Efficient generation of male germ-like cells derived during co-culturing of adipose-derived mesenchymal stem cells with Sertoli cells under retinoic acid and testosterone induction

被引:21
作者
Luo, Yanxia [1 ]
Xie, Lili [1 ]
Mohsin, Ali [1 ]
Ahmed, Waqas [1 ]
Xu, Chenze [1 ]
Peng, Yan [1 ]
Hang, Haifeng [1 ]
Zhuang, Yingping [1 ]
Chu, Ju [1 ]
Guo, Meijin [1 ]
机构
[1] East China Univ Sci & Technol, State Key Lab Bioreactor Engn, 130 Meilong Rd, Shanghai 200237, Peoples R China
关键词
Adipose-derived mesenchymal stem cells; Germ cell; Sertoli cell; Signaling pathways; Retinoic acid; Testosterone; Synergistic effect; SIGNALING PATHWAY; RESTORE SPERMATOGENESIS; SELF-RENEWAL; MUSE CELLS; DIFFERENTIATION; TESTIS; TISSUE; PROLIFERATION; EXPRESSION; PROMOTES;
D O I
10.1186/s13287-019-1181-5
中图分类号
Q813 [细胞工程];
学科分类号
摘要
BackgroundAdipose-derived mesenchymal stem cells (ADMSCs) are considered an efficient and important candidate for male infertility treatment because they contain pluripotent stem cells, which can differentiate into all cells from three germ layers. However, the efficient generation of male germ-like cell (MGLCs) is one of the key issues, and little is known about the mechanisms underlying generation of MGLCs. Herein, we attempt to improve the efficient generation of MGLCs derived during co-culturing of rat ADMSCs with SCs under retinoic acid (RA) and testosterone (T) treatment.MethodsADMSCs isolated from male SD rat were induced into generation of MGLCs by using respective methods in vitro. Transwell insert system was used for co-culturing. Busulfan-induced non-obstructive azoospermia rat mode was used to evaluate spermatogenic recovery ability of treated ADMSCs. Besides, the relative gene expression level was detected by reverse transcription PCR, quantitative RT-PCR. The relative protein expression level was detected by western blot (WB) and immunostaining analysis.ResultsThe results showed that ADMSCs co-cultured with TM4 cells under RA and T induction enhanced the formation of bigger and tightly packed MGLCs feature colonies in vitro. Moreover, the expression of male germ cell-related markers (Oct4, Stella, Ddx4, Dazl, PGP9.5, Stra8, and ITG6) is significantly upregulated in TM4 cell-co-cultured ADMSCs in vitro and in busulfan-treated rat testis after injecting TM4 cell-treated ADMSCs for 2months. Comparatively, the ADMSCs treated by TM4 cell with RA and T exhibited the highest expression of male germ cell-related markers. RA- and T-treated TM4 cell showed fewer dead cells and higher cytokine secretion than untreated groups. The protein expression level of TGF-SMAD2/3, JAK2-STAT3, and AKT pathways in ADMSCs co-cultured with TM4 cells under RA and T was higher than others. Whereas, downregulation of male germ cell-related marker expression subsequently inhibited the phosphorylation of SMAD2/3, JAK2, STAT3, and AKT.ConclusionThese results suggested that TM4 cells could efficiently stimulate in vitro generation of MGLCs during co-culturing of ADMSCs under RA and T treatment. Conclusively, the ADMSCs co-cultured with TM4 cell under RA and T induction stimulate the efficient generation of MGLCs in vitro through activating TGF-SMAD2/3, JAK2-STAT3, and AKT pathways. Among them, JAK2-STAT3 and AKT pathways are being first reported to show involvement of in vitro generation of MGLCs during ADMSC co-culturing with SCs.
引用
收藏
页数:18
相关论文
共 68 条
[1]   Underlying Mechanisms that Restore Spermatogenesis on Transplanting Healthy Niche Cells in Busulphan Treated Mouse Testis [J].
Anand, Sandhya ;
Bhartiya, Deepa ;
Sriraman, Kalpana ;
Mallick, Alpna .
STEM CELL REVIEWS AND REPORTS, 2016, 12 (06) :682-697
[2]   Chemoablated mouse seminiferous tubular cells enriched for very small embryonic-like stem cells undergo spontaneous spermatogenesis in vitro [J].
Anand, Sandhya ;
Patel, Hiren ;
Bhartiya, Deepa .
REPRODUCTIVE BIOLOGY AND ENDOCRINOLOGY, 2015, 13
[3]   Endogenous, very small embryonic-like stem cells: critical review, therapeutic potential and a look ahead [J].
Bhartiya, Deepa ;
Shaikh, Ambreen ;
Anand, Sandhya ;
Patel, Hiren ;
Kapoor, Sona ;
Sriraman, Kalpana ;
Parte, Seema ;
Unni, Sreepoorna .
HUMAN REPRODUCTION UPDATE, 2017, 23 (01) :41-76
[4]   SPERMATOGENESIS FOLLOWING MALE GERM-CELL TRANSPLANTATION [J].
BRINSTER, RL ;
ZIMMERMANN, JW .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1994, 91 (24) :11298-11302
[5]  
Chen J, 2013, TISSUE ENG PART B-RE, V19, P516, DOI [10.1089/ten.TEB.2012.0672, 10.1089/ten.teb.2012.0672]
[6]   Infertility in men: Recent advances and continuing controversies [J].
De Kretser, DM ;
Baker, HWG .
JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM, 1999, 84 (10) :3443-3450
[7]   Muse Cells Provide the Pluripotency of Mesenchymal Stem Cells: Direct Contribution of Muse Cells to Tissue Regeneration [J].
Dezawa, Mari .
CELL TRANSPLANTATION, 2016, 25 (05) :849-861
[8]   Differentiation of human male germ cells from Wharton's jelly-derived mesenchymal stem cells [J].
Dissanayake, D. M. A. B. ;
Patel, H. ;
Wijesinghe, P. S. .
CLINICAL AND EXPERIMENTAL REPRODUCTIVE MEDICINE-CERM, 2018, 45 (02) :75-81
[9]   TGF-β superfamily: how does it regulate testis development [J].
Fan, Yun-Shu ;
Hu, Yan-Jun ;
Yang, Wan-Xi .
MOLECULAR BIOLOGY REPORTS, 2012, 39 (04) :4727-4741
[10]   Human induced pluripotent stem cells and male infertility: an overview of current progress and perspectives [J].
Fang, Fang ;
Li, Zili ;
Zhao, Qian ;
Li, Honggang ;
Xiong, Chengliang .
HUMAN REPRODUCTION, 2018, 33 (02) :188-195