Fig. 2
Addition of EGF and LIF effectively accelerated SSCs transformation. a The morphology of newly isolated SSCs maintained on MEF feeder with medium1 after 1 passage was exhibited. b, SSCs colonies on MEF feeder with medium 1 for 4 passages, and medium 1 was replaced with medium 2, were exhibited. c After another 3 passages in medium 2, some GSPCs colonies appeared (red arrow heads). d The representative GSPCs colonies after long-term culture were exhibited. e-l The expression of NANOG, SSEA1 and MVH in stable GSPCs was detected using IF (e. PLZF, f. NANOG, g. DAPI, h. merge; i. SSEA1, j. MVH (arrows indicate a few MVH+ cells), k. DAPI, l. merge). m The expression levels of Gfrα1, α6-integrin, Plzf, Mvh, Nanog, Sox2, Klf4, Oct4 and Gapdh in SSCs and GSPCs were determined using RT-PCR (M. marker, 1. SSCs, 2. GSPCs). n Alkaline phosphatase activity of stable GSPCs derived from SSCs cultured in medium 2 was detected. o GSPCs derived in medium 2 induced teratoma in nude mice as early as 8–10 days, while SSCs cultured in medium 1 could not induce teratoma. p The conversion ratio of SSCs cultured in medium 1 was statistically analyzed. q The strategy to make germline specific GFP mice and isolate GFP labeled SSCs. r Tracing the formation of GFP labeled GSPCs under medium 2 condition. The total cells from mTmGfl/+ mice indiscriminately expressed tomato (left), while SSCs from mTmGfl/fl;Ddx4-Cre+ mice specifically expressed GFP (middle), and when they transformed in medium 2, GFP signal was only observed in GSPCs (right). Scale bar = 20 μm