Dataset Information

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

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.

SUBMITTER: Manzini S

PROVIDER: S-EPMC4653244 | biostudies-other | 2015 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

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

Manzini Stefano S Viiri Leena E LE Marttila Suvi S Aalto-Setälä Katriina K

Stem cell reviews and reports 20151201 6

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, Nucleofe ...[more]

PMID: 26341105

Similar Datasets

Project description:Human-induced pluripotent stem cells (iPSCs) are derived from differentiated somatic cells using defined factors and provide a renewable source of autologous cells for cell therapy. Many reprogramming methods have been employed to generate human iPSCs, including the use of integrating vectors and non-integrating vectors. Maintenance of the genomic integrity of iPSCs is highly desirable if the cells are to be used in clinical applications. Here, using the Affymetrix Cytoscan HD array, we investigated the genomic aberration profiles of 19 human cell lines: 5 embryonic stem cell (ESC) lines, 6 iPSC lines derived using integrating vectors (“integrating iPSC lines”), 6 iPSC lines derived using non-integrating vectors (“non-integrating iPSC lines”), and the 2 parental cell lines from which the iPSCs were derived. The genome-wide copy number variation (CNV), loss of heterozygosity (LOH) and mosaicism patterns of integrating and non-integrating iPSC lines were investigated. The maximum sizes of CNVs in the genomes of the integrating iPSC lines were 20 times higher than those of the non-integrating iPSC lines. Moreover, the total number of CNVs was much higher in integrating iPSC lines than in other cell lines. The average numbers of novel CNVs with a low degree of overlap with the DGV and of likely pathogenic CNVs with a high degree of overlap with the ISCA database were highest in integrating iPSC lines. Different SNP calls revealed that, using the parental cell genotype as a reference, integrating iPSC lines displayed more single nucleotide variations and mosaicism than did non-integrating iPSC lines. This study describes the genome stability of human iPSCs generated using either a DNA-integrating or non-integrating reprogramming method, of the corresponding somatic cells, and of hESCs. Our results highlight the importance of using a high-resolution method to monitor genomic aberrations in iPSCs intended for clinical applications to avoid any negative effects of reprogramming or cell culture. 19 human cell lines: 5 embryonic stem cell (ESC) lines, 6 iPSC lines derived using integrating vectors (“integrating iPSC lines”), 6 iPSC lines derived using non-integrating vectors (“non-integrating iPSC lines”), and the 2 parental cell lines from which the iPSCs were derived.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets