell; improved; in vitro Model; in vivo; induced pluripotent stem cell; interest; lead exposure; leydig interstitial cell; male; men; novel; overexpression; phase 2 study; polycarbonate plastic; prospective; reproductive; sertoli cell; shear stress; sperm cell; toxicant

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.

描述（由申请人提供）：在过去的50年里，已经开发和销售了87，000多种化学品。其中绝大多数尚未在人类或动物中测试潜在的毒性作用。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。尽管动物研究提供了重要信息，但它们可能与人类的接触或接触效应并不十分接近。有一个未满足的需要，具有成本效益的生物测定系统，模拟和评估人类接触及其生殖影响的系统，前瞻性调查潜在的有毒物质。本研究的总体目标是开发一种功能性的体外人睾丸细胞精子发生模型，作为生殖毒理学的临床相关产品。第一代产品的开发是通过我们从成年人供体（hSertCs）分离和繁殖原代支持细胞以及在体外操纵原始生殖细胞（PGCs）、人胚胎干细胞（hESC）和诱导多能干细胞（iPSC）以产生单倍体细胞的能力实现的。自2009年5月以来，hSertCs已通过与Lonza（Walkersville，MD）的合作关系提供给全球研究人员，并已用于在transwell板的涂层插入物中创建血-睾丸屏障（BTB）模型。通过沉默和过表达编码生殖细胞特异性细胞质RNA结合蛋白的基因，我们已经能够调节人类生殖细胞的形成和发育进程，促进减数分裂的后期阶段和单倍体配子的发育。在第一阶段，我们的目标是建立一个模型，使用原代人Sertoli，Leydig，和管周肌样细胞形成一个功能（BTB）在一个封闭的中空纤维系统模拟生理剪切应力条件，并进行分析和毒理学研究的细胞。第一阶段有三个具体目标。 1.建立一个体外人睾丸支持细胞三维模型。2.开发检测和定量睾丸模型中细胞的方法。3.证明生殖毒素对人类睾丸细胞活力的影响。在第二阶段，我们打算在模型中驱动诱导多能干细胞（iPSC）和精原干细胞（SSC）分化为单倍体状态，并测试毒素对这一过程的影响。通过这一努力，我们希望确定生殖毒性测试的可简化、可靠和相关的终点，然后建立结果标志物和检测方法，使FDA能够批准该模型以及所有新药申请所需的其他毒理学测试。对更相关和改进的生殖毒理学测试的需求代表了一个重要的商业机会，特别是在制药行业。生物测定产品最初将在收费服务的基础上提供给工业界。这一努力可能导致在体外复制正常人类精子发生的能力，并为严重不育的无精子症男性（包括癌症幸存者）重新生成成熟精子。 公共卫生相关性：在过去的50年里，已经开发和分发了超过87，000种化学品。其中绝大多数尚未在人类或动物中测试潜在的毒性作用。有一个未得到满足的需要，更符合成本效益的生物测定系统，将模拟和评估人类接触及其生殖影响的系统，前瞻性调查潜在的有毒物质。本研究的总体目标是开发一种功能性的体外人睾丸细胞精子发生模型，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毒性暴露，降低男性不育症的发生率，潜在地降低睾丸癌的发病率，并为市场创造更安全的药物。此外，我们对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，以潜在地为严重不育的无精子症男性（包括癌症幸存者）重新生成成熟精子。