Journal of Reproduction & Infertility

Journal of Reproduction & Infertility

A Comparative Analysis of Culture Systems with Human Amniotic Mesenchymal Stem Cells

Authors
Mahdi Jahanbakhsh 1
Fatemeh Asgari 2
Roya Hassani 3
Morteza Koruji 3
1 Stem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Tehran, Iran
2 Avicenna Fertility Center, Avicenna Research Institute, ACECR, Tehran, Iran
3 Stem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Tehran, Iran; Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
Abstract
Background: A “indirect co-culture using mesh” system is commonly employed to maintain spermatogenesis in cancer patients undergoing chemotherapy and radiation. This study aimed to investigate the co-culturing of mouse spermatogonial stem cells (SSCs) with human amniotic mesenchymal stem/stromal cells (hAMSCs) in an optimized environment. Methods: SSCs from 3-6-day-old mice (n=10) were indirectly co-cultured with hAMSCs via mesh for two weeks. Three groups evaluated: control, SSCs with conditioned media, and SSCs indirectly co-cultured with hAMSCs. Gene expression analyzed for Plzf, c-kit, Sycp3, and Prm1. Immunohistochemistry assessed Plzf, and flow cytometry evaluated c-kit and Plzf. Results: Showed a twofold increase in Plzf-positive cells after 14 days of culture (76.47%, P≤0.05), with a significant elevation in Plzf gene expression observed in the conditioned media group (188.1 ± 65%, P≤0.05). Conversely, the expression of the c-kit gene decreased significantly in both the conditioned media and “indirect co-culture using mesh” groups. Notably, Sycp3 and Prm1 expression levels significantly increased in the conditioned media group compared to the control. These findings suggest the potential of conditioned media as a novel feeder for promoting in vitro mouse spermatogenesis. Conclusion: Our results demonstrate that the inclusion of growth factors, such as GDNF and BMP-4, along with conditioned media and an “indirect co-culture using mesh” system utilizing meshes with SSCs, significantly enhances SSC proliferation and differentiation. The optimized conditions media provided by hAMSCs offer a superior feeder compared to traditional “indirect co-culture using mesh” systems for promoting both the proliferation and differentiation of SSCs.
Keywords
Amniotic mesenchymal stem cells
differentiation
placenta
Proliferation
  1. Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, et al. Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod. 2003;69(2):612-6.
  2. Talebi A, Gilani MAS, Koruji M, Ai J, Rezaie MJ, Navid S, et al. Colonization of mouse spermatogonial cells in modified soft agar culture system utilizing nanofibrous scaffold: a new approach. Galen Med J. 2019;8:e1319.
  3. Blatt J. Pregnancy outcome in long‐term survivors of childhood cancer. Med Pediatr Oncol. 1999;33(1):29-33.
  4. Ghorbani S, Eyni H, Khosrowpour Z, Salari Asl L, Shabani R, Nazari H, et al. Spermatogenesis induction of spermatogonial stem cells using nanofibrous poly (l‐lactic acid)/multi‐walled carbon nanotube scaffolds and naringenin. Polym Adv Technol. 2019;30(12):3011-25.
  5. Matulewicz RS, Meeks JJ. JAMA patients page. Testicular mass. JAMA. 2016;316(5):557.
  6. Oatley MJ, Kaucher AV, Yang QE, Waqas MS, Oatley JM. Conditions for long-term culture of cattle undifferentiated spermatogonia. Biol Reprod. 2016;95(1):14.
  7. De Rooij DG. The nature and dynamics of spermatogonial stem cells. Development. 2017;144(17):3022-30.
  8. Kanatsu-Shinohara M, Shinohara T. Spermatogonial stem cell self-renewal and development. Annu Rev Cell Dev Biol. 2013;29:163-87.
  9. Kanatsu-Shinohara M, Inoue K, Lee J, Yoshimoto M, Ogonuki N, Miki H, et al. Generation of pluripotent stem cells from neonatal mouse testis. Cell. 2004;119(7):1001-12.
  10. Hamidabadi HG, Bojnordi MN. Co-culture of mouse spermatogonial stem cells with sertoli cell as a feeder layer, stimulates the proliferation and spermatogonial stemness profile. Middle East Fertil Soc J. 2018;23(2):107-11.
  11. Ghorbanlou M, Abdanipour A, Shirazi R, Malekmohammadi N, Shokri S, Nejatbakhsh R. Indirect co-culture of testicular cells with bone marrow mesenchymal stem cells leads to male germ cell-specific gene expressions. Cell J. 2019;20(4):505-12.
