Human Hepatocytes with Drug Metabolic Function Induced from Fibroblasts by Lineage Reprogramming

Abstract

Obtaining fully functional cell types is a major challenge for drug discovery and regenerative medicine. Currently, a fundamental solution to this key problem is still lacking. Here, we show that functional human induced hepatocytes (hiHeps) can be generated from fibroblasts by overexpressing the hepatic fate conversion factors HNF1A, HNF4A, and HNF6 along with the maturation factors ATF5, PROX1, and CEBPA. hiHeps express a spectrum of phase I and II drug-metabolizing enzymes and phase III drug transporters. Importantly, the metabolic activities of CYP3A4, CYP1A2, CYP2B6, CYP2C9, and CYP2C19 are comparable between hiHeps and freshly isolated primary human hepatocytes. Transplanted hiHeps repopulate up to 30% of the livers of Tet-uPA/Rag2(-/-)/γc(-/-) mice and secrete more than 300 μg/ml human ALBUMIN in vivo. Our data demonstrate that human hepatocytes with drug metabolic function can be generated by lineage reprogramming, thus providing a cell resource for pharmaceutical applications.

Direct somatic lineage conversion

“…For mouse cells, the factor combination Hnf4a, Foxa1, Foxa2/Foxa3 [43] or Gata4, Hnf1a, Foxa3 [44] showed similar results and the combination FOXA3, HNF1A, HNF4A was effective in human cells although the maturation stage of induced hepatic cells may not be equivalent [45]. Of note, another study by Du et al claimed that a combination of HNF1A, HNF4A and HNF6 together with the factors ATF5, PROX1 and CEBPA was required to generate more mature hepatocytes [46]. These findings confirm modest inter-species differences in regulation of transcriptional programmes for lineage differentiation and further suggest that the complexity of an organism is partially reflected in lineage identity specification on the cellular level.…”

A simpler twist of fate

“…His initial efforts to directly turn skin cells into liver cells through the forced expression of master regulator genes alone yielded cells that failed to perform key, liverlike functions. Then, during a second round of screening, he identified additional genes that could complete the reprogramming 5 . He calls them maturation factors -genes that are unimportant for initiating the conversion but crucial for obtaining functionally mature cells.…”

In vivo reprogramming of hepatic myofibroblasts into hepatocytes attenuates liver fibrosis: back to the future?

“…More recently, some laboratories obtained HLCs by means of direct reprogramming of adult fibroblasts through overexpression of specific hepatocyte-related transcription factors in order to circumvent the induced pluripotent state (13)(14)(15)(16). Murine fibroblasts have been reprogrammed to HLCs by either combining transduction of Hnf1α, Gata4, and Foxa3 with p19(Arf) inactivation (13) or through ectopic expression of HNF4, Foxa1, Foxa2 or Foxa3 (14).…”