Figure 1.

Establishment of a Rapid and Highly Efficient Cell Reprogramming System by Modified Factors

(A) Schematic diagram of construction of modified reprogramming factors, including OCT4-YAP^TAD (Oy), SOX2-YAP^TAD (Sy), and NANOG-YAP^TAD (Ny). Coding sequences of murine Oct4, Sox2, Nanog were fused with murine Yap transcription activation domain (TAD) (amino acids 275–489) in the C terminus directly to generate Oct4-YAP^TAD (Oy), Sox2-YAP^TAD (Sy), and Nanog-YAP^TAD (Ny) expression constructs, respectively. The modified factors were then cloned into pMXs-retroviral vector.

(B) HEK293 cells were transfected in a 24-well plate with various expression plasmids along with a pGL4.2-basic-6XCR4 vector and Renilla control vector using the calcium phosphate precipitation method. Thirty-six hours posttransfection, cells were lysed for the measurement of luciferase activity. Firefly luciferase activities were normalized based on the Renilla activity. ^∗∗∗p < 0.001 versus control groups. NS, not significant.

(C) Schematic diagram describing the procedure for the generation of iPSCs induced by the modified factor combination OySyNyK. Oct4-GFP MEFs were used to generate iPSCs.

(D) Statistical summary showing the different efficiencies of GFP-positive colony formation induced by OSNK or OySyNyK. In this assay, OySyNyK- and OSNK-induced OCT4-GFP-positive colonies were counted at day 7 and day 16, respectively. ^∗∗∗p < 0.001 versus control groups.

(E) Dynamics of the percentage of GFP-positive cells in OSNK- or OySyNyK-infected MEFs during somatic cell reprogramming. ^∗∗∗p < 0.001 versus control groups.

(F/F′ and G/G′) Fluorescence (F and G) and bright-field (F′ and G′) images showing OySyNyK-induced iPSCs at passage 1 (F) or passage 10 (G).

(H) qRT-PCR assays showing the expression levels of endogenous pluripotency markers Oct4, Sox2, and Nanog in OSNK- and OySyNyK-transduced MEFs at different time points. Both iPSCs and ESCs were included for the comparison. OSNK- and OySyNyK-transduced MEFs groups were compared to MEF groups. ^∗∗∗p < 0.001 versus control groups.

Data from (B), (D), (E), and (H) are representative of at least three independent experiments (mean and SD of triplicate assays). Groups were compared using the Student’s t test. N.S, not significant; ^∗∗∗p < 0.001 versus control groups.