Determination of Feeder Cell‐Based Cellular Niches Supporting the Colonization and Maintenance of Spermatogonial Stem Cells from Prepubertal Domestic Cat Testes

Abstract

Recently, isolation and in vitro culture of putative spermatogonial stem cells (SSCs) in the domestic cat have been conducted. However, the cellular niche conditions that facilitate the establishment and long-term maintenance of feline SSCs (FSSCs) have not been described. Therefore, we investigated the type of feeder cells used to stimulate colony formation and growth of FSSCs among the various factors in the FSSC niche. Spermatogonial stem cells isolated from feline testes were cultured on mitotically inactivated testicular stromal cells (TSCs) derived from cats, dogs and mice, and mouse embryonic fibroblasts (MEFs). The formation and growth of colonies derived from SSCs cultured on each type of feeder cell were identified at passage 0, and the morphology, alkaline phosphatase (AP) activity and expression of SSC-specific genes in surviving colonies were investigated at passage 4. Among these diverse feeder cells, TSCs from cat showed the greatest colony formation, growth and maintenance of FSSCs, and SSC colonies cultured by passage 4 showed a typical dome-shaped morphology, AP activity and expression of SSC-specific genes (NANOG, OCT4, SOX2 and CD9). Accordingly, these results demonstrate that feline TSCs could be used as feeder cells to support the establishment and maintenance of SSCs from domestic cats.

Molecular markers of putative spermatogonial stem cells in the domestic cat

“…It is possible that some species‐specificity issues in the growth factors used herein, including GDNF, may have precluded adequate support of stem cell activity of cultured cat germ cells. Herein, cat germ cells did not proliferate in culture for longer periods than the timeframe reported in previous studies (Han et al., ; Tiptanavattana et al., ). While Han et al.…”

“…While Han et al. () reported that the use of TSCs as feeders in culture could provide a niche for the persistence of cat SSCs, the analysis of the resulting colonies for stem cell activity was not convincing. Therefore, the requirements for in vitro culture of cat SSCs require further investigation.…”

Determination phase at transition of gonocytes to spermatogonial stem cells improves establishment efficiency of spermatogonial stem cells in domestic cats

“…This result, therefore, confirms the importance of the G/SSC phase transition and also possibly the negative effects of other testicular cells (differentiated germ cells and supporting testicular cells) on successful SSC culture [ 22 ]. However, maintaining SSC activities in vitro remains problematic for long-term culture of SSCs in domestic cats [ 27 , 50 ]. Further study is required to underpin the mechanisms that essentially contribute to the multifaceted fate of SSC properties during SSC culture.…”

Screening of Integrin Heterodimers Expressed Functionally on the Undifferentiated Spermatogonial Stem Cells in the Outbred ICR Mice

“…Their self-renewal is infinitely maintained on the seminiferous tubule basement membrane (STBM) with defined ECM components in vivo (15). Nevertheless, technical difficulties in constructing the artificial STBM has led to the use of a cellular niche based on feeder cells during the in vitro culture of SSCs (16)(17)(18). However, a small population of SSCs located in the seminiferous tubule of the testis derived from diverse species did not sufficiently expand under these conditions (19).…”