title: Highly efficient transduction of the green fluorescent protein gene in human umbilical cord blood stem cells capable of cobblestone formation in long-term cultures and multilineage engraftment of immunodeficient mice.
abstract: Purified CD34(+) and CD34(+)CD38(-) human umbilical cord blood (UCB) cells were transduced with the recombinant variant of Moloney murine leukemia virus (MoMLV) MFG-EGFP or with SF-EGFP, in which EGFP expression is driven by a hybrid promoter of the spleen focus-forming virus (SFFV) and the murine embryonic stem cell virus (MESV). Infectious MFG-EGFP virus was produced by an amphotropic virus producer cell line (GP+envAm12). SF-EGFP was produced in the PG13 cell line pseudotyped for the gibbon ape leukemia virus (GaLV) envelope proteins. Using a 2-day growth factor prestimulation, followed by a 2-day, fibronectin fragment CH-296-supported transduction, CD34(+) and CD34(+)CD38(-) UCB subsets were efficiently transduced using either vector. The use of the SF-EGFP/PG13 retroviral packaging cell combination consistently resulted in twofold higher levels of EGFP-expressing cells than the MFG-EGFP/Am12 combination. Transplantation of 10(5) input equivalent transduced CD34(+) or 5 x 10(3) input equivalent CD34(+)CD38(-) UCB cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in median engraftment percentages of 8% and 5%, respectively, which showed that the in vivo repopulating ability of the cells had been retained. In addition, mice engrafted after transplantation of transduced CD34(+) cells using the MFG-EGFP/Am12 or the SF-EGFP/PG13 combination expressed EGFP with median values of 2% and 23% of human CD45(+) cells, respectively, which showed that the NOD/SCID repopulating cells were successfully transduced. EGFP+ cells were found in all human hematopoietic lineages produced in NOD/SCID mice including human progenitors with in vitro clonogenic ability. EGFP-expressing cells were also detected in the human cobblestone area-forming cell (CAFC) assay at 2 to 6 weeks of culture on the murine stromal cell line FBMD-1. During the transduction procedure the absolute numbers of CAFC week 6 increased 5- to 10-fold. The transduction efficiency of this progenitor cell subset was similar to the fraction of EGFP+ human cells in the bone marrow of the NOD/SCID mice transplanted with MFG-EGFP/Am12 or SF-EGFP/PG13 transduced CD34(+) cells, ie, 6% and 27%, respectively. The study thus shows that purified CD34(+) and highly purified CD34(+)CD38(-) UCB cells can be transduced efficiently with preservation of repopulating ability. The SF-EGFP/PG13 vector/packaging cell combination was much more effective in transducing repopulating cells than the MFG-EGFP/Am12 combination.
authors.affiliation: Institute of Hematology, Erasmus University Rotterdam, The Netherlands; and the Department of Cryobiology and Cell Therapy, Institut de Recerca Oncologica, Barcelona, Spain.
authors.forename: P B
authors.initials: PB
authors.lastname: van Hennik
authors.forename: M M
authors.initials: MM
authors.lastname: Verstegen
authors.forename: M F
authors.initials: MF
authors.lastname: Bierhuizen
authors.forename: A
authors.initials: A
authors.lastname: Limón
authors.forename: A W
authors.initials: AW
authors.lastname: Wognum
authors.forename: J A
authors.initials: JA
authors.lastname: Cancelas
authors.forename: J
authors.initials: J
authors.lastname: Barquinero
authors.forename: R E
authors.initials: RE
authors.lastname: Ploemacher
authors.forename: G
authors.initials: G
authors.lastname: Wagemaker
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay / Q000379:methods; D005312:Fetal Blood / Q000166:cytology; D018014:Gene Transfer Techniques; D006085:Graft Survival; D049452:Green Fluorescent Proteins; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000502:physiology*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D051379:Mice; D016513:Mice, SCID
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins