title: Derivation and characterization of a transgene-free human induced pluripotent stem cell line and conversion into defined clinical-grade conditions.
abstract: Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic genes remaining in actively transcribed regions of the genome, limit their potential for use in human therapeutic applications. Additionally, non-human antigens derived from stem cell reprogramming or differentiation into therapeutically relevant derivatives preclude these hiPSCs from being used in a human clinical context. In this video, we present a procedure for reprogramming and analyzing factor-free hiPSCs free of exogenous transgenes. These hiPSCs then can be analyzed for gene expression abnormalities in the specific intron containing the lentivirus. This analysis may be conducted using sensitive quantitative polymerase chain reaction (PCR), which has an advantage over less sensitive techniques previously used to detect gene expression differences. Full conversion into clinical-grade good manufacturing practice (GMP) conditions, allows human clinical relevance. Our protocol offers another methodology--provided that current safe-harbor criteria will expand and include factor-free characterized hiPSC-based derivatives for human therapeutic applications--for deriving GMP-grade hiPSCs, which should eliminate any immunogenicity risk due to non-human antigens. This protocol is broadly applicable to lentiviral reprogrammed cells of any type and provides a reproducible method for converting reprogrammed cells into GMP-grade conditions.
authors.affiliation: Department of Molecular and Medical Pharmacology, University of California, Los Angeles (UCLA).
authors.forename: Jason P
authors.initials: JP
authors.lastname: Awe
authors.affiliation: Department of Molecular and Medical Pharmacology, University of California, Los Angeles (UCLA).
authors.forename: Agustin
authors.initials: A
authors.lastname: Vega-Crespo
authors.affiliation: Department of Molecular and Medical Pharmacology, University of California, Los Angeles (UCLA); Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles (UCLA); jbyrne@mednet.ucla.edu.
authors.forename: James A
authors.initials: JA
authors.lastname: Byrne
mesh_terms: D002454:Cell Differentiation / Q000235:genetics; D003584:Cytological Techniques / Q000379:methods*; D015870:Gene Expression; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000502:physiology*; D016086:Lentivirus / Q000235:genetics; D019076:Transgenes