Trans differentiating human adipose-derived mesenchymal stem cells into male germlike cells utilizing Rabbit Sertoli cells: An experimental study


Background: Mesenchymal stem cells (MSCs) are deemed as potential new therapeutic agents for infertility treatment and adipose tissue (AT) becomes a potential MSCs source. To direct MSCs through the differentiation process properly, an environment comparable to the in vivo niche might be indispensable.

Objective: This study aims to differentiate human AT-derived MScs (hAD-MScs) into male germ-like cells in vitro using a combination of rabbit Sertoli cells conditioned medium (SCCM), bone morphogenetic protein 4, and retinoic acid.

Materials and Methods: MScs were isolated from human ATs of fertile and infertile donors. The verified MScs were differentiated using a 2-step protocol; the first step included 20 ng/ml bone morphogenetic protein 4 treatment. The second step was performed utilizing 1 μM retinoic acid and/or SCCM. The morphological changes and the expression of germ cell (GC)-specific markers: octamer-binding transcription factor- 4; stimulated by retinoic-acid-8, synaptonemal complex protein-3, and protamine-1 were assessed in the treated cells using quantitative polymerase chain reaction.

Results: Induction of hAD-MScs resulted in the upregulation of GC-specific genes where SCCM treatment showed the highest expression. The synaptonemal complex protein-3 and protamine-1 gene expression was detected after 19 and 26 days of induction, respectively. PRM1 was detected in hAD-MScs cultured in SCCM earlier than in other treated groups. The treated cells became more elongated-like spindles and formed aggregates.

Conclusion: hAD-MScs differentiated to GC lineage exhibited the ability to express GC-specific markers under in vitro conditions, and rabbit’s Sertoli cells can be used for inducing transdifferentiation of hAD-MScs into germ-like cells.

Key words: Adipose tissue-derived mesenchymal stem cell, Bone morphogenetic protein 4, Germ-line cells, Retinoic acid, Sertoli cells.

[1] Ghaem Maghami R, Mirzapour T, Bayrami A. Differentiation of mesenchymal stem cells to germlike cells under induction of Sertoli cell-conditioned medium and retinoic acid. Andrologia 2018; 50: e12887.

[2] Nagamatsu G, Hayashi K. Stem cells, in vitro gametogenesis and male fertility. Reproduction 2017; 154: F79–F91.

[3] Zhankina R, Baghban N, Askarov M, Saipiyeva D, Ibragimov A, Kadirova B, et al. Mesenchymal stromal/stem cells and their exosomes for restoration of spermatogenesis in non-obstructive azoospermia: A systemic review. Stem Cell Res Ther 2021; 12: 229.

[4] Mouka A, Tachdjian G, Dupont J, Drévillon L, Tosca L. In vitro gamete differentiation from pluripotent stem cells as a promising therapy for infertility. Stem Cells Dev 2016; 25: 509–521.

[5] Majidi F, Bamehr H, Shalchian Z, Kouchakian MR, Mohammadzadeh N, Khalili A. Differentiation of human umbilical cord mesenchymal stem cell into germ-like cell under effect of co-culture with testicular cell tissue. Anat Histol Embryol 2020; 49: 359–364.

[6] De Felici M. The formation and migration of primordial germ cells in mouse and man. Molecular mechanisms of cell differentiation in gonad development. Cham: Springer; 2016.

[7] Irie N, Sybirna A, Surani MA. What can stem cell models tell us about human germ cell biology? Curr Top Dev Biol 2018; 129: 25–65.

[8] Madeja ZE, Pawlak P, Piliszek A. Beyond the mouse: Non-rodent animal models for study of early mammalian development and biomedical research. Int J Dev Biol 2019; 63: 187–201.

[9] Banco B, Grilli G, Giudice C, Tomas Marques A, Cotti Cometti S, Visigalli G, et al. Immunophenotyping of rabbit testicular germ and Sertoli cells across maturational stages. J Histochem Cytochem 2016; 64: 715–726.

[10] Grieco V, Banco B. Rabbit sertoli cells: Immunohistochemical profile from neonatal to adult age. Methods Mol Biol 2018; 1748: 37–47.

