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      Generation of KCL027 research grade human embryonic stem cell line carrying a mutation in the HTT gene

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          Abstract

          The KCL027 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (43 trinucleotide repeats; 21 for the normal allele). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays.

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          Derivation and feeder-free propagation of human embryonic stem cells under xeno-free conditions.

          Dusko Ilic, Emma Stephenson, Victoria Wood … (2012)
          Human embryonic stem (hES) cells hold great potential for cell therapy and regenerative medicine because of their pluripotency and capacity for self-renewal. The conditions used to derive and culture hES cells vary between and within laboratories depending on the desired use of the cells. Until recently, stem cell culture has been carried out using feeder cells, and culture media, that contain animal products. Recent advances in technology have opened up the possibility of both xeno-free and feeder-free culture of stem cells, essential conditions for the use of stem cells for clinical purposes. To date, however, there has been limited success in achieving this aim. Protocols were developed for the successful derivation of two normal and three specific mutation-carrying (SMC) (Huntington's disease and myotonic dystrophy 1) genomically stable hES cell lines, and their adaptation to feeder-free culture, all under xeno-free conditions.
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            Derivation and propagation of human embryonic stem cell lines from frozen embryos in an animal product-free environment.

            Laureen Jacquet, Y Dajani, Peter Braude … (2012)
            The protocols described here are comprehensive instructions for deriving human embryonic stem (hES) cell lines in xeno-free conditions from cryopreserved embryos. Details are included for propagation, cryopreservation and characterization. Initial derivation is on feeder cells and is followed by adaptation to a feeder-free environment; competent technicians can perform these simplified methods easily. From derivation to cryopreservation of fully characterized initial stocks takes 3-4 months. These protocols served as the basis for standard operating procedures (SOPs), with both operational and technical components, that we set to meet good manufacturing practice (GMP) and UK regulatory body requirements for derivation of clinical-grade cells. As such, these SOPs are currently used in our current GMP compliant facility to derive hES cell lines ab initio, in an animal product-free environment; these lines are suitable for research and potentially for clinical use in cell therapy. So far, we have derived eight clinical-grade lines, which will be freely available to the scientific community after submission/accession to the UK Stem Cell Bank.
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              Strategy for the creation of clinical grade hESC line banks that HLA-match a target population

              Laureen Jacquet, Emma Stephenson, Robert Collins … (2012)
              Here, we describe a pre-derivation embryo haplotyping strategy that we developed in order to maximize the efficiency and minimize the costs of establishing banks of clinical grade hESC lines in which human leukocyte antigen (HLA) haplotypes match a significant proportion of the population. Using whole genome amplification followed by medium resolution HLA typing using PCR amplification with sequence-specific primers (PCR-SSP), we have typed the parents, embryos and hESC lines from three families as well as our eight clinical grade hESC lines and shown that this technical approach is rapid, reliable and accurate. By employing this pre-derivation strategy where, based on HLA match, embryos are selected for a GMP route on day 3–4 of development, we would have drastically reduced our cGMP laboratory running costs.
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                Author and article information

                Contributors
                Dusko Ilic
                Journal
                Journal ID (nlm-ta): Stem Cell Res
                Journal ID (iso-abbrev): Stem Cell Res
                Title: Stem Cell Research
                Publisher: Elsevier
                ISSN (Print): 1873-5061
                ISSN (Electronic): 1876-7753
                Publication date PMC-release: 1 March 2016
                Publication date (Print): March 2016
                Volume: 16
                Issue: 2
                Pages: 274-277
                Affiliations
                [a ]Stem Cell Laboratories, Division of Women's Health, Faculty of Life Sciences and Medicine, King's College London and Assisted Conception Unit, Guys' Hospital, London, United Kingdom
                [b ]Histology Laboratory, Wolfson Centre for Age-Related Diseases, Faculty of Life Sciences and Medicine, King's College London, London, United Kingdom
                Author notes
                [* ]Corresponding author. dusko.ilic@ 123456kcl.ac.uk
                Article
                Publisher ID: S1873-5061(16)00018-0
                DOI: 10.1016/j.scr.2016.01.008
                PMC ID: 4823767
                PubMed ID: 27345982
                SO-VID: e562d67d-e661-4132-a9b6-70d6e8a7467f
                Copyright © © 2016 University of Texas at Austin Dell Medical School
                License:

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                History
                Date received : 31 December 2015
                Date revision received : 7 January 2016
                Date accepted : 12 January 2016
                Categories
                Subject: Lab Resource: Stem Cell Line

                ScienceOpen disciplines: Molecular medicine
                Data availability:
                ScienceOpen disciplines: Molecular medicine

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