117 Zeinab Narimanpour 1 , Maryam Nazm Bojnordi 1,2* , Hatef Ghasemi Hamidabadi 1,2 1. Department of Anatomy & Cell Biology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. 2. Immunogenic Research Center, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. * Corresponding Author: Maryam Nazm Bojnordi, PhD. Address: Department of Anatomy & Cell Biology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. Phone: +98 (912) 8102359 E-mail: bojnordi@modares.ac.ir An Efcient in Vitro Culture Sysem to Amplify Sper - matogonia Stem Cell Markers Background: Proliferation of Spermatogonial Stem Cells (SSCs) can be a treatment for infertile men. Here, we design an efcient method based on culturing in the presence of Sertoli cells to improve the expression level of some specifc spermatogonia sem cell genes during two weeks pos culture. Materials and Methods: Cells were derived from neonatal (2-6 days old) mice teses and were cultured in DMEM medium with FBS. The colonization of cultured SSCs in days 4, 7, and 14 of culture was counted via phase-contras microscope and Image J software. Methyl Thiazolyl Tetrazolium (MTT) tes was performed to evaluate the viability of cultured SSCs in days 3, 7, and 14 of culture. The expression level and the alteration pattern of specifc spermatogonial markers, i.e. Stra8, DAZL, and Piwill2 was examined via real-time Polymerase Chain Reaction (PCR) during two weeks pos culture. Results: The number and the diameters of colonies showed a signifcant increase in cultured cells. MTT results proved the higher viability of tesicular cells during the culture period. The results of ALP saining detected a positive reaction in spermatogonia colonies. Real-time PCR data showed that culturing SSCs in the presence of intersitial cells of the tesis, amplifed the level and alteration pattern of specifc spermatogonia sem cells genes benefcial in the enrichment of SSCs propagation. Conclusion: Providing a similar culture environment to tesicular niche increases viability, forms SSCs colonies, and regulates the level and alteration pattern of spermatogonia sem cell genes. A B S T R A C T Keywords: Spermatogonial sem cells, Proliferation, Viability, Colony formation, Gene Citation Narimanpour Z, Nazm Bojnordi M, Ghasemi Hamidabadi H. An Efcient in Vitro Culture Sysem to Amplify Spermatogonia Stem Cell Markers. Research in Molecular Medicine. 2020; 8(3):117-124. https://doi.org/10.32598/rmm.8.3.2 https://doi.org/10.32598/rmm.8.3.2 Use your device to scan and read the artcle online Article info: Received: 25 May 2020 Revised: 17 Jun 2020 Accepted: 10 Jul 2020 Introduction permatogonial Stem Cells (SSCs) transport genetic information from one generation to the next one via spermatogenesis and sperm production [1]. They are located in the basal lamina of seminiferous tubules of the tesis, surrounded by cellular network consised of intersitial cells that act as an SSCs feeder lay- er and regulates nutrition and proliferation of SSCs [2, 3]. Some elements, especially glial cell-derived neuro- trophic factor (GDNF) mediates the survival and prolif- eration of SSCs [4, 5]. It seems that GDNF is an essential factor for in vitro propagation and colony formation of SSCs [6, 7]. The niche of tesis includes extracellular ma- S Article Type: Research Article