Background: Ex-vivo gene therapy is an interesting alternative to orthotropic liver transplantation (LT) for treating metabolic-liver diseases. However, the therapeutic efficiency of this method is hampered by the limited amount of auto-transplantable cells. Stem cell transplantation has been proposed as an attractive alternative approach to restore liver mass and function. Recent progress has been reported on the generation of induced pluripotent stem cells (iPSC) from somatic cells. We aimed to reprogram diseased human hepatocytes to pluripotency and to restore the missing metabolic function by ex-vivo gene therapy.
Methods: Hepatocytes from a patient with Familial Hypercholesterolemia who underwent LT were reprogrammed into iPSC using a single, multicistronic and excisable lentiviral vector expressing the four transcription factors, Oct4, Klf4, Sox2, and cMyc. LDL-uptake was then restored by the transduction of the human LDL-receptor using a lentivector. We established a differentiation protocol under well-defined culture conditions, alternating between normal and hypoxic O2-concentrations, with a cocktail of hepatocyte-specific growth factors.
Results: Hepatocyte-derived iPSC appeared indistinguishable from human embryonic stem cells as for: colony morphology, growth properties, expression of pluripotency-associated transcription factors, surface markers, differentiation-potential in 3D and in vivo teratoma assays. These cells were able to directly differentiate into definitive endoderm, hepatic progenitors, and mature hepatocytes, and to restore the missing metabolic function by transgenesis. Karyotype analysis of these cells did not show any gross genomic differences between original and reprogrammed cells. Methylome analysis showed that iPSC exhibited a methylation pattern similar to the cells they were reprogrammed from.
Conclusion: We show, for the first time, the reprogramming of diseased human hepatocytes to pluripotency. Differentiation into mature hepatocytes resulted more efficient than that with fibroblast-derived iPS cells. The generation of diseased hepatocytederived human iPSC lines thus provides the basis for the study of liver disease pathogenesis, as well as safety, efficacy and clinical potential assessments of liver-derived human iPSC cells which are intrinsically more prone to hepatocyte differentiation. Thanks to this, such technology provides a potentially unlimited reservoir of cells generating genetically corrected liver cells for the treatment of human liver diseases, via auto-transplantation of genetically modified hepatocytes, avoiding LT and lifelong immunosuppression.