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Melatonin Promotes Differentiation of Human Spermatogonial Stem Cells Cultured on Three-Dimensional Decellularized Human Testis Matrix Publisher Pubmed



Salem M1 ; Khadivi F2 ; Feizollahi N1 ; Khodarahmian M1 ; Saedi Marghmaleki M1 ; Ayub S1 ; Chegini R1 ; Bashiri Z3 ; Abbasi Y4 ; Naji M5 ; 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. Medical Plants Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Iran
  3. 3. Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
  4. 4. Department of Neural and Pain Sciences, School of Dentistry, Program in Neuroscience, Center to Advance Chronic Pain Research, University of Maryland Baltimore, Baltimore, MD, United States
  5. 5. Urology and Nephrology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Source: Urology journal Published:2024


Abstract

PURPOSE: The use of 3D (3-Dimensional) culture systems supported cell-to-cell and cell-to-extracellular matrix (ECM) interactions, proliferation, and differentiation of SSCs (Spermatogonial stem cells). The potential advantages of ECM-based scaffolds for in vitro spermatogenesis have been indicated in human and animal experiments. Furthermore, the strong antioxidant and anti-inflammatory activities of melatonin have improved in vitro manipulation of human SSCs in culture conditions. MATERIALS AND METHODS: SSCs were isolated from the testis of three dead-brain patients and then propagated for four weeks. The characterization of SSC colonies was done using real-time PCR (Polymerase chain reaction), ICC (Immunocytochemistry), and xenotransplantation to mice model. Decellularization of the human testis was performed using 0.3% sodium dodecyl sulfate (SDS) solution and 1% Triton X-100. Also, various characterizations of DTM (Decellularized testicular matrix ) were carried out using histological staining and DNA content analysis. The optimum dose of melatonin was selected by MTT (Methyl thiazol tetrazolium). SSCs were cultured in 4 groups: control, melatonin, ECM, and ECM-melatonin in a differentiation medium for four weeks. The expression of differentiation genes was evaluated by real-time polymerase chain reaction. In addition, the viability of cultured cells was assessed by MTT assay. RESULTS: The results of ICC and real-time PCR showed the expression of undifferentiated SSC markers (PLZF and GRFA1) in SSC colonies following the 2D culture of isolated SSCs. The presence of testicular ECM components after different staining methods; and the reduction of DNA content confirmed the proper decellularization process. Germ cell apoptosis significantly decreased in melatonin and ECM groups, and the higher viability of SSCs was seen in the ECM-melatonin group. The relative expression of GFRA1 and PRM2 decreased and increased in ECM and ECM-melatonin groups, respectively. CONCLUSION: Our study showed that the addition of melatonin to the human naturally-derived ECM scaffold could provide a suitable platform for inducing the differentiation and preserving the viability of SSCs.