[11] Ziaeipour S, Ahrabi B, Naserzadeh P, Aliaghaei A, Sajadi E, Abbaszadeh H-A, et al. Effects of Sertoli cell transplantation on spermatogenesis in azoospermic mice. Cell Physiol Biochem 2019; 52: 421–434.

[12] Afsartala Z, Rezvanfar MA, Hodjat M, Tanha S, Assadollahi V, Bijangi K, et al. Amniotic membrane mesenchymal stem cells can differentiate into germ cells in vitro. In Vitro Cell Dev Biol Anim 2016; 52: 1060–1071.

[13] Volarevic V, Bojic S, Nurkovic J, Volarevic A, Ljujic B, Arsenijevic N, et al. Stem cells as new agents for the treatment of infertility: Current and future perspectives and challenges. Biomed Res Int 2014; 2014: 507234.

[14] Choudhery MS, Badowski M, Muise A, Pierce J, Harris DT. Donor age negatively impacts adipose tissue-derived mesenchymal stem cell expansion and differentiation. J Transl Med 2014; 12: 8.

[15] Camilleri ET, Gustafson MP, Dudakovic A, Riester SM, Garces CG, Paradise CR, et al. Identification and validation of multiple cell surface markers of clinical-grade adipose-derived mesenchymal stromal cells as novel release criteria for good manufacturing practice-compliant production. Stem Cell Res Ther 2016; 7: 107.

[16] Liu H, Chen M, Liu L, Ren S, Cheng P, Zhang H. Induction of human adipose-derived mesenchymal stem cells into germ lineage using retinoic acid. Cell Reprogram 2018; 20: 127–134.

[17] Ghatreh Samani K, Eliyasi Dashtaki M, Alaei Sh, Saki Gh. Differentiation potential of adipose tissue-derived mesenchymal stem cells into germ cells with and without growth factors. Andrologia 2021; 53: e13892.

[18] Braunig P, Glanzner WG, Rissi VB, Goncalves PBD. The differentiation potential of adipose tissue-derived mesenchymal stem cells into cell lineage related to male germ cells. Arq Bras Med Vet Zootec 2018; 70: 160–168.

[19] Bakhmet EI, Tomilin AN. Key features of the POU transcription factor Oct4 from an evolutionary perspective. Cell Mol Life Sci 2021; 78: 7339–7353.

[20] Lee W-Y, Lee R, Park H-J, Do JT, Park C, Kim J-H, et al. Characterization of male germ cell markers in canine testis. Anim Reprod Sci 2017; 182: 1–8.

[21] Jodar M, Oliva R. Protamine alterations in human spermatozoa. Genetic damage in human spermatozoa. New York: Springer; 2014.

[22] Liu Y, Niu M, Yao C, Hai Y, Yuan Q, Liu Y, et al. Fractionation of human spermatogenic cells using STA-PUT gravity sedimentation and their miRNA profiling. Sci Rep 2015; 5: 8084.

[23] Dissanayake D, Patel H, Wijesinghe PS. Differentiation of human male germ cells from Wharton’s jelly-derived mesenchymal stem cells. Clin Exp Reprod Med 2018; 45: 75–81.

[24] Ma Z, Qin M, Liang H, Chen R, Cai S, Huang Z, et al. Primary cilia-dependent signaling is involved in regulating mesenchymal stem cell proliferation and pluripotency maintenance. J Mol Histol 2020; 51: 241–250.

[25] Mayere C, Neirijnck Y, Sararols P, Rands CM, Stevant I, Kuhne F, et al. Single cell transcriptomics reveal temporal dynamics of critical regulators of germ cell fate during mouse sex determination. FASEB J 2021; 35: e21452.

[26] Guo J, Grow EJ, Mlcochova H, Maher GJ, Lindskog C, Nie X, et al. The adult human testis transcriptional cell atlas. Cell Res 2018; 28: 1141–1157.

[27] Ghorbanlou M, Abdanipour A, Shirazi R, Malekmohammadi N, Shokri S, Nejatbakhsh R. Indirect coculture of testicular cells with bone marrow mesenchymal stem cells leads to male germ cell-specific gene expressions. Cell J 2019; 20: 505–512.