Simplified isolation and enrichment of spermatogonial stem-like cells from pubertal domestic cats (Felis catus).

The Journal of veterinary medical science / the Japanese Society of Veterinary Science

PubMedID: 26074411

Tiptanavattana N, Techakumphu M, Tharasanit T. Simplified isolation and enrichment of spermatogonial stem-like cells from pubertal domestic cats (Felis catus). J Vet Med Sci. 2015;.
The efficiency of spermatogonial stem cell (SSC) isolation and culture from pubertal donors is currently poor primarily, because of contamination with other testicular cells. This study aimed to purify SSC-like cells using different extracellular matrixes and a discontinuous gradient density. In experiment 1, testes (n=6) were analyzed for histology and SSC-related protein expressions (laminin, SSEA-4, DDX-4 and GFRa-1). After enzymatic digestion, the cell suspension was plated onto either a laminin- or gelatin-coated dish. The number of SSC-like cells was determined at 15, 30 and 60 min of culture (experiment 2). Experiment 3 was performed to test whether or not the additional step of Percoll gradient density centrifugation could really improve purification of SSC-like cells. Testicular histology revealed complete spermatogenesis with laminin expression essentially at the basal lamina of the seminiferous tubules. SSEA-4 and GFRa-1 co-localized with DDX-4 in the spermatogonia. The relative percentage of SSC-like cells, as determined by cells expressing SSEA-4 (59. 42 ± 2. 18%) and GFRa-1 (42. 70 ± 1. 28%), revealed that the highest SSC-like cell purity was obtained with the 15-min laminin-coated dish compared with other incubation times and gelatin treatment (P<0. 05). Percoll treatment prior to laminin selection (15 min) significantly improved SSC-like cell recovery (91. 33 ± 0. 14%, P<0. 001) and purity (83. 82 ± 2. 05% for SSEA-4 and 64. 39 ± 1. 51% for GFRa-1, P<0. 05). These attached cells demonstrated a typical SSC-like cell morphology and also expressed POU5F1, RET and ZBTB16 mRNA. In conclusion, double enrichment with Percoll gradient density centrifugation and laminin plating highly enriched the SSC-like cells population.