Rapid and Efficient Conversion of Integration-Free Human Induced Pluripotent Stem Cells to GMP-Grade Culture Conditions

Kyle M. Loh,Jens Durruthy-Durruthy,Ioannis Karakikes,James Byrne,Vittorio Sebastiano,Patrick C. Lee,Amander Clark,Jason Awe,Cyril Y. Ramathal,Elias T. Zambidis,Andrew R. Hoffman,Julia D. Heidmann,Joseph C. Wu,Saravanan Karumbayaram,Sharon F. Briggs,Renee A. Reijo Pera

doi:10.1371/journal.pone.0094231

Abstract

Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs) will be realized. Nonetheless, clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP). Optimally, derivation of hiPSCs should be rapid and efficient in order to minimize manipulations, reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here, we provide an optimized, fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity, purity, stability and safety at a GMP facility and cryopreserved. To our knowledge, as a proof of principle, these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes.

Highlights

Derivation of human induced pluripotent stem cells from virtually any adult tissue provides numerous opportunities for development of therapeutic strategies for multiple pathologies that involve tissue degeneration [1,2]
The clinical application of human induced pluripotent stem cells (hiPSCs) requires that their production must meet Good Manufacturing Practice (GMP)-compatible standards and that the derivation of specified cell derivatives from pluripotent stem cells is performed under GMP conditions
Previous federal oversight has indicated that the conversion of pluripotent stem cell lines from a research-grade environment to a GMP-grade environment is potentially an accepted practice as long as rigorous tests are run on the converted lines to make sure that no detectable contamination of pathogens of animal origin can be found in the cells

Summary

Introduction

Derivation of human induced pluripotent stem cells (hiPSCs) from virtually any adult tissue provides numerous opportunities for development of therapeutic strategies for multiple pathologies that involve tissue degeneration [1,2]. Nonintegrative DNA-based methods of inducing pluripotency including episomal vectors [4] and minicircles [5] have been developed, it is difficult to exclude the possibility of integration of very small fragments of DNA. Extensive passaging is required to dilute out exogenous vectors, which increases opportunities for the acquisition of mutations. Other proposed non-integrative methods like protein-based reprogramming [6] or Sendai virus [7] are very inefficient (protein-based) or require extensive passaging to remove residual viral expression (Sendai virus). It has been shown that modified mRNAs can be used to efficiently derive footprint-free iPSCs [8,9]

Methods

Results

Conclusion