Development of Novel Testicular Tissue Organ Culture Systems to Induce in vitro Spermatogenesis

开发新型睾丸组织器官培养系统来诱导体外精子发生

• 批准号: 10254274
• 负责人: Kien T.D. Tran
• 金额: $ 3.96万
• 依托单位: MAGEE-WOMEN'S RES INST AND FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10254274
• 关键词: Adult; Autologous; Back; Biological; Cells; Chemotherapy and/or radiation; Child; Clinic; Cryopreservation; Cryopreserved Tissue; Data Set; Development; Devices; Engineering; Failure; Fertility; Fertilization; Freezing; Fresh Tissue; Future; Gases; Gel; Genes; Germ Cells; Goals; Haploidy; Home; Human; In Vitro; Individual; Infertility; Knowledge; Laboratories; Liquid substance; Macaca mulatta; Maintenance; Male Infertility; Malignant Neoplasms; Medical; Meiosis; Membrane; Methods; Microfluidic Microchips; Microfluidics; Monitor; Monkeys; Mus; Natural regeneration; Necrosis; Neonatal; Organ Culture Techniques; Patients; Phenotype; Pilot Projects; Primates; Process; Production; Puberty; Pump; Quality of life; Reporting; Reproducibility; Reproduction; Reproductive Technology; Research; Rhesus; Risk; Rodent; Sampling; Sepharose; Somatic Cell; Spermatids; Spermatocytes; Spermatogenesis; Spermatogonia; System; Technology; Testicular Tissue; Testing; Testis; Tissue Transplantation; Tissue-Specific Gene Expression; Tissues; Translating; Undifferentiated; acrosin; base; boys; cancer cell; cancer therapy; design; differential expression; experimental study; fertility preservation; human tissue; in vivo; male; neonatal mice; next generation; nonhuman primate; novel; offspring; polycarbonate; polydimethylsiloxane; prepuberty; sertoli cell; single-cell RNA sequencing; sperm cell; sperm cryopreservation; sperm function; stem cells; tissue culture

Development of Novel Testicular Tissue Organ Culture Systems to Induce in vitro Spermatogenesis Spermatogonial stem cells (SSCs) have a tremendous capacity to regenerate spermatogenesis under proper conditions. Chemotherapy or radiation treatments for cancers or other conditions can destroy the stem cell pool and cause permanent infertility. The only option to preserve the fertility of prepubertal male patients is testicular tissue cryopreservation. When those tissues are thawed, testicular tissue organ culture is an approach to mature those tissues to produce sperm outside of the body. Takehiko Ogawa’s lab produced mouse sperm and offspring from cultures of fresh neonatal testes that were maintained for several months in a pump-driven microfluidic (MF) device. To overcome the bulkiness of the MF device, the same laboratory invented a pumpless microfluidic (PL) device. However, sperm and offspring have not been produced using the PL devices; spermatogenesis was not reported from cryopreserved tissues in the PL system, and the system has not been tested in any other species. This study aims to fill the gaps in knowledge by generating mouse sperm and offspring using the PL devices in cultures of fresh and cryopreserved testes, and to translate this technology to non-human primate and/or human testicular tissues. In addition, to overcome several pitfalls in the PL design, I developed and tested a novel PDMS-roof transwell (PRT) culture system, comprised of a polycarbonate-membrane-transwell and a polydimethylsiloxane (PDMS) roof, to ease device production and simplify sample loading and culture maintenance. The efficiency of in vitro spermatogenesis using the PRT device was similar to the PL device in cultures of fresh neonatal mouse testes (>70% tubules showed Acrosin- GFP+ post-meiotic cells within 1 month in culture). In the PRT system, I confirmed VASA-positive germ cells in 81.6±6.63%, SALL4-positive undifferentiated spermatogonia in 70.1±10.21%, STRA8-positive differentiating spermatogonia in 50.0±14.01%, SYCP3-positive spermatocytes in 75.9±9.2%, and SOX9-positive Sertoli cells in 99.08±0.9% of tubules. A similar experiment using cryopreserved neonatal mouse testes in both culture systems is underway. In a pilot study on immature rhesus macaque testicular tissues using the PRT system, I observed VASA-positive germ cell retention in 90.3% of tubules after 3 months in culture. More detailed analyses are in process. Unlike mouse, monkeys and humans have a prolonged prepubertal period before the initiation of spermatogenesis and there is limited information about the mechanisms that regulate prepubertal to pubertal to adult developmental transitions in testicular somatic cells or germ cells. I will exploit our unique pipeline of healthy rhesus and human testicular tissues from prepubertal, pubertal and adult individuals and perform high-throughput single-cell RNA sequencing to identify a subset of genes with differential expression at these developmental stages. This will provide a novel dataset that will help guide our efforts to develop a simplified, efficient ex vivo testicular tissue organ culture system and translate it toward the human clinic.

