Amidi Fardin, Ataie Nejad Nahid, Agha Hoseini Marziyeh, Nayernia Karim, Mazaheri Zohreh, Yamini Nazila, Saeednia Sara
Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran.
Department of Obstetrics and Gynecology, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran.
In Vitro Cell Dev Biol Anim. 2015 Nov;51(10):1093-101. doi: 10.1007/s11626-015-9929-4. Epub 2015 Oct 1.
Human umbilical Wharton's jelly-derived mesenchymal stem cells (HWJMSCs) are the best candidate to get plentiful stem cells and differentiate them to germ cells under appropriate conditions to treat infertility. We sought to determine under which conditions HWJMSCs could form male germ cells in vitro. So, HWJMSCs were differentiated to male germ cells under a mixture of bone morphogenetic protein-4 (BMP-4) and testicular and placental culture condition (TCC and PCC) medium followed by retinoic acid for 21 d. In the present study, the HWJMSCs were obtained from Wharton's jelly of umbilical cords of male neonates delivered by cesarean section. At the third passage, mesenchymal stem cell markers and differentiation to osteocytes and adipocytes were investigated. Then, HWJMSCs were induced to differentiate into male germ cells in the presence of BMP-4, all-trans retinoic acid, PCC, and TCC for 21 d. The profile of c-Kit, DDX4, Piwil2, and Dazl gene expression was evaluated by qPCR and ICC. Data was analyzed by ANOVA test. After 3 wk of treatment with different reagents, the morphology of these spindle-like cells changed to shiny clusters and germ cell-specific markers in mRNA were upregulated in both TCC + retinoic acid (RA) and BMP-4 + RA. Induction of HWJMSCs with TCC in the presence of RA resulted in significant upregulation (P ≤ 0.05) of all germ cell-specific genes (c-Kit 2.6795 ± 0.75, DDX4 4.3188 ± 1.18, Piwil2 4.9962 ± 1.55, Dazl 6.1199 ± 0.78) compared to control and PCC + RA. Our results indicated that TCC and RA are involved in human germ cell development. Moreover, BMP signaling also induced differentiation. Our findings provide a novel effective approach for generation of germ cells in vitro and studying the interaction of germ cells with their niche. Our work represents an essential step toward gaining knowledge of the molecular properties of HWJMSCs in the field of cell therapy. We demonstrated that under a suitable situation, HWJMSCs have the ability to differentiate into germ cells and this provides an excellent pattern to study infertility cause and cure.
人脐带华通氏胶间充质干细胞（HWJMSCs）是获取大量干细胞并在适当条件下将其分化为生殖细胞以治疗不孕症的最佳选择。我们试图确定HWJMSCs在哪些条件下可以在体外形成雄性生殖细胞。因此，将HWJMSCs在骨形态发生蛋白-4（BMP-4）与睾丸和胎盘培养条件（TCC和PCC）培养基的混合物中分化为雄性生殖细胞，随后用视黄酸处理21天。在本研究中，HWJMSCs取自剖宫产分娩的男性新生儿脐带的华通氏胶。在第三代时，研究了间充质干细胞标志物以及向骨细胞和脂肪细胞的分化情况。然后，在BMP-4、全反式视黄酸、PCC和TCC存在的情况下，将HWJMSCs诱导分化为雄性生殖细胞21天。通过qPCR和ICC评估c-Kit、DDX4、Piwil2和Dazl基因表达谱。数据采用方差分析进行分析。用不同试剂处理3周后，这些纺锤状细胞的形态变为有光泽的细胞簇，并且在TCC + 视黄酸（RA）和BMP-4 + RA中，mRNA中的生殖细胞特异性标志物均上调。在RA存在的情况下用TCC诱导HWJMSCs导致所有生殖细胞特异性基因（c-Kit 2.6795±0.75，DDX4 4.3188±1.18，Piwil2 4.9962±1.55，Dazl 6.1199±0.78）与对照组和PCC + RA相比显著上调（P≤0.05）。我们的结果表明，TCC和RA参与人类生殖细胞发育。此外，BMP信号传导也诱导分化。我们的研究结果为体外生成生殖细胞以及研究生殖细胞与其微环境的相互作用提供了一种新的有效方法。我们的工作是在细胞治疗领域了解HWJMSCs分子特性的重要一步。我们证明，在合适的条件下，HWJMSCs有能力分化为生殖细胞，这为研究不孕症的病因及治疗提供了一个极好的模式。
