Fast and efficient multitransgenic modification of human pluripotent stem cells.
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.
1 Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO) , Department of Cardiac, Thoracic, Transplantation, and Vascular Surgery, 30625 Hannover, Germany .
Kristin
K
Schwanke
Sylvia
S
Merkert
Henning
H
Kempf
Susann
S
Hartung
Monica
M
Jara-Avaca
Christian
C
Templin
Gudrun
G
Göhring
Axel
A
Haverich
Ulrich
U
Martin
Robert
R
Zweigerdt
D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D004351:Drug Resistance / Q000235:genetics; D005822:Genetic Vectors / Q000378:metabolism; D006321:Heart / Q000000981:diagnostic imaging; D006801:Humans; D007457:Iodine Radioisotopes / Q000737:chemistry; D051379:Mice; D023421:Models, Animal; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D011877:Radionuclide Imaging; D051381:Rats; D013552:Swine; D027981:Symporters / Q000235:genetics / Q000378:metabolism; D019076:Transgenes / Q000235:genetics*; D024723:Ventricular Myosins / Q000235:genetics / Q000378:metabolism; D000099209:Sodium-Iodide Symporters
D007457:Iodine Radioisotopes; D027981:Symporters; D024723:Ventricular Myosins; D000099209:Sodium-Iodide Symporters