title: Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming.
abstract: The production of megakaryocytes (MKs)--the precursors of blood platelets--from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 10(5) mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology.
authors.affiliation: Department of Haematology, University of Cambridge and NHS Blood and Transplant, Long Road, Cambridge CB2 0PT, UK.|The Anne McLaren Laboratory, Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute and Department of Surgery, University of Cambridge, West Forvie Site, Robinson Way, Cambridge CB2 0SZ, UK.|Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Tennis Court Road, Cambridge CB2 1QR, UK.
authors.forename: Thomas
authors.initials: T
authors.lastname: Moreau
authors.affiliation: Department of Haematology, University of Cambridge and NHS Blood and Transplant, Long Road, Cambridge CB2 0PT, UK.|Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Tennis Court Road, Cambridge CB2 1QR, UK.
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Evans
authors.affiliation: Human Genetics, Wellcome Trust Sanger Institute, Genome Campus, Hinxton CB10 1RQ, UK.
authors.forename: Louella
authors.initials: L
authors.lastname: Vasquez
authors.affiliation: Department of Haematology, University of Cambridge and NHS Blood and Transplant, Long Road, Cambridge CB2 0PT, UK.
authors.forename: Marloes R
authors.initials: MR
authors.lastname: Tijssen
authors.affiliation: Human Genetics, Wellcome Trust Sanger Institute, Genome Campus, Hinxton CB10 1RQ, UK.
authors.forename: Ying
authors.initials: Y
authors.lastname: Yan
authors.affiliation: The Anne McLaren Laboratory, Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute and Department of Surgery, University of Cambridge, West Forvie Site, Robinson Way, Cambridge CB2 0SZ, UK.
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Trotter
authors.affiliation: Department of Haematology, University of Cambridge and NHS Blood and Transplant, Long Road, Cambridge CB2 0PT, UK.|Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Tennis Court Road, Cambridge CB2 1QR, UK.
authors.forename: Daniel
authors.initials: D
authors.lastname: Howard
authors.affiliation: Department of Haematology, University of Cambridge and NHS Blood and Transplant, Long Road, Cambridge CB2 0PT, UK.|Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Tennis Court Road, Cambridge CB2 1QR, UK.
authors.forename: Maria
authors.initials: M
authors.lastname: Colzani
authors.affiliation: Department of Haematology, University of Cambridge and NHS Blood and Transplant, Long Road, Cambridge CB2 0PT, UK.|Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Tennis Court Road, Cambridge CB2 1QR, UK.
authors.forename: Meera
authors.initials: M
authors.lastname: Arumugam
authors.affiliation: Department of Haematology, University of Cambridge and NHS Blood and Transplant, Long Road, Cambridge CB2 0PT, UK.|Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Tennis Court Road, Cambridge CB2 1QR, UK.
authors.forename: Wing Han
authors.initials: WH
authors.lastname: Wu
authors.affiliation: Department of Haematology, University of Cambridge and NHS Blood and Transplant, Long Road, Cambridge CB2 0PT, UK.|Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Tennis Court Road, Cambridge CB2 1QR, UK.
authors.forename: Amanda
authors.initials: A
authors.lastname: Dalby
authors.affiliation: Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Tennis Court Road, Cambridge CB2 1QR, UK.
authors.forename: Riina
authors.initials: R
authors.lastname: Lampela
authors.affiliation: Department of Haematology, University of Cambridge and NHS Blood and Transplant, Long Road, Cambridge CB2 0PT, UK.|Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Tennis Court Road, Cambridge CB2 1QR, UK.
authors.forename: Guenaelle
authors.initials: G
authors.lastname: Bouet
authors.affiliation: Department of Haematology, University of Cambridge and NHS Blood and Transplant, Long Road, Cambridge CB2 0PT, UK.|Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Tennis Court Road, Cambridge CB2 1QR, UK.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Hobbs
authors.affiliation: Department of Haematology, University of Cambridge and NHS Blood and Transplant, Long Road, Cambridge CB2 0PT, UK.|Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Tennis Court Road, Cambridge CB2 1QR, UK.
authors.forename: Dean C
authors.initials: DC
authors.lastname: Pask
authors.affiliation: Institute of Cardiovascular Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors.forename: Holly
authors.initials: H
authors.lastname: Payne
authors.affiliation: Institute of Cardiovascular Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors.forename: Tatyana
authors.initials: T
authors.lastname: Ponomaryov
authors.affiliation: Institute of Cardiovascular Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors.forename: Alexander
authors.initials: A
authors.lastname: Brill
authors.affiliation: Human Genetics, Wellcome Trust Sanger Institute, Genome Campus, Hinxton CB10 1RQ, UK.
authors.forename: Nicole
authors.identifier: http://orcid.org/0000-0003-1095-3852
authors.initials: N
authors.lastname: Soranzo
authors.affiliation: Department of Haematology, University of Cambridge and NHS Blood and Transplant, Long Road, Cambridge CB2 0PT, UK.
authors.forename: Willem H
authors.initials: WH
authors.lastname: Ouwehand
authors.affiliation: The Anne McLaren Laboratory, Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute and Department of Surgery, University of Cambridge, West Forvie Site, Robinson Way, Cambridge CB2 0SZ, UK.|Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Tennis Court Road, Cambridge CB2 1QR, UK.
authors.forename: Roger A
authors.initials: RA
authors.lastname: Pedersen
authors.affiliation: Department of Haematology, University of Cambridge and NHS Blood and Transplant, Long Road, Cambridge CB2 0PT, UK.|Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Tennis Court Road, Cambridge CB2 1QR, UK.
authors.forename: Cedric
authors.initials: C
authors.lastname: Ghevaert
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics* / Q000378:metabolism; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D049109:Cell Proliferation; D065150:Cellular Reprogramming; D015925:Cryopreservation / Q000379:methods; D050982:GATA1 Transcription Factor / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008533:Megakaryocytes / Q000166:cytology* / Q000378:metabolism; D046228:Microarray Analysis; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D051789:Proto-Oncogene Protein c-fli-1 / Q000235:genetics* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D000073942:T-Cell Acute Lymphocytic Leukemia Protein 1; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D050982:GATA1 Transcription Factor; D049452:Green Fluorescent Proteins; D051789:Proto-Oncogene Protein c-fli-1; D011518:Proto-Oncogene Proteins; D000073942:T-Cell Acute Lymphocytic Leukemia Protein 1; C078433:FLI1 protein, human; C494688:GATA1 protein, human; C067143:TAL1 protein, human