Naeemi Sahar, Sabetkish Shabnam, Kiani Mohammad Javad, Dehghan Amin, Kajbafzadeh Abdol-Mohammad
Pediatric Urology and Regenerative Medicine Research Center, Section of Tissue Engineering and Stem Cells Therapy, Children's Hospital Medical Center, Tehran University of Medical Sciences, Tehran, Iran.
School of Mechanical Engineering, Iran University of Science and Technology, Tehran, Iran.
Cell Tissue Bank. 2023 Mar;24(1):153-166. doi: 10.1007/s10561-022-10024-6. Epub 2022 Jul 6.
This study was designed to provide both ex-vivo and in-vivo methods for the extraction and expansion of spermatogonial stem cells (SSCs).
For in-vivo experiments, azoospermic mouse model was performed with Busulfan. Isolation, culture, and characterization of neonate mouse SSC were also achieved. We performed an in-vivo injection of labeled SSCs to the testes with azoospermia. In ex-vivo experiments, extracted SSCs were seeded on the fabricated scaffold consisting of hyaluronic acid (HA) and decellularized testis tissues (DTT). Immunofluorescence staining with PLZF, TP1, and Tekt 1 was performed for SSCs differentiation and proliferation.
Several studies demonstrated efficient spermatogenic arrest in seminiferous tubules and proved the absence of spermatogenesis. Transplanted SSCs moved and settled in the basement covering the seminiferous tubules. Most of the cells were positive for Dil, after 4 weeks. An epithelium containing spermatogonia-like cells with Sertoli-like, and Leydig cells were evident in the seminiferous tubules of biopsies, and the IHC staining was significantly positive, 4 weeks after injection of SSCs. The results of the ex-vivo experiments showed positive staining for all markers, which was significantly enhanced in scaffolds of ex-vivo experiments compared with in-vitro seeded scaffolds.
Ex-vivo SSC differentiation and proliferation using cell-seeded microfluidic testis scaffolds maybe effective for treatment of the azoospermia.
本研究旨在提供体外和体内提取及扩增精原干细胞（SSCs）的方法。
体内实验采用白消安建立无精子症小鼠模型。同时实现了新生小鼠SSCs的分离、培养和鉴定。我们将标记的SSCs体内注射到无精子症小鼠的睾丸中。体外实验中，将提取的SSCs接种到由透明质酸（HA）和去细胞睾丸组织（DTT）制成的支架上。用PLZF、TP1和Tekt 1进行免疫荧光染色，以检测SSCs的分化和增殖情况。
多项研究表明曲细精管内精子发生有效停滞，并证实无精子生成。移植的SSCs移动并定居在覆盖曲细精管的基底膜上。4周后，大多数细胞Dil呈阳性。注射SSCs 4周后，活检曲细精管中可见含有类精原细胞样细胞、支持细胞样细胞和间质细胞的上皮，免疫组化染色呈显著阳性。体外实验结果显示，所有标志物染色均呈阳性，与体外接种支架相比，体外实验支架中的阳性染色显著增强。
使用接种细胞的微流控睾丸支架进行体外SSCs分化和增殖可能对无精子症的治疗有效。
