Transdifferentiation of human fibroblasts to hepatocyte-like cells by transcription factors

Introduction: Liver transplantation as a treatment for late stage liver disease is limited by the availability of suitable donor organs. In order to help affected patients, the development of liver cell therapy may significantly augment therapeutic options (1). However, primary hepatocytes from healthy donors are limited in clinical application by their inability to be expanded in vitro and by the need of immunosuppression after allogenic transplantation. Liver cells derived from more readily available human cells represent an attractive alternative cell source. Recently, hepatic (trans-)differentiation by lentiviral expression of transcription factors has been demonstrated in murine hepatic precursor cells (2) and fibroblasts (3,4). Achieving a similar transdifferentiation of human non-hepatic cells would be an important step towards liver cell therapy. In this project, expression of the hepatic transcription factors FOXA3, GATA4, HNF1a and HNF4a in human fibroblasts resulted in the generation of induced hepatocyte-like (iHep) cells that displayed several characteristics of primary human hepatocytes.

Results: Human fibroblasts were transduced with lentiviral vectors coding for FOXA3, GATA4, HNF1a and HNF4a. After eleven days of culturing, fibroblasts were converted to iHep cells and were analyzed for morphology, hepatic marker expression, global gene expression and hepatic function. iHep cells were epithelial and angular, resembling primary hepatocytes in cell culture. The hepatic marker genes albumin, α-fetoprotein, α-1-antitrypsin, APOB, TAT, TDO, and CYP1A2 were induced in iHep cells. TDO and CYP1A2 expression levels were in the range of primary hepatocytes. The global gene expression pattern of iHep cells had similarities to primary hepatocytes as well as to untransduced fibroblasts. In contrast to untransduced fibroblasts, iHep cells were shown to secrete albumin, at 1% the rate of primary hepatocytes. iHep cells exhibited significantly higher CYP1A2 activity than untransduced fibroblasts, reaching 10% of the activity in primary hepatocytes.

Conclusion and further plans:

These results indicate that expression of FOXA3, GATA4, HNF1a and HNF4a can convert human fibroblasts to hepatocyte-like cells that share several characteristics with primary hepatocytes. This concept has been shown in murine fibroblasts before, and we demonstrate it the first time in human fibroblasts. Liver engraftment of iHep cells in an appropriate mouse model could enable them to reach a more mature hepatocyte-like phenotype. Consequently, we transplanted the iHep cells that we generated into immunodeficient Fah-/- mice to analyze engraftment and in vivo function. This experiment is currently ongoing.

References:

(1) Heo J et al. (2006). Hepatology 44, 1478–1486.

(2) Iacob R et al. (2011) Stem Cell Res. 6, 251–61.

(3) Huang P et al. (2011) Nature 475, 386–9.

(4) Sekiya S, Suzuki A. (2011) Nature 475, 390–3.