Adhesome-targeting shRNA modulates inferred cell fate trajectories and cell-cell communication during iPSC reprogramming

Structural properties of cell-adhesive surfaces influence induced pluripotent stem cell (iPSC) reprogramming efficiency, yet their influence on cell fate transitions remains poorly characterized. Here, we investigated the collective role of genes encoding proteins mediating cell-cell and cell-ECM adhesions and their effectors—collectively termed the “adhesome”—in cell fate reprogramming. Using an RNAi screen targeting 103 dynamically regulated adhesome genes, we found that elevated adhesome gene expression generally suppresses reprogramming. Among these, shRNAs targeting both major transcript isoforms of SHROOM3 significantly enhanced iPSC generation in a human inducible cell line and in primary cells. Single-cell RNA-seq analysis suggests that SHROOM3 knockdown alters computationally inferred developmental trajectories through a neural crest-like intermediate, destabilizes alternative fates (e.g., smooth muscle-like populations), reduces transcriptional noise in pluripotency genes, and reduces fibronectin signaling. Furthermore, fibronectin surface coating was a potent inhibitor of iPSC reprogramming, which was abrogated upon SHROOM3 knockdown.