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Three-Dimensional Co-Culture of Human Spermatogonial Stem Cells With Sertoli Cells in Soft Agar Culture System Supplemented by Growth Factors and Laminin Publisher Pubmed



Jabari A1 ; Sadighi Gilani MA8 ; Koruji M2 ; Gholami K3 ; Mohsenzadeh M4 ; Rastegar T1 ; Khadivi F1 ; Ghanami Gashti N1 ; Nikmahzar A1 ; Mojaverrostami S1 ; Talebi A5, 6 ; Ashouri Movassagh S1, 7 ; Rezaie MJ9 ; Abbasi M1
Authors
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Authors Affiliations
  1. 1. Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
  2. 2. Cellular and Molecular Research Center & Department of Anatomical Sciences, Iran University of Medical Sciences, Tehran, Iran
  3. 3. Gametogenesis Research Center, Kashan University of Medical Sciences, Kashan, Iran
  4. 4. Iranian Tissue Bank and Research Center of Tehran University of Medical Sciences, Tehran, Iran
  5. 5. School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran
  6. 6. Sexual Health and Fertility Research Center, Shahroud University of Medical Sciences, Shahroud, Iran
  7. 7. Human and Animal Cell Bank, Iranian Biological Resource Center (IBRC), ACECR, Tehran, Iran
  8. 8. Department of Urology, School Medicine, Tehran University of Medical Sciences, Tehran, Iran
  9. 9. Department of Embryology, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran

Source: Acta Histochemica Published:2020


Abstract

Application of a three-dimensional (3D) culture system for in vitro proliferation and differentiation of human spermatogonial stem cells (SSCs) is a useful tool for the investigation of the spermatogenesis process and the management of male infertility particularly in prepubertal cancer patients. The main purpose of this study was to investigate the proliferation of human SSCs co-cultured with Sertoli cells in soft agar culture system (SACS) supplemented by Laminin and growth factors. Testicular cells were isolated from testes of brain-dead patients and cultured in two-dimensional (2D) culture system for 3 weeks. After 3 weeks, functional SSCs were evaluated by xenotransplantation and also identification of cells was assessed by immunocytochemistry, flow cytometry, and RT-PCR. Then, SSCs and Sertoli cells were transferred to the upper layer of SACS for 3 weeks. After 3 weeks, the number of colonies and the expression of specific SSCs and Sertoli cell markers, as well as apoptotic genes were evaluated. Our results showed that transplanted SSCs, migrated into the basement membrane of seminiferous tubules of recipient mice. The expression of PLZF, α6-Integrin, and Vimentin proteins in SSCs and Sertoli cells were observed in 2D and 3D culture systems. The expression rate of PLZF, α6-Integrin, Bcl2, and colony number in SACS supplemented by Laminin and growth factors group were significantly higher than non-supplemented groups (P ≤ 0.01), but the expression rate of c-kit and Bax in supplemented group were significantly lower than non-supplemented groups (P ≤ 0.05). This 3D co-culture system decreased apoptosis and increased propagation of human SSCs. Therefore, this designed system can be utilized to increase the proliferation of human SSCs in prepubertal male cancer and azoospermic men to obtain an adequate SSCs number to outotransplant success and in vitro spermatogenesis. © 2020
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