A Comparative View on Easy to Deploy non-Integrating Methods for Patient-Specific iPSC Production.

Authors:
Manzini S, Viiri LE, Marttila S, Aalto-Setälä K
In:
Source: Stem Cell Reports
Publication Date: (2015)
Issue: epub: 1-9
Research Area:
Stem Cells
Cells used in publication:
Fibroblast, dermal(NHDF-Ad), human adult
Species: human
Tissue Origin: dermal
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Platform:
Nucleofector™ I/II/2b
4D-Nucleofector™ X-Unit
Experiment

Authors tested 4 different transfection methods for generation of patient specific iPS cell lines (Sendai Virus, Nucleofection, NEON, Lipofectamine 3000). 6x10e5 human fibroblasts (from patients) were transfected with P2 and program EO-114 plus 3µg DNA of reprogram cocktail (pCXLE-hOCT3, pCXLE-hSK, pCXLE-hUL, pCXLE-EBNA1)

Abstract

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.