Abstract
BackgroundProlonged, altered hematopoietic reconstitution is commonly observed in patients undergoing myeloablative conditioning and bone marrow and/or mobilized peripheral blood-derived stem cell transplantation. We studied the reconstitution of myeloid and lymphoid compartments after the transplantation of autologous CD34+ bone marrow cells following gamma irradiation in cynomolgus macaques.ResultsThe bone marrow cells were first transduced ex vivo with a lentiviral vector encoding eGFP, with a mean efficiency of 72% ± 4%. The vector used was derived from the simian immunodeficiency lentivirus SIVmac251, VSV-g pseudotyped and encoded eGFP under the control of the phosphoglycerate kinase promoter. After myeloid differentiation, GFP was detected in colony-forming cells (37% ± 10%). A previous study showed that transduction rates did not differ significantly between colony-forming cells and immature cells capable of initiating long-term cultures, indicating that progenitor cells and highly immature hematopoietic cells were transduced with similar efficiency. Blood cells producingeGFP were detected as early as three days after transplantation, and eGFP-producing granulocyte and mononuclear cells persisted for more than one year in the periphery.ConclusionThe transplantation of CD34+ bone marrow cells had beneficial effects for the ex vivo proliferation and differentiation of hematopoietic progenitors, favoring reconstitution of the T- and B-lymphocyte, thrombocyte and red blood cell compartments.
Highlights
Prolonged, altered hematopoietic reconstitution is commonly observed in patients undergoing myeloablative conditioning and bone marrow and/or mobilized peripheral blood-derived stem cell transplantation
The CD34+ cells were transduced by coculture for 24 h with the lentiviral vector (MOI = 100) in medium supplemented with SCF, Flt3-L, IL-3 and IL-6
After 24 hours of culture with the lentiviral vector, some of the purified CD34+ cells were cultured for 14 days in semi-solid medium containing SCF, GM-CSF, IL-3 and EPO to allow the myeloid differentiation of colony-forming cells (CFC), whereas some cells were cocultured for 35 days on a layer of murine fibroblasts of the M2-10B4 cell line and were cultured for 14 days on semi-solid medium containing SCF, GM-CSF, IL-3 and EPO, for the identification of long-term cultureinitiating cells (LTC-IC)
Summary
Prolonged, altered hematopoietic reconstitution is commonly observed in patients undergoing myeloablative conditioning and bone marrow and/or mobilized peripheral blood-derived stem cell transplantation. Gene therapy strategies hold promise for the treatment of hematopoietic disorders. All hematopoietic lineages, including polymorphonuclear cells, monocytes, lymphocytes and natural killer cells, and hematopoietic stem cells (HSC) – which are capable of self-renewal and pluripotent differentiation – have been targeted for transduction with therapeutic genes. Retroviral vectors present the major advantage of integrating the transferred DNA stably into the genome of target cells, which is passed on to progeny. They cannot infect and integrate into non dividing cells[1]. Vectors derived from HIV1[15,16], HIV-2[17], FIV[18] and equine infectious anemia virus (EIAV)have been tested[19]
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