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Transduction of human CD34+cord blood cells prior to ex vivo expansion does not alter their ability to generate hematopoiesis in vitro and in vivo


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A. Chapel*, M. Bensidhoum*, JPh. Laporte*, D. Thierry, S. Bouchet*, C. Demarguay*, J. Frik*, F. Cuvelier*, E. Tisserand*, N. Fanzonne, M. Lopez*, L. Douay*, G. Wagemaker*,P. Gourmelon*, NC. Gorin, laboratoire de Thérapie Cellulaire et de Radioprotection Accidentelle, UPRES 1638, Centre Claude Bernard, Paris, Institut de Protection et de Sûreté Nucléaire, Département de protection de la Santé de l'Homme et de Dosimétrie, Section Autonome de Radiobiologie Appliquée à la Médecine, Fontenay aux Roses, INSERM U 76, CHU St Antoine Paris, Service des Maladies du sang, Hôpital St Antoine, Paris, France. Institute of hematology, Erasmus University of Rotterdam, PO Box 1738, 3000 DR Rotterdam, Netherlands Congrès ASH, Orlando (USA), 5-9/12/2001

Résumé

In order to subsequently track expanded cells progeny in the blood or bone marrow we have developed a retroviral gene transfer method to transduce GFP gene into CD34+ umbilical cord cells. Qualitative and quantitative analysis of the expansion products were performed in vitro and in vivo in the NOD/SCID mouse model. Umbilical cord blood CD34+were pre-stimulated for 48 hours in serum free medium (105 c/ml, TPO, SCF, Flt-3L 100ng/ml). The cells were then transduced twice for 48 hours in the presence of supernatant of PG13 cell line producing the GFP. After GFP labelling CD34+ cells were expanded for 14 days in serum free medium (104 c/ml, TPO, SCF, Flt-3L 100ng/ml, G-CSF 10ng/ml). The functions of expanded cells were analysed using clonogenic assays (CFC, LTC-IC, E-LTC4C) and the production of the transgene was evaluated in clonogenic cells through fluorescent analysis. The percentage of transduced cells was up to 90% of the CD34+ cord blood. A high level (97%) of CD34+ cells was preserved. Clonogenecity assays showed that up to 95% of CFU-GM and 100% of BFU-E were fluorescent After expansion the transgene was expressed up to 85% of cells as well as in up to 80% of CFU-GM, up to 100% of BFU-E, and up to 90% of LTC-IC (40% of E-LTC-IC). The level of expansion were up to 1700 fold for total cells, 200 fold for CD34+, 57 fold for CFU-GM, 3 fold for BFU-E, 5 fold for LTC-IC. These levels as well as the differentiation pattern of the cells were similar to the control (expansion without GFP labelling). In vivo the long-term engraftment of the expanded cells was evaluated in bone marrow, spleen, and liver of NOD-SCID mice. Up to 107 cells per mouse were grafted and the animals (n : 24) were analysed 4 to 9 weeks later. Expanded cells engrafted in NOD-SCID mice with the same frequency and tissue distribution whether they are transduced or non-transduced as evidenced by FACS analysis of human CD 45+ cells . In conclusion, this method allows a high degree of GFP transduction in C034+ cord blood cells without impairing their ability to expand and to generate progeny. Preliminary studies are being set up in order to implement phase I/II clinical protocols that will test ex vivo expanded cord blood unit to transplant adult or pediatric patients. The transduction protocol we developed would allow to track blood or marrow cells originated in patients by cord blood stem cells after in vitro expansion.