Fig. 2

TECs can expand spermatogonial stem cells (SSCs) in culture. a Quantification of GDNF by ELISA in conditioned media from TECs, LuECs and LiECs after treatment with the indicated concentration of FGF-2 for 2 days, n = 3. Bar = 100 μm. Data are presented as the mean ± s.e.m. b Representative bright-field (BF) and GFP images of 2D cultures of GFP⁺ SSCs co-cultured with STO cells or TECs plus exogenous GDNF and FGF-2 on days 1 and 8 after seeding. Quantification of stem cell numbers 8 days after co-culture with STO or TECs and the average colony area are shown on the right, n = 3. Data are presented as the mean ± s.e.m. Bar = 100 μm. *P < 0.05. c Images of 2D co-cultures of GFP⁺Thy1.2⁺ SSCs with TECs and immunostained with the germ cell marker DDX4. Inset: bar = 10 μm. d The 3D spheroid co-cultures of GFP⁺Thy-1.2⁺ SSCs with TECs or LuECs were generated with Matrigel but no exogenous GDNF or FGF-2. GFP⁺ Spheroid co-cultures were immunostained with DDX4 (red), Oct4 (white) and DAPI (blue) at day 15. Quantification of the average diameter and total number of colonies is shown on the right; n = 3. All data are presented as the mean ± s.e.m. Bar = 50 μm, *P < 0.05. **P < 0.01. e Representative BF images merged with GFP images of serially passaged GFP⁺Thy-1.2⁺ SSCs co-cultured with TECs at the indicated passage number (1st, 2nd, 3rd or 4th) or day (D). Quantification of colony number and mean colony diameter at the indicated passage number is shown on the right. Data shown are representative of six independent experiments. Two-tailed unpaired T-test