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HJEs (passage 1) were cultivated in a growth culture medium containing 50% primary culture medium (Advanced DMEM/F-12 (Life Technologies), 20% fetal bovine serum (Eurobio), Glutamax 1X (Life Technologies), 1% penicillin/streptomycin (Life Technologies) and 50% conditioned medium from L-WRN cell culture, secreting proliferation factors, Wnt3a, R-Spondin 3 and Noggin (CRL-3276, ATCC) supplemented with a ROCK pathway inhibitor (Y-27632, Biotechne, 10 M) and a TGFBR1 inhibitor (SB431542, Biotechne, 10 M) [36]

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