  12. Asgari F, Asgari HR, Najafi M, Eftekhari BS, Vardiani M, Gholipourmalekabadi M, et al. Optimization of decellularized human placental macroporous scaffolds for spermatogonial stem cells homing. J Mater Sci Mater Med. 2021;32(5):47.
  13. Parolini O, Caruso M. Preclinical studies on placenta-derived cells and amniotic membrane: an update. Placenta. 2011;32 Suppl 2:S186-95.
  14. Koruji M, Movahedin M, Mowla SJ, Gourabi H, Arfaee A. Efficiency of adult mouse spermatogonial stem cell colony formation under several culture conditions. In Vitro Cell Dev Biol Anim. 2009;45(5-6):281-9.
  15. Baazm M, Mashayekhi FJ, Babaie S, Bayat P, Beyer C, Zendedel A. Effects of different Sertoli cell types on the maintenance of adult spermatogonial stem cells in vitro. In Vitro Cell Dev Biol Anim. 2017;53(8):752-8.
  16. Parolini O, Alviano F, Bagnara GP, Bilic G, Bühring HJ, Evangelista M, et al. Concise review: isolation and characterization of cells from human term placenta: outcome of the first international workshop on placenta derived stem cells. Stem Cells. 2008;26(2):300-11.
  17. Hofer HR, Tuan RS. Secreted trophic factors of mesenchymal stem cells support neurovascular and musculoskeletal therapies. Stem Cell Res Ther. 2016;7(1):131.
  18. Ma S, Xie N, Li W, Yuan B, Shi Y, Wang Y. Immunobiology of mesenchymal stem cells. Cell Death Differ. 2014;21(2):216-25.
  19. Caplan AI, Dennis JE. Mesenchymal stem cells as trophic mediators. J Cell Biochem. 2006;98(5):1076-84.
  20. Kouchakian MR, Koruji M, Najafi M, Moniri SF, Asgari A, Shariatpanahi M, et al. Human umbilical cord mesenchymal stem cells express cholinergic neuron markers during co-culture with amniotic membrane cells and retinoic acid induction. Med J Islam Repub Iran. 2021;35:129.
  21. Shabani R, Ashtari K, Behnam B, Izadyar F, Asgari H, Asghari Jafarabadi M, et al. In vitro toxicity assay of cisplatin on mouse acute lymphoblastic leukaemia and spermatogonial stem cells. Andrologia. 2016;48(5):584-94.
  22. Eslahi N, Hadjighassem MR, Joghataei MT, Mirzapour T, Bakhtiyari M, Shakeri M, et al. The effects of poly L-lactic acid nanofiber scaffold on mouse spermatogonial stem cell culture. Int J Nanomed. 2013;8:4563-76.
  23. Navid S, Abbasi M, Hoshino Y. The effects of melatonin on colonization of neonate spermatogonial mouse stem cells in a three-dimensional soft agar culture system. Stem Cell Res Ther. 2017;8(1):233.
  24. Gholami K, Pourmand G, Koruji M, Sadighigilani M, Navid S, Izadyar F, et al. Efficiency of colony formation and differentiation of human spermatogenic cells in two different culture systems. Reprod Biol. 2018;18(4):397-403.
  25. Nayernia K. Stem cells derived from testis show promise for treating a wide variety of medical conditions. Cell Res. 2007;17(11):895-7.
  26. Fayomi AP, Orwig KE. Spermatogonial stem cells and spermatogenesis in mice, monkeys and men. Stem Cell Res. 2018;29:207-14.
  27. Tiwari JN, Tiwari RN, Kim KS. Zero-dimensional, one-dimensional, two-dimensional and three-dimensional nanostructured materials for advanced electrochemical energy devices. Prog Mater Sci. 2012;57(4):724-803.
  28. Ibtisham F, Honaramooz A. Spermatogonial stem cells for in vitro spermatogenesis and in vivo restoration of fertility. Cells. 2020;9(3):745.
  29. Azizi H, Niazi Tabar A, Skutella T. Successful transplantation of spermatogonial stem cells into the seminiferous tubules of busulfan-treated mice. Reprod Health. 2021;18(1):189.
  30. Maghen L, Shlush E, Gat I, Filice M, Barretto TA, Jarvi K, et al. Human umbilical perivascular cells (HUCPVCs): a novel source of mesenchymal stromal-like (MSC) cells to support the regeneration of the testicular niche. Reproduction. 2017;153(1):85-95.
  31. Chen B, Wang YB, Zhang ZL, Xia WL, Wang HX, Xiang ZQ, et al. Xeno-free culture of human spermatogonial stem cells supported by human embryonic stem cell-derived fibroblast-like cells. Asian J Androl. 2009;11(5):557-65.
