Fig. 6

Human SSCs can be maintained and expanded in vitro with human ECs. a Representative brigh-tfield images of human testicular cells from fresh and frozen testis tissue cultured with or without human ECs after Dil-Ac-LDL (red) uptake on the indicated days. Bar = 100 μm. b Characterization of SSC colonies generated in human testicular cell and endothelial cell co-cultures. Colonies were immunostained with the human SSC markers, CD49f and SSEA4, Bar = 50 μm. c Bright-field and GFP-merged images at day 2 (inset) and day 40 after transplantation of PKH-labeled human SSC colonies (green) expanded in co-cultures with ECs, into busulfan-treated immunodeficient Nude mice. Red arrows indicate PKH-labeled SSC colonies in the seminiferous tubules. Transplanted PKH-labeled human SSC colonies (green) were visible in the cavity of seminiferous tubules at day 2 after microinjection. d Representative immunofluorescence images of cross-sections of a seminiferous tubule from Nude mice 40 days after transplantation with PKH-labeled human SSC colonies (green). Sections were immunostained with SSEA4 (red). Bar = 50 μm. e Relative expression of secreted factors by antibody arrays in media conditioned by LuEC, LiEC, TEC and TECs expressing constitutively active NFATc1 (caNFATc1) during Matrigel capillary tube formation. f Representative bright-field images of human testicular cells showing SSC colonies in feeder-free cultures in media containing SDF-1, MIP-2, IGFBP-2, GDNF and FGF-2 at the indicated days, bar = 50 μm. g Quantification of SSC numbers over time in feeder-free cultures with the indicated growth factors. h Quantification of SSC numbers in co-culture with human TECs and addition of either FGF-2 alone or FGF-2 and GDNF at the indicated passages. Values are mean ± s.e.m, n = 3. Data shown are representative of three independent experiments