Small molecule supplementation potentiates miRNA mediated direct reprogramming of human fibroblasts into functional hepatocytes

Abstract

BackgroundHepatic transcription factors such as Hnf4α, Foxa1, Foxa2 or Foxa3 and Gata4 have successfully been used for directly reprogram the fibroblasts into hepatic lineages (iHeps). Although it has several advantages such as devoid of teratoma formation, robust conversion rate and potential in vivo reprograming to repair injured tissues, majority of the direct reprograming that employed genetic manipulations to modify the expression of tissue‐specific transcription factors have limitation in downstream clinical applications. Thus, it is critical to develop a new method for reprograming fibroblasts to hepatocytes.HypothesisWe hypothesized that the combination of small molecules such as epigenetic modifiers and GSK‐inhibitor, and miRNAs could successfully be used in direct differentiation of fibroblasts to iHeps.AimThe aim of this study was to directly reprogram the human fibroblasts into functional iHeps using small molecules that regulate reprogramming of somatic cells and miRNAs.MethodsHepatocyte specific miRNAs were selected from liver fibrosis, regulation of mesenchymal to epithelial transition (MET) and regulation of liver specific transcription factors HNF4α and FOXa2 miRNA datasets. Primary human fibroblasts grown to 80% confluent were transfected with either miRNA cocktail and/or small molecules. Transfected cells were cultured in hepatocyte growth media to generate iHeps. Albumin, E‐cad, HNF4α and FOXa2 expressions were detected by flow cytometry, immunostaining and qPCR analyses to establish iHeps. The iHeps mixed with human vascular endothelial cells (HUVEC) in collagen were implanted in partial‐hepatectomized immune‐compromised mice. Explants collected were characterized 4 weeks following implantation. Human albumin level was estimated by ELISA in iHeps implanted mouse serum.ResultsmiRNA cocktail alone significantly downregulated Snail1 and vimentin, and upregulation E‐cad specific mRNA abundance. Although small molecules do not exert any effect, its supplementation along with miRNA cocktail drastically downregulated Snail1 and vimentin, and upregulated E‐cad abundance. Albumin, E‐cad, HNF4α and FOXa2 expression established iHeps lineage. Integration of iHeps with vascular structures were present in liver (shown by arrows in Figure ), while human albumin (control vs iHeps: 0.78 ± 0.01 vs 63.8 ± 0.2 ng/ml) was detected in serum of mice implanted with iHeps.ConclusionsWe conclude that: 1) the down‐regulation of Snail1 and vimentin, and upregulation E‐cad establish MET; 2) the small molecule supplementation potentiate the miRNA cocktail induced MET; and 3) the direct reprograming of human fibroblast using miRNA along with small molecule supplementation generate a viable and functional iHeps.SpeculationWe speculate that small molecules along with miRNA cocktail could potentially be used to treat liver fibrosis in vivo.Support or Funding InformationFunding was provided by the Institute of Bioengineering and Nanotechnology(Biomedical Research Council, Agency for Science, Technology and Research, Singapore). VMR was supported by NIH grants DK112085 and DK104791.H&E staining of sections of explants harvested from partially hepatectomized mouse modelFigure 1