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

Purified CD34(+) and CD34(+)CD38(-) human umbilical cord blood (uds) c ells were transduced with the recombinant variant of Moloney murine le ukemia virus (MoMLV) MFG-EGFP or with SF-EGFP. in which EGFP expressio n is driven by a hybrid promoter of the spleen focus-forming virus (SF FV) and the murine embryonic stem cell virus (MESV). Infectious MFG-EG FP virus was produced by an amphotropic virus producer cell line (GP+e nvAm12). SF-EGFP was produced in the PG13 cell line pseudotyped for th e gibbon ape leukemia virus (GaLV) envelope proteins. Using a a-day gr owth 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-EGF P/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 dia betic/severe combined immunodeficient (NOD/SCID) mice resulted in medi an engraftment percentages of 8% and 5%, respectively, which showed th at 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 expresse d EGFP with median values of 2% and 23% of human CD45(+) cells, respec tively, which showed that the NOD/SCID repopulating cells were success fully 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 transduc tion procedure the absolute numbers of CAFC week 6 increased 5- to 10- fold.,The transduction efficiency of this progenitor cell subset was s imilar to the fraction of EGFP(+) human cells in the bone marrow of th e NOD/SCID mice transplanted with MFG-EGFP/Am12 or SF-EGFP/PG13 transd uced CD34+ cells, ie, 6% and 27%, respectively. The study thus shows t hat purified CD34(+) and highly purified CD34(+)CD38(-) UCB cells can be transduced efficiently with preservation of repopulating ability. T he SF-EGFP/PG13 vector/packaging cell combination was much more effect ive in transducing repopulating cells than the MFG-EGFP/Am12 combinati on. (C) 1998 by The American Society of Hematology.