A Novel Feeder-Free Culture System for Expansion of Mouse Spermatogonial Stem Cells

Abstract

Spermatogonial stem cells (SSCs, also called germline stem cells) are self-renewing unipotent stem cells that produce differentiating germ cells in the testis. SSCs can be isolated from the testis and cultured in vitro for long-term periods in the presence of feeder cells (often mouse embryonic fibroblasts). However, the maintenance of SSC feeder culture systems is tedious because preparation of feeder cells is needed at each subculture. In this study, we developed a Matrigel-based feeder-free culture system for long-term propagation of SSCs. Although several in vitro SSC culture systems without feeder cells have been previously described, our Matrigel-based feeder-free culture system is time- and cost- effective, and preserves self-renewability of SSCs. In addition, the growth rate of SSCs cultured using our newly developed system is equivalent to that in feeder cultures. We confirmed that the feeder-free cultured SSCs expressed germ cell markers both at the mRNA and protein levels. Furthermore, the functionality of feeder-free cultured SSCs was confirmed by their transplantation into germ cell-depleted mice. These results suggest that our newly developed feeder-free culture system provides a simple approach to maintaining SSCs in vitro and studying the basic biology of SSCs, including determination of their fate.

Optimization of Matrigel-based culture for expansion of neural stem cells

“…Matrigel was a promising candidate because it is commonly used for adherent cultures of pluripotent stem cells, such as human ESCs and human induced pluripotent stem cells. Recently, our group demonstrated that mouse spermatogonial stem cells can also be expanded on Matrigel and maintained in culture for a long time (Choi et al 2014).…”

“…Because of supporting self-renewability and pluripotency, Matrigel is mostly used for in vitro feeder-free cultures of mouse and human embryonic stem cells (ESCs; Xu et al 2001;Amit et al 2004;Ludwig et al 2006). Our recent study showed that spermatogonial stem cells (also called germline stem cells), unipotent spermproducing stem cells in the testis, can be successfully expanded in Matrigel-based feeder-free culture in vitro (Choi et al 2014). Matrigel has been used to enhance survival and differentiation of neural cells derived from mouse and human ESCs Uemura et al 2010;Ramos-Hryb et al 2013).…”

Biomodification of PCL Scaffolds with Matrigel, HA, and SR1 EnhancesDe NovoEctopic Bone Marrow Formation Induced by rhBMP-2

“… 33 Matrigel promotes in vitro cell differentiation, but it also provides a critical 3D structure for stem cell culture that allows stem cell expansion without the need for feeder layers. 34 , 35 We exploited these characteristics by introducing Matrigel into the precast thin PCL scaffolds as a way to mimic the in vivo ECM for controlled release of biologics and temporal regulation of cell proliferation and differentiation. The inclusion of Matrigel was critical for the development of hematopoiesis, because the scaffolds without a Matrigel coating showed lower CFUs within the induced ossicles (group 2) when compared with ossicles induced by scaffolds coated with Matrigel (groups 3–5).…”

p53 inhibits the proliferation of male germline stem cells from dairy goat cultured on poly‐L‐lysine

“…During development, extracellular matrix signalling is dynamic, constantly remoulded by proteases and changes in the expression of its different protein components, that initiate or alter various biological processes (Duband & Thiery, 1982;George, Georges-Labouesse, Patel-King, Rayburn, & Hynes, 1993;Page-McCaw, Ewald, & Werb, 2007). Extracellular matrix was also used in stem cell culture for its ability to either induce differentiation or for its characteristics that sustain stem cells, and in some cases, it can replace the feeder cells (Choi et al, 2014;Engler et al, 2006;RasiGhaemi et al, 2019). Poly-L-lysine, which is commonly used to enhance cell adhesion to the surface of cell culture dish (Babic et al, 2008), has several unique biological properties, like its ability to aid cell membrane permeability in murine tumour cells (Debnath, Mukherjee, Karan, Debnath, & Chatterjee, 2018).…”