Wisp1 is a key regulator of extracellular matrix genes. (A) Wisp1 regulates expression of several ECM genes. Expression of Tgfbi, Igfbp5, Dkk2, Nov, and Ccl20 were dramatically changed upon Wisp1 knockdown. Uninfected and 4F-infected MEFs were transfected with siWisp1 for 2 d and total RNAs were harvested for RT-qPCR analysis of different ECM genes. Error bars represent two independent experiments with duplicate wells. (B) Knockdown of Nov, Dkk2, and Tgfbi significantly enhances iPSC generation. MEFs were transduced with 4F at Day 0 and transfected with siRNAs at Day 5 post-infection. GFP+ colonies were quantified at around Days 11–13. Error bars represent three independent experiments with triplicate wells. (**) P < 0.01. (C) Overexpression of Wisp1-regulated ECM genes compromises reprogramming. The indicated ECM genes were cloned into pMX retroviral vectors. MEFs were transduced with 4F plus the indicated ECM genes and GFP+ colonies were quantified at around Days 11–13. Data were normalized to pMX-RFP-transduced cells. Error bars represent three independent experiments with triplicate wells. (**) P < 0.01. (D) Addition of recombinant ECM proteins compromises reprogramming. Purified recombinant TGFBI, DKK2, NOV, and CCL20 were added at a final concentration of 100 ng/mL to cultures of 4F-MEFs undergoing reprogramming. GFP+ colonies were quantified at Days 11–13. Error bars represent two independent experiments with triplicate wells. (*) P < 0.05.
