Journal Article

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

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

2014

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Adult / Animals / Biology and Life Sciences / Cell culture / Cell Culture Techniques - methods / Cell Culture Techniques - standards / Cell Differentiation / Cell Line / Cellular Reprogramming - drug effects / Cryopreservation / Deoxyribonucleic acid / Derivation / DNA / Embryoid Bodies / Embryos / Female / Fibroblasts / Fibroblasts - cytology / Fibroblasts - drug effects / Gene Expression Profiling / Germ Layers - cytology / Good Manufacturing Practice / Green Fluorescent Proteins - genetics / Growth / Humans / Induced Pluripotent Stem Cells - cytology / Induced Pluripotent Stem Cells - drug effects / Induced Pluripotent Stem Cells - transplantation / Infant, Newborn / Integration / Kruppel-Like Transcription Factors - genetics / Male / Messenger RNA / Methods / Mice / Mice, SCID / Middle Aged / mRNA / Mutation / Octamer Transcription Factor-3 - genetics / Physiological aspects / Pluripotency / Primary Cell Culture / Proto-Oncogene Proteins c-myc - genetics / RNA, Messenger - chemical synthesis / RNA, Messenger - isolation & purification / RNA, Messenger - pharmacology / RNA-Binding Proteins - genetics / Skin / Skin - cytology / SOXB1 Transcription Factors - genetics / Stem cells / Teratoma - etiology / Teratoma - pathology / Therapeutic applications / Transfection