Deciphering Transcription Factor Function to Engineer Mature and Functional iPSC-derived Hepatocytes

Abstract

The growing interest in differentiating induced pluripotent stem cells (iPSCs) into hepatocytes (iHeps) is driven by the shortage of liver donors for transplantations and the scarcity of primary human hepatocytes (PHHs) for toxicology studies. Thus far, iHeps have recapitulated many functions of PHHs, but current protocols yield iHeps with immature gene expression and epigenetic signatures. Transcription factors (TFs) have pioneering roles in coordinating gene expression networks during development, and the inability of current protocols to recapitulate correct TF expression throughout the differentiation protocol likely contributes to some of the functional deficiencies of iHeps. To address these limitations, we employed TFome-wide open reading frame (ORF) overexpression, CRISPR activation (CRISPRa), and CRISPR knock-out (CRISPRko) screens in both minimal and complete hepatic medias to identify factors that promote iHep maturity. iPSCs were lentivirally transduced with libraries of either ORFs or guide RNAs (gRNAs) and differentiated to iHeps. Subsequently, cells were immunostained for ALB and sorted to isolate cells based on ALB levels. This process generated a list of ~45 TFs putatively implicated in driving mature liver phenotypes when overexpressed, including TFs with known roles in liver development. Validation experiments by RNA-Seq corroborated the screen and confirmed the ability of most hits to upregulate the expression of maturation markers, including upregulation of ALB and SERPINA1 over 30- and 100-fold compared with negative controls, respectively. Further, scRNA-Seq demonstrated that the TF perturbations push the cells towards a more homogenously mature population, and functional experiments show that hit TFs produced iHeps with increased secretion of the liver-specific proteins. To identify TFs that have both synergistic maturation effects, top validated hits were studied in combination using a selective removal approach. Three TF isoforms (PPARG_3, NR5A2_1, and KLF7_2 (PNK)) uniquely led to augmented hepatic transcriptomic profiles when overexpressed together relative to each TF alone. Further studies have shown that PNK-iHeps enable long-term, functionally mature phenotypes in vitro in a variety of contexts including in other iPSC lines, other iHep complete protocols, iHep minimal protocols, and in 3D culture. Overall, we have developed a platform for screening iHep maturity and elucidated factors that improve hepatic maturity in a meaningful way for in vitro disease modeling and toxicology applications.