Fig 2: SSC propagation on a GDNF-secreting STO feeder layer.(a) A culture of a line of SSCs harvested from 6–8 days old C57BL/6 mouse testes. The morphology of SSC clumps formed at Day 1, 7 and Day 90. Scale bar = 50 µm; (b) The growth curve calculated from a line of SSCs (combined) supported by GDNF-expressing STO feeders with the addition of 20 ng/ml GDNF, 1 ng/ml FGF2 and 150 ng/ml GFRA1 (GDNF-OE STO with GDNF, GFRA1, FGF2), a line by GDNF-expressing STO feeders with 1ng/ml FGF2 (GDNF-OE STO with FGF2), and a line by normal STO feeders with GDNF, GFRA1, FGF2 (STO with GDNF, GFRA1, FGF2). The 25-day culture data were collected through three wells of each individual line of SSCs, the error bars indicate the means ± SD.

Fig 3: Studying a protein associated with the GDNF pathway to explore the possible effect of ADSC. Western immunoblots of p-ERK1/2 and total ERK for B6D2F1 (normal mouse), untreated MBP1 (sham mouse), and ADSC-treated MBP1 (mouse treated with ADSC) at the end of the 4-week test period after ADSC transplantation. ADSC: adipose-derived stem cell; ERK: extracellular signal-regulated kinase; GDNF: glial cell-derived neurotrophic factor.

Fig 4: Karyotype analysis and SSC associated gene expression.(a) A metaphase spread and Karyotype analysis of SSCs cultured on GDNF-expressing STO feeders (GDNF-OE Culture) and normal STO feeder (Regular Culture). There was no obvious difference when based on a sum of thirty cells. Scale Bar = 10 µm; (b,c) quantitative PCR analysis of expression of self-renewal and differentiation associated genes and cell cycle associated genes, the error bars indicate the means ± SD; (d) The cell number for SSC growing for one week after the replacement of the GDNF-expressing STO feeders by normal STO feeder with or without growth factors, p < 0.05. From left to right, a culture with every-other-day refreshment of GDNF, FGF2, GFRA1, a culture with every-other-day refreshment of FGF2, and a culture without recombinant growth factors (serum-free medium only).

Fig 5: miniSINEUP-GDNF RNA Increases Selectively GDNF Protein Expression In Vitro(A) Schematic representation of miniSINEUP-GDNF structure, with the SINEUP BD overlapping the 5' UTR of the Gdnf mRNA. (B and C) Effect of miniSINEUP-GDNF RNA on endogenous GDNF protein levels in two cell lines, the Neuro2a (B) and the C8-D1A astrocytes (C) (F2, 12 = 8.09, p < 0.001, n = 5 for Neuro2a; and F2, 9 = 15.69, p < 0.01, n = 4 for astrocytes). miniSINEUP-GDNF transfected in cells increases GDNF proteins levels without altering Gdnf mRNA levels. Both miniSINEUP RNA with two different BD (-40/+4 and the -14/+4, respectively) were expressed in transfected cells, but not in CTRL cells. (D) miniSINEUP-GDNF increases extracellular GDNF secreted by cells, as measured by an ELISA (n = 8). (E) Neuro2a cells were transfected with a pLVX-TetOne vector expressing the miniSINEUP-GDNF. After 24 h post-transfection, cells were treated with different concentrations of doxocycline for 24 h to evaluate the correlation between miniSINEUP RNA levels and GDNF protein expression. The induction of the miniSINEUP-GDNF RNA expression by doxocycline (F4, 29 = 3.45, p < 0.05) provokes an increase in GDNF protein levels (F4, 20 = 3.52, p < 0.05, n = 3). (F) Off-target effects of miniSINEUP-GDNF were studied. After a BLAST analysis, the protein and mRNA levels of three possible off-targets were measured with western blot and qRT-PCR, namely, DAD1, CBX4, and LRRC14. miniSINEUP-GDNF did not alter protein or mRNA levels of these potential off-targets (n = 3). Data represent means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired t test with Welch’s correction (D and F) or one-way ANOVA followed by Dunnet’s multiple comparison test was used (B, C, and E).