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Fast and efficient multitransgenic modification of human pluripotent stem cells


Schwanke, Kristin; Merkert, Sylvia; Kempf, Henning; Hartung, Susann; Jara-Avaca, Monica; Templin, Christian; Göhring, Gudrun; Haverich, Axel; Martin, Ulrich; Zweigerdt, Robert (2014). Fast and efficient multitransgenic modification of human pluripotent stem cells. Human Gene Therapy. Methods, 25(2):136-153.

Abstract

Human pluripotent stem cells (hPSCs) represent a prime cell source for pharmacological research and regenerative therapies because of their extensive expansion potential and their ability to differentiate into essentially all somatic lineages in vitro. Improved methods to stably introduce multiple transgenes into hPSCs will promote, for example, their preclinical testing by facilitating lineage differentiation and purification in vitro and the subsequent in vivo monitoring of respective progenies after their transplantation into relevant animal models. To date, the establishment of stable transgenic hPSC lines is still laborious and time-consuming. Current limitations include the low transfection efficiency of hPSCs via nonviral methods, the inefficient recovery of genetically engineered clones, and the silencing of transgene expression. Here we describe a fast, electroporation-based method for the generation of multitransgenic hPSC lines by overcoming the need for any preadaptation of conventional hPSC cultures to feeder-free conditions before genetic manipulation. We further show that the selection for a single antibiotic resistance marker encoded on one plasmid allowed for the stable genomic (co-)integration of up to two additional, independent expression plasmids. The method thereby enables the straightforward, nonviral generation of valuable multitransgenic hPSC lines in a single step. Practical applicability of the method is demonstrated for antibiotic-based lineage enrichment in vitro and for sodium iodide symporter transgene-based in situ cell imaging after intramyocardial cell infusion into explanted pig hearts.

Abstract

Human pluripotent stem cells (hPSCs) represent a prime cell source for pharmacological research and regenerative therapies because of their extensive expansion potential and their ability to differentiate into essentially all somatic lineages in vitro. Improved methods to stably introduce multiple transgenes into hPSCs will promote, for example, their preclinical testing by facilitating lineage differentiation and purification in vitro and the subsequent in vivo monitoring of respective progenies after their transplantation into relevant animal models. To date, the establishment of stable transgenic hPSC lines is still laborious and time-consuming. Current limitations include the low transfection efficiency of hPSCs via nonviral methods, the inefficient recovery of genetically engineered clones, and the silencing of transgene expression. Here we describe a fast, electroporation-based method for the generation of multitransgenic hPSC lines by overcoming the need for any preadaptation of conventional hPSC cultures to feeder-free conditions before genetic manipulation. We further show that the selection for a single antibiotic resistance marker encoded on one plasmid allowed for the stable genomic (co-)integration of up to two additional, independent expression plasmids. The method thereby enables the straightforward, nonviral generation of valuable multitransgenic hPSC lines in a single step. Practical applicability of the method is demonstrated for antibiotic-based lineage enrichment in vitro and for sodium iodide symporter transgene-based in situ cell imaging after intramyocardial cell infusion into explanted pig hearts.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Clinic for Cardiology
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:April 2014
Deposited On:12 Feb 2015 13:10
Last Modified:12 Aug 2017 06:31
Publisher:Mary Ann Liebert
ISSN:1946-6536
Additional Information:This is a copy of an article published in the Human Gene Therapy. Methods © 2014 copyright Mary Ann Liebert, Inc.; Human Gene Therapy. Methods is available online at: http://online.liebertpub.com
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1089/hgtb.2012.248
PubMed ID:24483184

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