title: Efficient episomal reprogramming of blood mononuclear cells and differentiation to hepatocytes with functional drug metabolism.
abstract: The possibility of converting cells from blood mononuclear cells (MNC) to liver cells provides promising opportunities for the study of diseases and the assessment of new drugs. However, clinical applications have to meet GMP requirements and the methods for generating induced pluripotent cells (iPCs) have to avoid insertional mutagenesis, a possibility when using viral vehicles for the delivery of reprogramming factors. We have developed an efficient non-integration method for reprogramming fresh or frozen blood MNC, maintained in an optimised cytokine cocktail, to generate induced pluripotent cells. Using electroporation for the effective delivery of episomal transcription factors (Oct4, Sox2, Klf4, L-Myc, and Lin28) in a feeder-free system, without any requirement for small molecules, we achieved a reprogramming efficiency of up to 0.033% (65 colonies from 2×10(5) seeded MNC). Applying the same cytokine cocktail and reprogramming methods to cord blood or fetal liver-derived CD34(+) cells, we obtained 148 iPS colonies from 10(5) seeding cells (0.148%). The iPS cell lines we generated maintained typical characteristics of pluripotent cells and could be successfully differentiated into hepatocytes with drug metabolic function.
authors.affiliation: Scottish National Blood Transfusion Service (SNBTS) Cell Therapy Group, Centre for Regenerative Medicine, The Chancellor's Building, 49 Little France Crescent, Edinburgh EH16 4SB, United Kingdom; Tissue Injury and Repair Group, MRC Centre for Regenerative Medicine, University of Edinburgh, EH16 4SB United Kingdom; Peking University Institute of Hematology, Peking University People's Hospital, Beijing, 100044 China.
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.affiliation: Tissue Injury and Repair Group, MRC Centre for Regenerative Medicine, University of Edinburgh, EH16 4SB United Kingdom.
authors.forename: Joanna
authors.initials: J
authors.lastname: Brzeszczynska
authors.affiliation: Scottish National Blood Transfusion Service (SNBTS) Cell Therapy Group, Centre for Regenerative Medicine, The Chancellor's Building, 49 Little France Crescent, Edinburgh EH16 4SB, United Kingdom.
authors.forename: Kay
authors.initials: K
authors.lastname: Samuel
authors.affiliation: Tissue Injury and Repair Group, MRC Centre for Regenerative Medicine, University of Edinburgh, EH16 4SB United Kingdom.
authors.forename: Jim
authors.initials: J
authors.lastname: Black
authors.affiliation: Tissue Injury and Repair Group, MRC Centre for Regenerative Medicine, University of Edinburgh, EH16 4SB United Kingdom.
authors.forename: Anwar
authors.initials: A
authors.lastname: Palakkan
authors.affiliation: MRC Centre for Reproductive Health, University of Edinburgh, 47, Little France Crescent, Edinburgh EH16 4TJ, United Kingdom.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Anderson
authors.affiliation: Scottish National Blood Transfusion Service (SNBTS) Cell Therapy Group, Centre for Regenerative Medicine, The Chancellor's Building, 49 Little France Crescent, Edinburgh EH16 4SB, United Kingdom. Electronic address: ronnie_gallagher@yahoo.co.uk.
authors.forename: Ronald
authors.initials: R
authors.lastname: Gallagher
authors.affiliation: Tissue Injury and Repair Group, MRC Centre for Regenerative Medicine, University of Edinburgh, EH16 4SB United Kingdom. Electronic address: j.a.ross@ed.ac.uk.
authors.forename: James A
authors.initials: JA
authors.lastname: Ross
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D065150:Cellular Reprogramming / Q000502:physiology*; D016207:Cytokines / Q000378:metabolism; D005312:Fetal Blood / Q000378:metabolism / Q000502:physiology*; D022781:Hepatocytes / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism / Q000502:physiology; D000090062:Kruppel-Like Factor 4; D007963:Leukocytes, Mononuclear / Q000378:metabolism / Q000502:physiology*; D010957:Plasmids / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D018952:Antigens, CD34; D016207:Cytokines; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D014157:Transcription Factors
keywords: Episomal; Hepatocyte; Pluripotent; Reprogramming; Stem cell