title: Increased gene transfer into human cord blood cells by centrifugation-enhanced transduction in fibronectin fragment-coated tubes.
abstract: We investigated whether transduction of human cord blood progenitor cells can be increased by spinoculation in fibronectin fragment CH-296 (FN)-coated tubes. Bicistronic vectors PA317/LgEIN, containing the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (neo) genes, and PG13/LgDIN, containing the dihydrofolate reductase and neo genes, were used to transduce CD34-enriched human cord blood cells. Transduction by spinoculation in FN-coated tubes (spin/FN+) was compared with spinoculation in noncoated tubes (spin/FN-) and transduction in plates coated with FN (plate/FN+). Antibody to TGF-beta was added to spin/FN+ to evaluate its impact on transduction. Using producer cell line PA317/LgEIN for transduction of CD34+ cord blood cells, FACS analysis for expression of EGFP revealed mean transduction of 30.6+/-4.3, 9.1+/-1.6, and 21.1+/-6.5% of CD34+ cells in the spin/FN+, spin/FN-, and plate/FN+ arms, respectively. Transduction of CD+CD38low cells was also higher in the spin/FN+ arm as compared with transduction in the spin/FN- arm. These results were corroborated by colony-forming assays. Antibody to TGF-beta did not further increase transduction. Using a different producer cell line, PG13/pLgDIN, a higher number of G418-resistant CFU-GM was observed in the spin/FN+ as compared with the plate/FN+ and spin/FN-arms. NOD/SCID mice were transplanted with transduced, CD34-enriched human cord blood cells, and persistence of transduced human cells was analyzed in the mice marrows after 6-8 weeks: 32.8, 6.0, and 23.9% human G418-resistant CFU-GM colonies were observed in the spin/FN+, spin/FN-, and plate/FN+ arms, respectively. These results suggest that spinoculation in FN-coated tubes increases transduction of early human cord blood progenitor cells as compared with spinoculation in noncoated tubes.
authors.affiliation: Bone Marrow Transplant Division, University of Wisconsin, Madison 53792, USA.
authors.forename: A
authors.initials: A
authors.lastname: Sanyal
authors.forename: F G
authors.initials: FG
authors.lastname: Schuening
mesh_terms: D000818:Animals; D000906:Antibodies / Q000502:physiology; D002470:Cell Survival; D002498:Centrifugation / Q000379:methods; D003114:Colony-Forming Units Assay; D005312:Fetal Blood / Q000166:cytology; D005353:Fibronectins / Q000502:physiology; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D014161:Transduction, Genetic; D016212:Transforming Growth Factor beta / Q000276:immunology
chemical_list: D000906:Antibodies; D005353:Fibronectins; D008164:Luminescent Proteins; D016212:Transforming Growth Factor beta; D049452:Green Fluorescent Proteins