Figure 2. Generation of secondary hiPS cells.

A. Experimental scheme depicting the generation of secondary hiPS cells. hiPS cells were differentiated in vitro as embryoid bodies for 7 days, then plated to adherent conditions. Fibroblast-like colonies were picked and expanded for at least three passages prior to undergoing re-induction by doxycycline.

B. Alkaline phosphatase staining of reprogrammable cells grown in the absence or presence of doxycycline. Doxycycline was withdrawn at day 21, and colonies were stained and counted at day 30.

C. Morphology and expression of OCT4 and Tra-1–81 in doxycycline-independent secondary hiPS cells.

D. Microarray analysis of gene expression between BJ fibroblasts, HUES8 hES cells, primary fibroblast-derived hiPS cells, and a resulting secondary hiPS clone. Shown are genes with >2-fold expression value between BJ fibroblasts and HUES8 hES cells.

E. In vitro differentiation of secondary hiPS cells into lineages from all three germ layers. Immunostaining for i) Tuj1, ii) cardiac troponin T, and iii) alpha-fetoprotein.

F. Temporal requirement of factor expression in hiPS-derived fibroblast-like cells. 10⁴ cells were plated per time-point and doxycycline was withdrawn daily from days 4 through 19. The number of hES-like colonies that expressed Tra-1–81 were counted at day 25.