Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Stem Cell and Regenerative Medicine Research Center & Department of Anatomy, Iran University of Medical Sciences, Tehran, Iran.
Acta Histochem. 2020 Dec;122(8):151627. doi: 10.1016/j.acthis.2020.151627. Epub 2020 Sep 28.
Spermatogonial stem cells (SSCs) are very sensitive to chemotherapy and radiotherapy, so male infertility is a great challenge for prepubertal cancer survivors. Cryoconservation of testicular cells before cancer treatment can preserve SSCs from treatment side effects. Different two-dimensional (2D) and three-dimensional (3D) culture systems of SSCs have been used in many species as a useful technique to in vitro spermatogenesis. We evaluated the proliferation of SSCs in 2D and 3D culture systems of platelet-rich plasma (PRP). testicular cells of four brain-dead patients cultivated in 2D pre-culture system, characterization of SSCs performed by RT-PCR, flow cytometry, immunocytochemistry and their functionality assessed by xenotransplantation to azoospermia mice. PRP prepared and dosimetry carried out to determine the optimized dose of PRP. After preparation of PRP scaffold, cytotoxic and histological evaluation performed and SSCs cultivated into three groups: control, 2D culture by optimized dose of PRP and PRP scaffold. The diameter and number of colonies measured and relative expression of GFRa1 and c-KIT evaluated by real-time PCR. Results indicated the expression of PLZF, VASA, OCT4, GFRa1 and vimentin in colonies after 2D pre-culture, xenotransplantation demonstrated proliferated SSCs have proper functionality to homing in mouse testes. The relative expression of c-KIT showed a significant increase as compared to the control group (: p < 0.05) in PRP- 2D group, expression of GFRa1 and c-KIT in PRP scaffold group revealed a significant increase as compared to other groups (**: p < 0.001). The number and diameter of colonies in the PRP-2D group showed a considerable increase (p < 0.01) as compared to the control group. In PRP- scaffold group, a significant increase (p < 0.01) was seen only in the number of colonies related to the control group. Our results suggested that PRP scaffold can reconstruct a suitable structure to the in vitro proliferation of SSCs.
精原干细胞（SSC）对化疗和放疗非常敏感，因此青春期前癌症幸存者的男性不育是一个巨大的挑战。在癌症治疗前冷冻保存睾丸细胞可以防止 SSC 受到治疗的副作用。已经在许多物种中使用不同的二维（2D）和三维（3D）培养系统来作为体外精子发生的有用技术。我们评估了富含血小板的血浆（PRP）的 2D 和 3D 培养系统中 SSC 的增殖。在 2D 预培养系统中培养了来自 4 位脑死亡患者的睾丸细胞，通过 RT-PCR、流式细胞术、免疫细胞化学进行 SSC 特征分析，并通过异种移植到无精子症小鼠中来评估其功能。制备 PRP 并进行剂量测定以确定 PRP 的最佳剂量。制备 PRP 支架后，进行细胞毒性和组织学评估，并将 SSC 培养到三组中：对照组、通过优化剂量的 PRP 进行的 2D 培养和 PRP 支架。测量了菌落的直径和数量，并通过实时 PCR 评估 GFRa1 和 c-KIT 的相对表达。结果表明，在 2D 预培养后，集落中表达了 PLZF、VASA、OCT4、GFRa1 和波形蛋白，异种移植证明增殖的 SSC 具有归巢到小鼠睾丸中的适当功能。与对照组相比，PRP-2D 组中 c-KIT 的相对表达显着增加（：p <0.05），PRP 支架组中 GFRa1 和 c-KIT 的表达与其他组相比显着增加（**：p <0.001）。与对照组相比，PRP-2D 组的菌落数量和直径显着增加（p <0.01）。在 PRP 支架组中，仅与对照组相比，菌落数量显着增加（p <0.01）。我们的结果表明，PRP 支架可以为 SSC 的体外增殖重建合适的结构。
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