title: Sustained expression of coagulation factor IX by modified cord blood-derived mesenchymal stromal cells.
abstract: BACKGROUND
  Hemophilia B patients are subject to frequent and spontaneous bleeding caused by a deficiency of clotting factor IX (FIX). Mesenchymal stromal cells (MSCs) have been used in cellular therapies as a result of their immunomodulatory properties, the ability to home to sites of injury and their amenability to various ex vivo modifications, including lentiviral-mediated gene transfer.
  
  
  METHODS
  MSCs were isolated from human umbilical cord blood and differentiated into adipogenic, chondrogenic and osteogenic lineages. A lentiviral DNA vector containing the human FIX gene was generated using traditional restriction enzyme digest and ligation techniques to generate viable replication-incompetent lentiviral particles that were used to transduce MSCs. Quantitative measurement of FIX expression was conducted using an enzyme-linked immunosorbent assay.
  
  
  RESULTS
  The over-expression of FIX was sustained in vitro at levels > 4 µg/10(6) cells/24 h and FIX coagulant activity was > 2.5 mIU/10(6) cells/24 h for the 6-week duration of study. Lentiviral modification of cells with a multiplicity of infection of 10 did not adversely affect the potential of cord blood (CB) MSCs to differentiate to adipocytes, chondrocytes and osteoblastic cells, and the expression of functional FIX was sustained after differentiation and was similar to that in nondifferentiated cells.
  
  
  CONCLUSIONS
  Modification of human CB MSCs with a lentiviral vector resulted in sustained high FIX expression in vitro after differentiation to adipogenic, chondrogenic and osteoblastic cells. These modified MSCs could have applications in cellular therapies for hemophilia B.
authors.affiliation: School of Biomedical Engineering, McMaster University, Hamilton, ON, Canada; Department of Biomedical Engineering, Jimma Institute of Technology, Jimma, Ethiopia.
authors.forename: Megan
authors.initials: M
authors.lastname: Dodd
authors.forename: Leah
authors.initials: L
authors.lastname: Marquez-Curtis
authors.forename: Anna
authors.initials: A
authors.lastname: Janowska-Wieczorek
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Hortelano
mesh_terms: D050156:Adipogenesis; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D020219:Chondrogenesis; D005164:Factor IX / Q000235:genetics* / Q000378:metabolism; D005312:Fetal Blood / Q000166:cytology*; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D002836:Hemophilia B / Q000235:genetics / Q000628:therapy; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D010314:Partial Thromboplastin Time; D014161:Transduction, Genetic
chemical_list: D005164:Factor IX
keywords: coagulation factor IX; hemophilia B; lentiviral vector; mesenchymal stromal cells