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

体外诱导精子发生的新型睾丸组织器官培养体系的建立 精原干细胞(SSCs)在适当的条件下具有巨大的再生生精能力。 条件。癌症或其他疾病的化疗或放射治疗会破坏干细胞。 并导致永久性不孕症。保留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 使这些组织成熟以在体外产生精子的方法。小川武彦的实验室生产 小鼠精子和来自新鲜新生儿睾丸培养物的后代在 泵驱动微流控(MF)装置。为了克服MF设备的笨重，同一实验室 发明了一种无泵微流控(PL)装置。然而，精子和后代并不是使用 PL装置；在PL系统中冷冻保存的组织中没有精子发生的报道，并且该系统 还没有在任何其他物种上进行过测试。这项研究旨在通过产生老鼠来填补知识空白 在新鲜和冷冻保存的睾丸培养中使用PL装置的精子和后代，并翻译 技术应用于非人类灵长类和/或人类睾丸组织。此外，要克服以下几个缺陷 在PL设计中，我开发并测试了一种新型的PDMS-屋顶Transwell(PRT)培养系统，该系统包括 聚碳酸酯-膜-转井和聚二甲基硅氧烷(PDMS)屋顶，以方便设备生产和 简化样本装载和培养维护。PRT体外生精效率的研究 在新鲜的新生小鼠睾丸培养中，该装置与PL装置相似(&gt；70%的小管显示顶体酶- GFP+减数分裂后细胞培养1个月内)。在PRT系统中，我确认了VASA阳性生殖细胞 81.6±6.63%，SALL4阳性未分化精原细胞占70.1±10.21%，STRA8阳性分化 精原细胞占50.0±14.01%，SYCP3阳性精母细胞占75.9±9.2%，支持细胞呈SOX9阳性 在99.08±0.9%的肾小管中。在两种培养中使用冷冻保存的新生小鼠睾丸的类似实验 系统正在进行中。在使用PRT系统对未成熟的恒河猴睾丸组织进行的初步研究中，我 培养3个月后，90.3%的肾小管有VASA阳性生殖细胞滞留。更详细 分析正在进行中。与老鼠不同，猴子和人类在青春期前有较长的青春期。 精子发生的启动，关于调节青春期前的机制的信息有限 从青春期到成年的睾丸体细胞或生殖细胞的发育转变。我将利用我们独一无二的 来自青春期前、青春期和成年个体的健康恒河猴和人类睾丸组织的管道 进行高通量单细胞RNA测序以确定差异表达基因的子集 在这些发育阶段。这将提供一个新的数据集，有助于指导我们开发 简化、高效的体外睾丸组织器官培养系统，并将其移植到人类临床。

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.