Fig. 5

Successful reprogramming of senescent human fibroblasts. a Adult human primary fibroblasts from a 50-year-old patient (F50) were passaged until they reached 46 population doublings (PDs), at which point the cells stopped dividing, and started exhibiting a senescent phenotype with an enlarged cellular morphology and 91% positivity for senescence-associated β-galactosidase (F50S). Scale bar, 200 µm. b Elongation of telomeres in iPSCs generated from F50 and F50S. The length of telomeres in the indicated cell lines was measured by qPCR analysis. Error bars, mean ± s.d. (n = 3). Prolonged fibroblast passaging of F50 resulted in a significant shortening of telomeres in F50S (***P < 0.001). P values were calculated using the unpaired two-tailed Student’s t test. c Immunofluorescent analysis showing expression of a panel of pluripotency markers in iPSC lines derived from F50S (I50S-1 and I50S-2). See Fig. 6b and Supplementary Figs. 11 and 12 for controls and iPSC lines derived from F50. Scale bar, 250 µm. d Immunostaining showing expression of the neuronal marker βIII-Tubulin (TUJ1) (ectoderm), and the endoderm-specific cytokeratin Endo-A in iPSCs derived from F50S subjected to directed differentiation. See Fig. 6d for directed differentiation of iPSC lines derived from F50. Scale bar, 250 µm. e Hematoxylin and eosin staining and immunofluorescent analysis of consecutive sections of teratomas derived from I50S-1 and I50S-2 showing histology and marker expression specific to ectoderm (βIII-Tubulin (TUJ1), neural tissues), mesoderm (vimentin, connective tissues), and endoderm (Endo-A, endothelium). See Fig. 6e for iPSC lines derived from F50. Scale bar, 250 µm