Direct reprogramming of human fibroblasts to hepatocyte-like cells by synthetic modified mRNAs.

Kamen P Simeonov,Hirdesh Uppal

doi:10.1371/journal.pone.0100134

Abstract

Direct reprogramming by overexpression of defined transcription factors is a promising new method of deriving useful but rare cell types from readily available ones. While the method presents numerous advantages over induced pluripotent stem (iPS) cell approaches, a focus on murine conversions and a reliance on retroviral vectors limit potential human applications. Here we address these concerns by demonstrating direct conversion of human fibroblasts to hepatocyte-like cells via repeated transfection with synthetic modified mRNAs. Hepatic induction was achieved with as little as three transcription factor mRNAs encoding HNF1A plus any two of the factors, FOXA1, FOXA3, or HNF4A in the presence of an optimized hepatic growth medium. We show that the absolute necessity of exogenous HNF1A mRNA delivery is explained both by the factor's inability to be activated by any other factors screened and its simultaneous ability to strongly induce expression of other master hepatic transcription factors. Further analysis of factor interaction showed that a series of robust cross-activations exist between factors that induce a hepatocyte-like state. Transcriptome and small RNA sequencing during conversion toward hepatocyte-like cells revealed global preferential activation of liver genes and miRNAs over those associated with other endodermal tissues, as well as downregulation of fibroblast-associated genes. Induced hepatocyte-like cells also exhibited hepatic morphology and protein expression. Our data provide insight into the process by which direct hepatic reprogramming occurs in human cells. More importantly, by demonstrating that it is possible to achieve direct reprogramming without the use of retroviral gene delivery, our results supply a crucial step toward realizing the potential of direct reprogramming in regenerative medicine.

Highlights

Direct reprogramming or conversion, where one cell type is directly converted into another without passage through a pluripotent intermediate, is an attractive source for valuable but unavailable cells, such as hepatocytes [1]
We have demonstrated that a combination of HNF1A along with two additional interchangeable factors is sufficient to reprogram human fibroblasts to a hepatocyte-like state
While our work was under review, two groups independently published on reprogramming human fibroblasts to hepatocytes using lentiviruses to deliver the desired factors [29,30]

Summary

Introduction

Direct reprogramming or conversion, where one cell type is directly converted into another without passage through a pluripotent intermediate, is an attractive source for valuable but unavailable cells, such as hepatocytes [1]. From basic and pharmaceutical research to cell therapy and regenerative medicine, cells derived by direct reprogramming offer near limitless potential [2,3,4]. Two major issues prevent the field from reaching full potential: First, while a variety of conversions have been discovered in mouse models [9,10,11,12,13], most encounter difficulty when applied to human cells, likely due to the differences in the transcriptional circuits controlling reprogramming in human and mouse [6]. For the field of direct reprogramming to reach full scientific and clinical relevance, these two issues must be resolved. We address the first of these problems by investigating the factors required to convert human neonatal fibroblasts to a hepatic fate and the second by relying on synthetic modified mRNAs to overexpress reprogramming factors without genomic modification

Methods

Results

Conclusion