title: Two new protocols to enhance the production and isolation of human induced pluripotent stem cell lines.
abstract: There are two critical stages in the retroviral reprogramming of somatic cells to produce human induced pluripotent stem cell (hiPSC) lines. One is the production of high titer virus required to reprogram somatic cells; the other is identification of true hiPSC colonies from heterogeneous cell populations, and their isolation and expansion to generate a sustainable, pluripotent stem cell line. Here we describe simple, time-saving methods to address the current difficulties at these two critical junctures. First, we have developed a method to increase the number of infectious viral units 600-fold. Second, we have developed a TRA-1-81-based positive selection column method for isolating "true" hiPSCs from the heterogeneous cell populations, which overcomes the labor-intensive and highly subjective method of manual selection of hiPSC colonies. We have used these techniques to produce 8 hiPSC lines from human fibroblasts and we believe that they are of considerable utility to researchers in the hiPSC field.
authors.affiliation: Wolfson Centre for Stem Cells, Tissue Engineering & Modelling, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.
authors.forename: Emily
authors.initials: E
authors.lastname: Dick
authors.forename: Elena
authors.initials: E
authors.lastname: Matsa
authors.forename: Jayson
authors.initials: J
authors.lastname: Bispham
authors.forename: Mojgan
authors.initials: M
authors.lastname: Reza
authors.forename: Michela
authors.initials: M
authors.lastname: Guglieri
authors.forename: Andrew
authors.initials: A
authors.lastname: Staniforth
authors.forename: Sue
authors.initials: S
authors.lastname: Watson
authors.forename: Rajendra
authors.initials: R
authors.lastname: Kumari
authors.forename: Hanns
authors.initials: H
authors.lastname: Lochmüller
authors.forename: Lorraine
authors.initials: L
authors.lastname: Young
authors.forename: David
authors.initials: D
authors.lastname: Darling
authors.forename: Chris
authors.initials: C
authors.lastname: Denning
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000502:physiology*; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009368:Neoplasm Transplantation
chemical_list: D015415:Biomarkers