Effect of ex vivo culture of CD34+ bone marrow cells on immune reconstitution of XSCID dogs following allogeneic bone marrow transplantation.
Successful genetic treatment of most primary immunodeficiencies or hematological disorders will require the transduction of pluripotent, self-renewing hematopoietic stem cells (HSC) rather than their progeny to achieve enduring production of genetically corrected cells and durable immune reconstitution. Current ex vivo transduction protocols require manipulation of HSC by culture in cytokines for various lengths of time depending upon the retroviral vector that may force HSC to enter pathways of proliferation, and possibly differentiation, which could limit their engraftment potential, pluripotentiality and long-term repopulating capacity. We have compared the ability of normal CD34(+) cells cultured in a standard cytokine cocktail for 18hours or 4.5 days to reconstitute XSCID dogs following bone marrow transplantation in the absence of any pretransplant conditioning with that of freshly isolated CD34(+) cells. CD34(+) cells cultured under standard gamma-retroviral transduction conditions (4.5 days) showed decreased engraftment potential and ability to sustain long-term thymopoiesis. In contrast, XSCID dogs transplanted with CD34(+) cells cultured for 18hours showed a robust T cell immune reconstitution similar to dogs transplanted with freshly isolated CD34(+) cells, however, the ability to sustain long-term thymopoiesis was impaired. These results emphasize the need to determine ex vivo culture conditions that maintain both the engraftment potential and "stem cell" potential of the cultured cells.
Kennedy
Douglas R
DR
Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
McLellan
Kyle
K
Moore
Peter F
PF
Henthorn
Paula S
PS
Felsburg
Peter J
PJ
D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000821:virology; D016026:Bone Marrow Transplantation; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation; D002478:Cells, Cultured / Q000187:drug effects / Q000637:transplantation; D004195:Disease Models, Animal; D004285:Dogs; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000627:therapeutic use*; D006085:Graft Survival; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology; D018380:Hematopoietic Stem Cell Transplantation; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000276:immunology; D053631:Interleukin Receptor Common gamma Subunit / Q000172:deficiency / Q000235:genetics* / Q000502:physiology; D016086:Lentivirus / Q000235:genetics; D008213:Lymphocyte Activation; D016131:Lymphocyte Subsets / Q000473:pathology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D016511:Severe Combined Immunodeficiency / Q000235:genetics / Q000473:pathology / Q000601:surgery / Q000628:therapy*; D013950:Thymus Gland / Q000473:pathology; D013997:Time Factors; D014182:Transplantation, Autologous
D018952:Antigens, CD34; D016298:Hematopoietic Cell Growth Factors; D007074:Immunoglobulin G; D053631:Interleukin Receptor Common gamma Subunit; D011993:Recombinant Fusion Proteins