Differentiation of spermatogonial stem cells by soft agar three-dimensional culture system

Abstract

Since proliferation and differentiation of spermatogonial stem cells (SSCs) in culture system provide successful transplantation in this study, culture of human SSCs was compared to SACS (soft agar culture system), gelatin and control groups. The cells were isolated from seminiferous tubules of nonazoospermia patients (NOA) and cultured in DMEM for 3 weeks. The presence of SSCs in culture system was confirmed by immunocytochemistry of GFR-a1 and ITGa6 antibodies. The proliferated cells were cultured in three mentioned groups in the presence of retinoic acid and Sertoli cells conditioned medium for another 2 weeks. The number of colonies in the SACS group was significantly higher than two other groups. Before 2 weeks of culture, only Oct4 expression was observed in testicular cells (2.32 ± 0.25). After 2 weeks, the expression of Oct4 in the gelatin group was higher than that of the SACS group on day 7. The maximum expression of Stra8 was observed in SACS and gelatin groups after 7 days, but its expression was significantly decreased after 14 days of culture (p < .05). The expression of Scp3 and Acrosin genes were higher after 14 days in the SACS group compared to other groups. SACS has positive effects on proliferation and differentiation of hSSCs.

Differentiation of neonate mouse spermatogonial stem cells on three-dimensional agar/polyvinyl alcohol nanofiber scaffold

“…Agar is a biocompatible, complex polysaccharide extracted from red sea weeds and was first used by Lin and colleagues (Lin et al 1975) to establish a 3D cell culture model (i.e., SACS) for characterizing clonal expansion of bone marrow cells. Since then several research groups have utilized SACS to investigate proliferation and differentiation of male germ cells in vitro (Stukenborg et al 2008(Stukenborg et al , 2009; Abu Elhija et al 2012; Reda et al 2016;Navid et al 2017;Gholami et al 2018aGholami et al , 2018bMohammadzadeh et al 2019). Application of SACS for in vitro spermatogenesis, however, was not devoid of pitfalls.…”

“…There are also studies evaluating the impact of 3D soft agar culture system (SACS) on the development of mouse testicular germ cells (Stukenborg et al 2008;Mahmoud 2012;Eslahi et al 2013). Recently, human testicular germ cells have been successfully induced to reach the final stages of spermatogenesis by embedding single-cell suspension from testicular tubules in soft agar (Mohammadzadeh et al 2019). It has been claimed that soft agar culture system produces a microenvironment that resembles the natural niche of SSCs in the seminiferous tubules and therefore reduces the risk of ischemia in a long-term testicular tissue culture (Weaver et al 1997;Zhang 2004).…”

Roles of Spermatogonial Stem Cells in Spermatogenesis and Fertility Restoration

“…Soft agar and agarose gel are the most common material used to establish the 3D culture system for human SSCs. A soft agar culture system has been shown to support the proliferation and differentiation of human SSCs ( 105 ). Another material that has been used in 3D culture systems for human SSCs is a polycaprolactone (PCL) nanofiber matrix ( 106 ).…”

Progress in germline stem cell transplantation in mammals and the potential usage

“…Many biological processes can be more accurately studied through the use of 3D culture system such as the spermatogenic process [ 74 ]. A novel 3D culture system is based on a soft agar culture system (SACS) [ 75 ], with two major constituents of SSCs niche, somatic cells [ 72 ] and the extracellular matrix (ECM) [ 76 ]. SACS makes it more efficient than some other 3D culture systems that are based on collagen, gelatin or matrigel, attributing to the thick layer for germ cells and supporting cells to embed in [ 77 ].…”