Abstract
Direct reprogramming using defined sets of transcription factors (TFs) is a recent strategy for generating induced hepatocytes (iHeps) from fibroblasts for use in regenerative medicine and drug development. Comprehensive studies detailing the regulatory role of TFs during this reprogramming process could help increase its efficiency. This study aimed to find the TFs with the greatest influences on the generation of iHeps from fibroblasts, and to further understand their roles in the regulation of the gene expression program. Here, we used systems biology approaches to analyze high quality expression data sets in combination with TF-binding sites data and protein-protein interactions data during the direct reprogramming of fibroblasts to iHeps. Our results revealed two main patterns for differentially expressed genes (DEGs): up-regulated genes were categorized as hepatic-specific pattern, and down-regulated genes were categorized as mesoderm- and fibroblast-specific pattern. Interestingly, hepatic-specific genes co-expressed and were regulated by hepatic-specific TFs, specifically Hnf4a and Foxa2. Conversely, the mesoderm- and fibroblast-specific pattern was mainly silenced by polycomb repressive complex 2 (PRC2) members, including Suz12, Mtf2, Ezh2, and Jarid2. Independent analysis of both the gene and core regulatory network of DE-TFs showed significant roles for Hnf4a, Foxa2, and PRC2 members in the regulation of the gene expression program and in biological processes during the direct conversion process. Altogether, using systems biology approaches, we clarified the role of Hnf4a and Foxa2 as hepatic-specific TFs, and for the first time, introduced the PRC2 complex as the main regulator that favors the direct reprogramming process in cooperation with hepatic-specific factors.
Highlights
Liver transplantation is the only approved strategy for the treatment of patients suffering from liver failure
Comparisons between samples were defined and normalized data were loaded into Flexarray software in order to detect differentially expressed genes (DEGs) and their Gene Expression Omnibus (GEO) platforms were downloaded for annotations [38]
We found that direct conversion of fibroblasts into induced hepatocytes (iHeps) are governed in parallel by hepatic-inducing factors and factors which take part in silencing fibroblast-specific genes
Summary
Liver transplantation is the only approved strategy for the treatment of patients suffering from liver failure. The limit in the number of liver donors, high cost, and rejection of transplanted tissue require alternative strategies for tackling liver diseases [1]. Cellular differentiation and direct reprogramming are two alternative strategies for the generation of hepatocytes. The differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is a multi-stage process that provides an unlimited number of hepatocytes, but poses risks for tumorigenesis, due to the possibility of residual undifferentiated cells [1,3,4]. The direct induction of somatic cells to a hepatic fate is a single step strategy for the generation of mature cells without tumorigenesis risks. Direct reprogramming is faster and safer in comparison with the differentiation of pluripotent stem cells (PSCs) into hepatocyte-like cells [5,6]
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