  32. Wei X, Jia Y, Xue Y, Geng L, Wang M, Li L, et al. GDNF-expressing STO feeder layer supports the long-term propagation of undifferentiated mouse spermatogonia with stem cell properties. Sci Rep. 2016;6:36779.
  33. Aponte PM, Soda T, Teerds KJ, Mizrak SC, van de Kant HJ, de Rooij DG. Propagation of bovine spermatogonial stem cells in vitro. Reproduction. 2008;136(5):543-57.
  34. Qiu P, Bai Y, Pan S, Li W, Liu W, Hua J. Gender depended potentiality of differentiation of human umbilical cord mesenchymal stem cells into oocyte‐Like cells in vitro. Cell Biochem Funct. 2013;31(5):365-73.
  35. 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(1):229.
  36. Xu Y, Hong Y, Xu M, Ma K, Fu X, Zhang M, et al. Role of keratinocyte growth factor in the differentiation of sweat gland‐like cells from human umbilical cord‐derived mesenchymal stem cells. Stem Cells Transl Med. 2016;5(1):106-16.
  37. Wang Y, Yin Y, Jiang F, Chen N. Human amnion mesenchymal stem cells promote proliferation and osteogenic differentiation in human bone marrow mesenchymal stem cells. J Mol Histol. 2015;46(1):13-20.
  38. Dadoune JP. New insights into male gametogenesis: what about the spermatogonial stem cell niche? Folia Histochem Cytobiol. 2007;45(3):141-7.
  39. He Z, Jiang J, Kokkinaki M, Dym M. Nodal signaling via an autocrine pathway promotes proliferation of mouse spermatogonial stem/ progenitor cells through Smad2/3 and Oct‐4 activation. Stem Cell. 2009;27(10):2580-90.
  40. Payne C, Braun RE. Histone lysine trimethylation exhibits a distinct perinuclear distribution in Plzf-expressing spermatogonia. Dev Biol. 2006;293(2):461-72.
  41. Blume‐Jensen P, Claesson‐Welsh L, Siegbahn A, Zsebo KM, Westermark B, Heldin C-H. Activation of the human c‐kit product by ligand‐induced dimerization mediates circular actin reorganization and chemotaxis. EMBO J. 1991;10(13):4121-8.
  42. Mauduit C, Hamamah S, Benahmed M. Stem cell factor/c-kit system in spermatogenesis. Hum Reprod Update. 1999;5(5):535-45.
  43. Li Y, Zhang Y, Zhang X, Sun J, Hao J. BMP4/Smad signaling pathway induces the differentiation of mouse spermatogonial stem cells via upregulation of Sohlh2. Anat Rec (Hoboken). 2014;297(4):749-57.
  44. Ozaki Y, Saito K, Shinya M, Kawasaki T, Sakai N. Evaluation of Sycp3, Plzf and Cyclin B3 expression and suitability as spermatogonia and spermatocyte markers in zebrafish. Gene Expr Patterns. 2011;11 (5-6):309-15.
  45. Zang ZJ, Ji SY, Zhang YN, Gao Y, Zhang B. Effects of saikokaryukotsuboreito on spermato-genesis and fertility in aging male mice. Chin Med J (Engl). 2016;129(7):846-53.
  46. Ziloochi Kashani M, Bagher Z, Asgari HR, Najafi M, Koruji M, Mehraein F. Differentiation of neonate mouse spermatogonial stem cells on three-dimensional agar/polyvinyl alcohol nanofiber scaffold. Syst Biol Reprod Med. 2020;66(3):202-15.
  47. Akmal M, Widodo MA, Sumitro SB, Purnomo BB. The important role of protamine in spermatogenesis and quality of sperm: A mini review. Asian Pac J Reprod. 2016;5(5):357-60.
  48. Haueter S, Kawasumi M, Asner I, Brykczynska U, Cinelli P, Moisyadi S, et al. Genetic vasectomy-Overexpression of Prm1‐EGFP fusion protein in elongating spermatids causes dominant male sterility in mice. Genesis. 2010;48(3):151-60.
  49. Gustafson EA, Seymour KA, Sigrist K, Rooij DG, Freiman RN. ZFP628 is a TAF4b-interacting transcription factor required for mouse spermio-genesis. Mol Cell Biol. 2020;40(7):e00228-19.
  50. Topraggaleh TR, Valojerdi MR, Montazeri L, Baharvand H. A testis-derived macroporous 3D scaffold as a platform for the generation of mouse testicular organoids. Biomater Sci. 2019;7(4):1422-36.
Journal of Reproduction & Infertility
Volume 26, Issue 4
Summer 2025
Pages 2013-2024