FIGURE 4.

BAMX-derived iNPCs are highly expandable while maintaining their NPC properties. A and Ai–Aiii, during conversion under culture conditions, the cells exhibited a cluster morphology with a bipolar appearance (Ai). Cluster-forming cells expressed nestin (Aii) and SOX2 (Aiii), whereas non-cluster cells expressed neither nestin nor SOX2 (a, nestin⁻/SOX2⁻ cells in non-cluster cells; b, nestin⁺/SOX2⁺ cells in cluster cells; a and b represent higher magnification views of Aii and Aiii). B–E, iNPCs are efficiently induced and proliferate following expression of Bcl-xL. Although neural precursor-like cells (cluster form) were observed under BAM-transduced conditions, the cluster size was smaller, and these cells did not proliferate (Bi, C, and E), unlike BAMX-transduced cells (Bii, C, and E). The number of iNPCs increased more than 90-fold compared with the first stage (E), and the number of cleaved caspase-3 (casp3)⁺ cells decreased following BAMX transduction (Biii–Biv and D). F–I, iNPCs were stably expandable without loss of self-renewing potential. J–M, after freezing and thawing at passage 22, iNPCs do not lose their self-renewal potential. Notably, no significant change was observed in the expression of NPC markers KI67/nestin (J and K) and SOX2⁺ after freezing and thawing (L and M, respectively). Error bars, S.E. *, p < 0.05, compared with BAM-transduced cells. Scale bar, 20 μm (Fig. 2, Bi and Bii; scale bar, 100 μm).