Cell-generated forces contribute to bottleneck during somatic cell reprogramming

Our study investigates the effects of cell adhesion and traction forces on reprogramming by utilizing a human fibroblast cell line (hiF-T) that can be reprogrammed by activating the expression of an OKSM (OCT4, KLF4, SOX2, C-MYC) gene cassette through the addition of doxycycline. We performed RNA interference experiments on 103 genes found in the ‘adhesome’ (encoding integrins, cadherins, and associated proteins) that are expressed and dynamically regulated (6-fold or more) across the reprogramming timeline. The impact of each shRNA on iPSC generation efficiency was assessed by immunostaining for TRA-1-60, a known pluripotency marker. From this RNAi screen, we identified the SHROOM3 knockdown as a potent reprogramming efficiency enhancer and generated RNA-seq timelines throughout reprogramming with or without shRNAs targeting SHROOM3 or a LacZ control. Gene co-expression network analysis highlights the existence of a critical state transition regulated through a SHROOM3-SRF signaling axis during late reprogramming, which coincides with cell-mediated force generation. Taken together, our data suggest that adhesome gene expression acts as a significant impediment to somatic cell reprogramming through the activation of mechano-signaling pathways associated with cell traction forces. These results provide important new insights into the role of cell adhesion and force generation during reprogramming to pluripotency and somatic cell fate transitions more broadly. Somatic reprogramming RNA-seq timelines with or without lentivirus expressing shRNA vectors targeting SHROOM3 or LacZ, a non-targeting control.