Abstract
Recently, it has been reported that human hepatocyte-like cells can be generated from fibroblasts by direct reprogramming technology. However, the conversion efficiency of human induced hepatocyte-like cells (hiHeps) is not high enough. In addition, comparative analysis with the existing models of hepatocytes, such as human iPS cell-derived hepatocyte-like cells and primary human hepatocytes, has not been sufficiently carried out. In this study, we screened hepatic transcription factors for efficient direct hepatic reprogramming and compared hepatic functions between hiHeps and other existing hepatocyte models. We found that human fibroblasts were efficiently converted into hiHeps by using a combination of ATF5, PROX1, FOXA2, FOXA3, and HNF4A (albumin+/alpha-1 antitrypsin+ cells = 27%, asialoglycoprotein receptor 1+ cells = 22%). The CYP expression levels and CYP activities in hiHeps were higher than those in human iPS cell-derived hepatocyte-like cells, but lower than those in short-term (4 hr) cultured primary human hepatocytes and primary human hepatocytes collected immediately after thawing. These results suggested that functional hiHeps could be efficiently generated by ATF5, PROX1, FOXA2, FOXA3, and HNF4A transduction. We believe that hiHeps generated by our method will be useful for the drug-discovery activities such as hepatotoxicity screening and drug metabolism tests.
Highlights
Hepatocyte-like cells differentiated from human iPS cells are expected to be applied for liver transplantation, drug metabolism tests, and hepatotoxicity screening
To perform a hepatic transcription factor screening for efficient direct hepatic reprogramming, we used LV vectors expressing the hepatic transcription factors ATF5, CEBPA, PROX1, FOXA2, FOXA3, HNF1A, HNF4A, HNF6, and GATA4 (9TFs)
To determine which of the 6 candidates (ATF5, PROX1, FOXA2, FOXA3, HNF1A, and HNF4A (6TFs)) were critical, we examined the effect of withdrawal of individual hepatic transcription factors from the pool of transduced candidate genes on the generation of human induced hepatocytelike cells (hiHeps) (Fig. 1C)
Summary
Hepatocyte-like cells differentiated from human iPS cells (iPS-Hepa) are expected to be applied for liver transplantation, drug metabolism tests, and hepatotoxicity screening. Several studies reported methods for the direct conversion of fibroblasts into hepatocyte-like cells without establishing iPS cells[3,4,5,6,7,8,9,10,11] Each of these methods uses a different combination of hepatic transcription factors for the direct reprogramming as described below. Because our ultimate goal is to apply hiHeps for drug-discovery research, we attempted to establish an efficient method for human iHeps, rather than mouse iHeps. In this decade, the differentiation technology of iPS-Hepa has been greatly improved. To investigate whether hiHeps have the potential to be utilized in drug-discovery studies, the expression of hepatic markers and hepatic functions of hiHeps were compared with those of existing hepatocyte models (human iPS-Hepa and PHH)
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