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

Abstract

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 ITGα6) is significantly upregulated in TM4 cell-co-cultured ADMSCs in vitro and in busulfan-treated rat testis after injecting TM4 cell-treated ADMSCs for 2 months. 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 TGFβ-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 TGFβ-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.Electronic supplementary materialThe online version of this article (10.1186/s13287-019-1181-5) contains supplementary material, which is available to authorized users.

Differentiation potential of adipose tissue‐derived mesenchymal stem cells into germ cells with and without growth factors

“…Similar studies were also carried out in the following years, and researchers reported the differentiation of adult stem cells into germ cells at different stages (Dissanayake et al, 2018;Salem et al, 2019). Many attempts have been made to improve the efficacy of differentiation and provide a similar niche to germ cells (Luo et al, 2019;Xie et al, 2015). The spermatogenesis happens within the seminiferous tubules of the testis, containing sertoli and germ cells.…”

“…with Sertoli cells and used RA and testosterone for the differentiation of stem cells into germ cells. They observed that spermatogenic markers were overexpressed in the co-cultured AT-MSCs, in the presence of RA and testosterone (Luo et al, 2019). RA is an initiator of meiosis in the gametogenesis of male and female mammalian and up-regulates the early and late meiosis markers during oogenesis and spermatogenesis; RA also is required for cell entry into the meiosis stage (Li & Clagett-Dame, 2009).…”

Genome editing approaches to augment livestock breeding programs

“…Mesenchymal stromal cells (MSCs) can be isolated from a variety of adult and neonatal tissues and can transdifferentiate into multiple cell lineages including PGCLCs. MSCs have been shown to express multiple germ cell markers following treatment with retinoic acid and/ or various growth factors (Dissanayake et al, 2018;Ghasemzadeh-Hasankolaei et al, 2014;Hua et al, 2009;Luo et al, 2019;Shirzeyli et al, 2017;Wei et al, 2016;Yan et al, 2015). Haploid spermatid-like cells have been observed from MSC-derived PGCLC cultures and transplants (Ghasemzadeh-Hasankolaei et al, 2016a,b;Shlush et al, 2017;Zhang et al, 2014).…”

Restoration of spermatogenesis in azoospermic mice by bone marrow mesenchymal stromal/stem cells conditioned medium

“…On the other hand, bone marrow-derived MSCs (BM-MSCs) and adipose tissue-derived MSCs (AT-MSCs) have recently been considered by many researchers. BM-MSCs and AT-MSCs are used to produce male germ cells in vitro (Ghasemzadeh-Hasankolaei et al, 2016;Luo et al, 2019) and in vivo (Karimaghai et al, 2018;Tamadon et al, 2015). Less invasive, less costly, powerful immunomodulatory effects, more proliferative potential, and more secretion of growth factors and cytokines are the most important priorities of AT-MSCs compared to BM-MSCs for cell therapy (Xu et al, 2017).…”