Akyash Fatemeh, Aflatoonian Reza, Farashahi-Yazd Ehsan, Hajizadeh-Tafti Fatemeh, Golzadeh Jalal, Tahajjodi Somayyeh Sadat, Aflatoonian Behrouz
Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Cell J. 2024 Aug 11;26(6):370-379. doi: 10.22074/cellj.2024.2012768.1419.
There are ethical and technical challenges in studying human germ cell development. Therefore, the aim of the study is differentiation of human embryonic stem cells (hESCs), as pluripotent cells, to the germ cells which is a valuable tool for studying molecular and cellular aspects of gametogenesis and understanding causes of infertility.
In this experimental study, two different complete media [Dulbecco's Modified Eagle Medium (DMEM)+20% fetal bovine serum (FBS) and embryoid bodies (EBs) medium; KOSR/HES without basic fibroblast growth factor (bFGF)] were used in the both of test groups using testicular cells derived conditioned medium (TCCM) and control groups spontaneously differentiated (SD). Thereby, EBs from hESCs (Yazd2; 46XY) were cultured in different conditions EB medium; EB medium and conditioned EB medium; EB medium, DMEM, and FBS without conditioning; EB medium, conditioned DMEM, and FBS medium. EBs were collected after 4, 7, and 14 days and their gene expression profiles were assessed and compared to hESCs, as day 0, using IF and relative reverse transcription quantitative polymerase chain reaction (RT-qPCR).
An increase in the gametogenesis gene expression level in TCCM groups was showed in comparison with SD groups. Additionally, immunostaining of differentiated cells in all groups showed gametogenesis (IVG).
Our findings showed that human TCCM could be used as a natural niche for male and female germ cell development. However, further studies are needed to define the factors and metabolites within the human TCCM.
研究人类生殖细胞发育存在伦理和技术挑战。因此，本研究的目的是将人类胚胎干细胞（hESCs）这种多能细胞分化为生殖细胞，这是研究配子发生的分子和细胞层面以及理解不孕原因的宝贵工具。
在本实验研究中，两个不同的完全培养基[杜氏改良 Eagle 培养基（DMEM）+20%胎牛血清（FBS）和胚状体（EBs）培养基；不含碱性成纤维细胞生长因子（bFGF）的 KnockOut 血清替代物/人胚胎干细胞培养基（KOSR/HES）]用于两个测试组，测试组使用睾丸细胞衍生条件培养基（TCCM），对照组自发分化（SD）。由此，将 hESCs（Yazd2；46XY）来源的 EBs 在不同条件下培养：EB 培养基；EB 培养基和条件 EB 培养基；未条件化的 EB 培养基、DMEM 和 FBS；EB 培养基、条件化 DMEM 和 FBS 培养基。在第 4、7 和 14 天收集 EBs，并使用免疫荧光（IF）和相对逆转录定量聚合酶链反应（RT-qPCR）评估其基因表达谱，并与第 0 天的 hESCs 进行比较。
与 SD 组相比，TCCM 组中配子发生基因表达水平有所增加。此外，所有组中分化细胞的免疫染色均显示有配子发生（IVG）。
我们的研究结果表明，人类 TCCM 可作为男性和女性生殖细胞发育的天然微环境。然而，需要进一步研究来确定人类 TCCM 中的因子和代谢产物。
