Generation of iPSCs as a Pooled Culture Using Magnetic Activated Cell Sorting of Newly Reprogrammed Cells.
Although significant advancement has been made in the induced pluripotent stem cell (iPSC) field, current methods for iPSC derivation are labor intensive and costly. These methods involve manual selection, expansion, and characterization of multiple clones for each reprogrammed cell sample and therefore significantly hampers the feasibility of studies where a large number of iPSCs need to be derived. To develop higher throughput iPSC reprogramming methods, we generated iPSCs as a pooled culture using rigorous cell surface pluripotent marker selection with TRA-1-60 or SSEA4 antibodies followed by Magnetic Activated Cell Sorting (MACS). We observed that pool-selected cells are similar or identical to clonally derived iPSC lines from the same donor by all criteria examined, including stable expression of endogenous pluripotency genes, normal karyotype, loss of exogenous reprogramming factors, and in vitro spontaneous and lineage directed differentiation potential. This strategy can be generalized for iPSC generation using both integrating and non-integrating reprogramming methods. Our studies provide an attractive alternative to clonal derivation of iPSCs using rigorously selected cell pools and is amenable to automation.
Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America.
Wenli
W
Yang
Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America.
Ying
Y
Liu
Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America.
Katherine J
KJ
Slovik
Division of Cardiology, Department of Medicine; Institute for Stem Cell Biology and Regenerative Medicine, Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, United States of America.
Joseph C
JC
Wu
Program in Regenerative Medicine and Stem Cell Biology, Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, WI United States of America.
Stephen A
SA
Duncan
Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America.
Daniel J
DJ
Rader
Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America.
Edward E
EE
Morrisey
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000502:physiology*; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology; D022781:Hepatocytes / Q000166:cytology / Q000502:physiology; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000502:physiology*; D051379:Mice