Zahiri Maria, Movahedin Mansoureh, Mowla Seyed Javad, Noruzinia Mehrdad, Koruji Morteza, Nowroozi Mohammad Reza, Asgari Fatemeh
Anatomical Science Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran.
Cell J. 2022 Aug 28;24(8):481-490. doi: 10.22074/cellj.2022.7888.
Objective: Epigenetic and genetic changes have important roles in stem cell achievements. Accordingly, the aim of this
study is the evaluation of the epigenetic and genetic alterations of different culture systems, considering their efficacy in
propagating human spermatogonial stem cells isolated by magnetic-activated cell sorting (MACS).
Materials and Methods: In this experimental study, obstructive azoospermia (OA) patient-derived spermatogonial cells were divided into two groups. The MACS enriched and non-enriched spermatogonial stem cells (SSCs) were cultured in the control and treated groups; co-culture of SSCs with Sertoli cells of men with OA, co-culture of SSCs with healthy Sertoli cells of fertile men, the culture of SSCs on PLA nanofiber and culture of testicular cell suspension. Gene-specific methylation by MSP, expression of pluripotency (NANOG, C-MYC and OCT-4), and germ cells specific genes (Integrin α6, Integrin β1, PLZF) evaluated. Cultured SSCs from the optimized group were transplanted into the recipient azoospermic mouse.
Results: The use of MACS for the purification of human stem cells was effective at about 69% with the culture of the testicular suspension, being the best culture system. Upon purification, the germ-specific gene expression was significantly higher in testicular cell suspension and treated groups (P≤0.05). During the culture time, gene-specific methylation patterns of the examined genes did not show any changes. Our data from transplantation indicated the homing of the donor-derived cells and the presence of human functional sperm.
Conclusion: Our in vivo and in vitro results confirmed that culture of testicular cell suspension and selection of
spermatogonial cells could be effective ways for purification and enrichment of the functional human spermatogonial cells. The epigenetic patterns showed that the specific methylation of the evaluated genes at this stage remained constant with no alteration throughout the entire culture systems over time.
目的：表观遗传和基因变化在干细胞研究成果中具有重要作用。因此，本研究旨在评估不同培养系统的表观遗传和基因改变，并考量其在扩增通过磁激活细胞分选（MACS）分离出的人类精原干细胞方面的效能。
材料与方法：在本实验研究中，将梗阻性无精子症（OA）患者来源的精原细胞分为两组。MACS富集和未富集的精原干细胞（SSCs）分别在对照组和处理组中培养；OA男性的SSCs与支持细胞共培养、与生育力正常男性的健康支持细胞共培养、SSCs在聚乳酸纳米纤维上培养以及睾丸细胞悬液培养。通过甲基化特异性PCR（MSP）检测基因特异性甲基化，评估多能性基因（NANOG、C-MYC和OCT-4）以及生殖细胞特异性基因（整合素α6、整合素β1、PLZF）的表达。将优化组培养的SSCs移植到无精子症受体小鼠体内。
结果：采用MACS纯化人类干细胞，在睾丸悬液培养时效率约为69%，是最佳培养系统。纯化后，睾丸细胞悬液组和处理组中生殖细胞特异性基因表达显著更高（P≤0.05）。在培养期间，所检测基因的基因特异性甲基化模式未显示任何变化。我们的移植数据表明供体来源细胞的归巢以及人类功能性精子的存在。
结论：我们的体内和体外研究结果证实，睾丸细胞悬液培养和精原细胞选择可能是纯化和富集功能性人类精原细胞的有效方法。表观遗传模式显示，在此阶段评估的基因特异性甲基化在整个培养系统中随时间保持恒定，未发生改变。
