A Comparative View on Easy to Deploy non-Integrating Methods for Patient-Specific iPSC Production.
Induced pluripotent stem cells (iPSCs) are routinely produced from dermal fibroblasts, with potential applications ranging from in vitro disease models to drug discovery and regenerative medicine. The need of eliminating the remaining reprogramming factors after iPSC production spurred the development of non-integrating viruses such as Sendai and other methods to deliver episomal vectors, which are progressively lost upon cell division. We compared four widespread methods (Sendai virus, Nucleofector, Neon transfection system and Lipofectamine 3000) to generate integration-free iPSC lines from primary human dermal fibroblasts (hDF) of three patients. Furthermore, we performed extensive characterization of the iPSC lines. We were able to produce iPSC lines with all tested methods with variable efficiency. Sendai virus method achieved the overall highest reprogramming rate, followed by electroporation-based methods Nucleofector and Neon transfection systems. Chemical-based Lipofectamine 3000 delivery resulted in the lowest number of iPSC colonies. We found the reprogramming rate to be intrinsically dependent on the individual hDFs but the amenability of each hDF to reprogramming showed consistency between methods. Regardless of the reprogramming strategy, iPSCs obtained did not reveal any significant differences in their morphology, expression of pluripotency markers, EB formation, karyotype or gene expression profiles.
BioMediTech, University of Tampere, 33014, Tampere, Finland.|Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy.
Stefano
S
Manzini
BioMediTech, University of Tampere, 33014, Tampere, Finland. leena.viiri@uta.fi.
Leena E
LE
Viiri
BioMediTech, University of Tampere, 33014, Tampere, Finland.
Suvi
S
Marttila
BioMediTech, University of Tampere, 33014, Tampere, Finland.|Heart Hospital, Tampere University Hospital, Tampere, Finland.
Katriina
K
Aalto-Setälä
D049109:Cell Proliferation; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000235:genetics*; D000067470:Cellular Reprogramming Techniques / Q000379:methods*; D018274:Electroporation; D005347:Fibroblasts / Q000166:cytology; D005822:Genetic Vectors; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D008055:Lipids / Q000494:pharmacology; D029082:Sendai virus / Q000378:metabolism; D014162:Transfection / Q000379:methods
D008055:Lipids; C086724:Lipofectamine
Electroporation; Episomal; Human dermal fibroblast; Induced pluripotent stem cells (iPSC); Plasmid; Reprogramming