Fig. 2. Morphological and phenotypic characteristics of cells derived from human SSCs by 3D-I system.

a Human SSCs (arrow, left panel) and inactivated Sertoli cells were co-cultured in the matrigel with the defined medium. Spermatocytes (arrows, middle panel) with condensed chromatins (inserted picture) emerged in the 3D-I system at day 10–15. Round spermatids (arrowheads, right panel) were seen in the 3D-I system at day 20. The data shown in a were presented from at least seven independent experiments. Scale bars = 10 μm. b Immunocytochemistry in situ showed the presence of SYCP3 and ACR proteins in the culture plates of human SSCs by 3D-I at day 20. The data shown in b were presented from at least three independent experiments. Scale bar = 10 μm. c Real-time PCR showed the relative expression levels of SYCP1, SYCP3, ACR, TNP1, TNP2, PRM1, and PRM2 to the housekeeping gene ACTB in the cells derived from human SSCs by 3D-I, 2D-I, 3D-C, 3D-N, and 2D-C at day 15. Notes: Post-hoc test method was used to compare the five groups. * indicated p < 0.05 and ** denoted p < 0.01. d Real-time PCR displayed the relative expression levels of SYCP1, SYCP3, ACR, TNP1, TNP2, PRM1, and PRM2 to the housekeeping gene ACTB in the cells derived from human SSCs after inducing for 5 days, 10 days, 15 days, and 20 days in 3D-I system. Notes: * indicated p < 0.05 and ** denoted p < 0.01 in the cells derived from human SSCs after induction for 10 days, 15 days, 20 days compared to 5 days. The results shown in c and d were presented from at least three independent experiments