Small Molecules Facilitate Single Factor-Mediated Hepatic Reprogramming

Abstract

Recent studies have shown that defined factors could lead to the direct conversion of fibroblasts into induced hepatocyte-like cells (iHeps). However, reported conversion efficiencies are very low, and the underlying mechanism of the direct hepatic reprogramming is largely unknown. Here, we report that direct conversion into iHeps is a stepwise transition involving the erasure of somatic memory, mesenchymal-to-epithelial transition, and induction of hepatic cell fate in a sequential manner. Through screening for additional factors that could potentially enhance the conversion kinetics, we have found that c-Myc and Klf4 (CK) dramatically accelerate conversion kinetics, resulting in remarkably improved iHep generation. Furthermore, we identified small molecules that could lead to the robust generation of iHeps without CK. Finally, we show that Hnf1α supported by small molecules is sufficient to efficiently induce direct hepatic reprogramming. This approach might help to fully elucidate the direct conversion process and also facilitate the translation of iHep into the clinic.

Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells

“…Some of the theoretical advantages of direct reprogramming strategies include the use of less time-consuming protocols, avoidance of the pluripotency stage and generation of cells with increased maturation profile for personalized medicine applications [3]. In fact, iHEPs were generated by direct reprogramming and were shown to be amenable for in vitro expansion and able to repopulate the liver when transplanted into mice [1,2,4].…”

Role of Hepatic-Specific Transcription Factors and Polycomb Repressive Complex 2 during Induction of Fibroblasts to Hepatic Fate

“…The significant presence of Klf4 in the regulation of DEGs and DE-TFs (Fig 2A and Fig 4A and 4B) supports the fact that Klf4 can be used as a candidate factor to increase the efficiency of the induction of hepatic fate from fibroblasts. For example, interestingly, Lim et al (2016) showed that using Klf4 and Myc in combination with hepatic-specific factors led to a significant increase in the efficiency of the generation of iHeps from fibroblasts [28]. Also, this study showed Klf4 and Myc are not crucial for the induction hepatocyte-like cells but increase the efficiency of the process.…”

“…Lim et al (2016) used a combination of Klf4 and Myc as their Yamanaka TFs along with Hnf4a as their hepatic-specific factor to drive the direct conversion of fibroblasts into iHeps. This group was also able to obtain iHeps from fibroblasts by combining small molecules with Hnf4a [28]. Despite the extensive use of TFs for the generation of hepatocyte-like cells through direct reprogramming, the molecular mechanisms by which different TFs regulate the gene expression program have not been well understood.…”

Automatic identification of small molecules that promote cell conversion and reprogramming

“…Recently, several methods relying fully or partially on drug treatment to enhance cell conversion have emerged 12 . Many of these use fibroblasts as the starting cell type, reprogrammed towards pluripotency 13,14 or trans-differentiated to specialized cell types including neurons 15 , endothelial cells 16 , pancreatic like cells 17 , cardiomyocytes 18 , hepatocytes 19 or other cell types 20,21,22 . Such studies provide a proof of principle for drug-based reprogramming, the exact mechanisms of which, however, are often poorly understood, making extensive trial-and-error unavoidable.…”