title: Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors.
abstract: BACKGROUND
  Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods.
  
  
  RESULTS
  We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration.
  
  
  CONCLUSIONS
  We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.
authors.affiliation: INSERM U 972, IFR 93, Bicêtre Hospital, and Paul Brousse Hospital, Villejuif F-94807, France.
authors.forename: Guanghua
authors.initials: G
authors.lastname: Yang
authors.forename: Karim
authors.initials: K
authors.lastname: Si-Tayeb
authors.forename: Sébastien
authors.initials: S
authors.lastname: Corbineau
authors.forename: Rémi
authors.initials: R
authors.lastname: Vernet
authors.forename: Régis
authors.initials: R
authors.lastname: Gayon
authors.forename: Noushin
authors.initials: N
authors.lastname: Dianat
authors.forename: Clémence
authors.initials: C
authors.lastname: Martinet
authors.forename: Denis
authors.initials: D
authors.lastname: Clay
authors.forename: Sylvie
authors.initials: S
authors.lastname: Goulinet-Mainot
authors.forename: Gérard
authors.initials: G
authors.lastname: Tachdjian
authors.forename: Gérard
authors.initials: G
authors.lastname: Tachdjian
authors.forename: Deborah
authors.initials: D
authors.lastname: Burks
authors.forename: Ludovic
authors.initials: L
authors.lastname: Vallier
authors.forename: Pascale
authors.initials: P
authors.lastname: Bouillé
authors.forename: Anne
authors.initials: A
authors.lastname: Dubart-Kupperschmitt
authors.forename: Anne
authors.initials: A
authors.lastname: Weber
mesh_terms: D016633:Apolipoprotein A-II / Q000235:genetics; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology*; D005434:Flow Cytometry; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008099:Liver / Q000166:cytology; D009928:Organ Specificity; D011401:Promoter Regions, Genetic / Q000235:genetics; D014161:Transduction, Genetic; D016662:Virus Integration / Q000502:physiology*
chemical_list: D016633:Apolipoprotein A-II; D015415:Biomarkers; D004279:DNA, Viral; D049452:Green Fluorescent Proteins; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human