title: Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette.
abstract: 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.
authors: Boston University Pulmonary Center, Boston, Massachusetts 02118, USA.
authors: Aba
authors: A
authors: Somers
authors: Jyh-Chang
authors: JC
authors: Jean
authors: Cesar A
authors: CA
authors: Sommer
authors: Amel
authors: A
authors: Omari
authors: Christopher C
authors: CC
authors: Ford
authors: Jason A
authors: JA
authors: Mills
authors: Lei
authors: L
authors: Ying
authors: Andreia Gianotti
authors: AG
authors: Sommer
authors: Jenny M
authors: JM
authors: Jean
authors: Brenden W
authors: BW
authors: Smith
authors: Robert
authors: R
authors: Lafyatis
authors: Marie-France
authors: MF
authors: Demierre
authors: Daniel J
authors: DJ
authors: Weiss
authors: Deborah L
authors: DL
authors: French
authors: Paul
authors: P
authors: Gadue
authors: George J
authors: GJ
authors: Murphy
authors: Gustavo
authors: G
authors: Mostoslavsky
authors: Darrell N
authors: DN
authors: Kotton
mesh_terms: 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