Urine-derived cells provide a readily accessible cell type for feeder-free mRNA reprogramming

Q. Francheteau,F. Chaubron,S. Knöbel,L. David,A. Gaignerie,C. Chariau,K. Si-Tayeb,N. Gaborit,L. Flippe,N. Lefort,A. Caillaud,A. Girardeau,V. Forest-Choquet,V. Francois–Campion,A. Derevier,M. Rousselle

doi:10.1038/s41598-018-32645-2

Abstract

Over a decade after their discovery, induced pluripotent stem cells (iPSCs) have become a major biological model. The iPSC technology allows generation of pluripotent stem cells from somatic cells bearing any genomic background. The challenge ahead of us is to translate human iPSCs (hiPSCs) protocols into clinical treatment. To do so, we need to improve the quality of hiPSCs produced. In this study we report the reprogramming of multiple patient urine-derived cell lines with mRNA reprogramming, which, to date, is one of the fastest and most faithful reprogramming method. We show that mRNA reprogramming efficiently generates hiPSCs from urine-derived cells. Moreover, we were able to generate feeder-free bulk hiPSCs lines that did not display genomic abnormalities. Altogether, this reprogramming method will contribute to accelerating the translation of hiPSCs to therapeutic applications.

Highlights

Human pluripotent stem cells, either embryonic stem cells derived from human blastocysts, or induced pluripotent stem cells derived from somatic cells, have the ability to differentiate into all cell types comprising an adult organism, offering a great promise for regenerative medicine
We noticed that the transfection efficiency was high in all three cell types, as represented by GFP expression (Fig. 1a), despite the nuclear GFP (nGFP) mRNA accounting for only 5% of the cocktail
As no transgenes were present after day 11 in this reprogramming protocol, human iPSCs (hiPSCs) lines were readily established and could be banked at early passages

Summary

Introduction

Human pluripotent stem cells (hPSCs), either embryonic stem cells (hESCs) derived from human blastocysts, or induced pluripotent stem cells (hiPSCs) derived from somatic cells, have the ability to differentiate into all cell types comprising an adult organism, offering a great promise for regenerative medicine. Schlaeger and colleagues assessed these factors and demonstrated that mRNA reprogramming is the most efficient method based on the number of iPS clones obtained per cells seeded[9] They have shown that mRNA reprogramming is the method with the least impact on genome stability. The original protocol required daily transfections of the reprogramming factors for 20 days This protocol was subsequently improved, requiring less than 12 transfections, and allowing feeder-free derivation of hiPSCs11–13, reducing the complexity of the protocol and paving the way for GMP production of hiPSCs. To date, the main source of cells for mRNA reprogramming is skin fibroblasts, a cell type that tolerates genomic rearrangements, that will be present in the fibroblasts and in the subsequent hiPSC lines[2]. Our work will extend the versatility of mRNA reprogramming method and help clear the remaining roadblocks to the therapeutic application of hiPSCs

Methods

Results

Conclusion