Figure 1. hPSCs-Derived hNCCs Prone to Generate NB.
(A) A schematic representation of the experiment. Lentiviral transgenes of FUW-TetO-ALKF1174L-t2A-tdTomato and FUW-TetO-MYCN-3×2A-NeoR were introduced to hPSCs for the controlled overexpression of ALKF1174L and MYCN. hPSCs were differentiated into hNCCs, and oncogenes were activated by Dox.
(B) Addition of Dox to the cultured hNCCs induces strong expression of MYCN, ALKF1174L, and lentiviral-dependent tdTomato (bottom, nuclear staining; middle, peripheral staining; and top, whole-cell fluorescence, respectively). Scale bars indicate 10 μm.
(C) Oncogene activation was monitored for gene overexpression using qRT-PCR.
(D) Oncogenes overexpression was confirmed by western blot. The Dox treatment also induced self-activation of ALK (pALK Tyr1604; Kelly NB cell line serve as positive control).
(E and F) hNCCs with activated oncogenes formed significantly more colonies in soft agar (E, n = 28), and (F) exhibited significantly high rates of cell proliferation.
(G) Tumor formation of Dox treated hNCCs following injection into flanks of immune-compromised mice (tumor growth visible by IVIS, right, and after postmortem dissection, right).
Data presented as means; error bars represent SD; *** p value < 0.0001; (A) and (E): t test; (F): two-way ANOVA.