Two new protocols to enhance the production and isolation of human induced pluripotent stem cell lines |
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| Authors: | Emily Dick Elena Matsa Jayson Bispham Mojgan Reza Michela Guglieri Andrew Staniforth Sue Watson Rajendra Kumari Hanns Lochmüller Lorraine Young David Darling Chris Denning |
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| Institution: | 1. Wolfson Centre for Stem Cells, Tissue Engineering & Modelling (STEM), Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK;2. NorthEast England Stem Cell Institute and Institute of Human Genetics, Newcastle University, International Centre for Life, Newcastle upon Tyne, NE1 3BZ , UK;3. Department of Cardiovascular Medicine, D Floor South Block, Queen''s Medical Centre, Nottingham, NG7 2UH, UK;4. Division of Pre-Clinical Oncology, School of Clinical Sciences, Queen''s Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK;5. Department of Haematological Medicine, King''s College, Guy''s, King''s and St Thomas’ School of Medicine, The Rayne Institute, 123 Coldharbour Lane, London, SE5 9NU, UK |
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| 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. |
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