睾丸组织器官体外培养体系的建立及其诱导精子发生的研究 精原干细胞（spermatogonialstemcells，SSCs）在适当的培养条件下具有巨大的再生精子的能力。 条件癌症或其他疾病的化疗或放射治疗会破坏干细胞 导致永久性不孕保持青春期前男性患者生育能力的唯一选择是 睾丸组织冷冻保存当这些组织解冻时，睾丸组织器官培养是一种 使这些组织成熟，在体外产生精子。小川武彦的实验室 小鼠精子和来自新鲜新生睾丸培养物的后代在一个 泵驱动的微流体（MF）装置。为了克服MF设备的笨重，同一实验室 发明了无泵微流体（PL）装置。然而，精子和后代还没有产生使用 PL器械;未报告PL系统中冻存组织的精子发生，并且该系统 尚未在其他物种中进行过测试。本研究的目的是填补知识的空白，通过生成鼠标 在新鲜和冷冻保存的睾丸培养物中使用PL装置的精子和后代，并将其翻译成 技术应用于非人类灵长类动物和/或人类睾丸组织。此外，为了克服几个陷阱， PL设计，我开发和测试了一种新的PDMS屋顶transwell（PRT）培养系统，包括一个 聚碳酸酯膜-transwell和聚二甲基硅氧烷（PDMS）顶盖，以简化装置生产， 简化样品装载和培养物维护。PRT体外精子发生效率的研究 装置在新鲜新生小鼠睾丸培养物中与PL装置相似（>70%的小管显示顶体酶， 培养物中1个月内的GFP+减数分裂后细胞）。在PRT系统中，我证实了VASA阳性生殖细胞在 81.6± 6.63%，SALL 4阳性未分化精原细胞70.1± 10.21%，STRA 8阳性分化精原细胞70.1± 10.21%，SALL 4阳性未分化精原细胞81.6± 6.63%，SALL 4阳性未分化精原细胞70.1 ± 10.21%，STRA 8阳性未分化精原细胞70.1 ± 10.21 精原细胞占50.0± 14.01%，SYCP 3阳性精母细胞占75.9± 9.2%，SOX 9阳性支持细胞 在99.08±0.9%的小管中。在两种培养物中使用冷冻保存的新生小鼠睾丸进行类似的实验 系统正在进行中。在使用PRT系统对未成熟恒河猴睾丸组织进行的初步研究中， 在培养3个月后观察到90.3%的小管中存在VASA阳性生殖细胞。更详细 分析正在进行中。与小鼠不同，猴子和人类在发育之前有一个较长的青春期前期。 启动精子发生和有有限的信息机制，调节青春期前 睾丸体细胞或生殖细胞从青春期到成年期的发育转变。我会利用我们独特的 来自青春期前、青春期和成年个体的健康恒河猴和人睾丸组织的管道， 进行高通量单细胞RNA测序以鉴定具有差异表达的基因子集 在这些发展阶段。这将提供一个新的数据集，将有助于指导我们的努力， 简化，有效的离体睾丸组织器官培养系统，并将其转化为人类临床。