Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette.
The development of methods to achieve efficient reprogramming of human cells while avoiding the permanent presence of reprogramming transgenes represents a critical step toward the use of induced pluripotent stem cells (iPSC) for clinical purposes, such as disease modeling or reconstituting therapies. Although several methods exist for generating iPSC free of reprogramming transgenes from mouse cells or neonatal normal human tissues, a sufficiently efficient reprogramming system is still needed to achieve the widespread derivation of disease-specific iPSC from humans with inherited or degenerative diseases. Here, we report the use of a humanized version of a single lentiviral "stem cell cassette" vector to accomplish efficient reprogramming of normal or diseased skin fibroblasts obtained from humans of virtually any age. Simultaneous transfer of either three or four reprogramming factors into human target cells using this single vector allows derivation of human iPSC containing a single excisable viral integration that on removal generates human iPSC free of integrated transgenes. As a proof of principle, here we apply this strategy to generate >100 lung disease-specific iPSC lines from individuals with a variety of diseases affecting the epithelial, endothelial, or interstitial compartments of the lung, including cystic fibrosis, α-1 antitrypsin deficiency-related emphysema, scleroderma, and sickle-cell disease. Moreover, we demonstrate that human iPSC generated with this approach have the ability to robustly differentiate into definitive endoderm in vitro, the developmental precursor tissue of lung epithelia.
Somers
Aba
A
Boston University Pulmonary Center, Boston, Massachusetts 02118, USA.
Jean
Jyh-Chang
JC
Sommer
Cesar A
CA
Omari
Amel
A
Ford
Christopher C
CC
Mills
Jason A
JA
Ying
Lei
L
Sommer
Andreia Gianotti
AG
Jean
Jenny M
JM
Smith
Brenden W
BW
Lafyatis
Robert
R
Demierre
Marie-France
MF
Weiss
Daniel J
DJ
French
Deborah L
DL
Gadue
Paul
P
Murphy
George J
GJ
Mostoslavsky
Gustavo
G
Kotton
Darrell N
DN
D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000235:genetics / Q000502:physiology; D004707:Endoderm / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*; D016086:Lentivirus / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction