The murine mAb used in this study were FITC- or PE-labeled CD3 (CA17.3G9), CD4 (CA13.1E4), CD8 (CA9.JD3), CD21 (CA2.1D6), and CD45RA (CA4.1D3) [42, 43]. to sustain long-term thymopoiesis. In contrast, XSCID dogs transplanted with CD34+ cells cultured for 18 hours 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 Rabbit polyclonal to Hsp22 vivo culture conditions that maintain both the engraftment potential and stem cell potential of the cultured cells. INTRODUCTION A prerequisite for any ex vivo NVP-TNKS656 approach to hematopoietic stem cell (HSC) gene therapy is the ability of the transduced HSC to successfully engraft in the recipient following their re-infusion. The major limitation of the ex vivo approach to gene therapy is the need for ex vivo manipulation of HSC with culturing in cytokines for various lengths of time depending upon the retroviral vector. CD34, a cell surface glycoprotein, is an antigen expressed on a subpopulation of hematopoietic cells that contain both stem cells, presumably pluripotent stem cells, and early committed progenitors that are capable of multilineage engraftment in humans, mice, nonhuman primates, and, more recently, dogs [1C8]. It is clear from a large body of clinical and experimental data that a population of cells within the CD34+ population is both pluripotent and capable of self-renewal, and selection based upon CD34 does not deplete the graft of significant numbers of HSCs. Therefore, enumeration of CD34+ cells is NVP-TNKS656 the current surrogate for determining the stem cell content of a human, nonhuman primate and canine bone marrow grafts and are the current targets for human and canine gene therapy involving diseases of the hematopoietic system. Because -retroviruses require active replication of the target cells for transduction, the typical -retroviral ex vivo transduction protocol used in human gene therapy clinical trials consists of a one to two day pre-stimulation in cytokines followed by three days of transductions to maximize the number of transduced cells [9C15]. Lentiviral vectors have gained considerable attention as potential vectors for HSC since they have been shown to be capable of transducing quiescent CD34+ cells, however maximal transduction occurs as the cell enters G1 of the cell cycle [16, 17]. Therefore, most studies use either an 18 hour (overnight) or 48 hour transduction protocol in the presence of various cytokine cocktails. Since HSCs are typically quiescent, ex vivo culture in cytokines that allow for gene transfer to occur may force HSCs to enter pathways of proliferation, and possibly differentiation, that could limit their engraftment potential, pluripotentiality and long-term repopulating capacity. Currently, the most controversial and important issue regarding the clinical use of ex vivo manipulated cells is the question of whether exhaustion of stem cells might result from growth factor stimulation ex vivo which has considerable significance for both gene therapy and HSC expansion protocols . Over the past few years, evidence has accumulated that cultured HSC have a reduced ability to engraft in murine, xenogeneic and large animal transplant models. These animal studies were performed in recipients following pre-transplant conditioning that is known to damage bone marrow stromal cells [19C21] and NVP-TNKS656 decrease HSC homing to or survival in the bone marrow following transplantation . Competitive repopulation studies in mice have revealed significantly reduced engraftment with expanded cells compared with freshly isolated bone marrow (BM) cells [23, 24]. Transplantation of ex vivo cultured autologous BM CD34+ cells in myeloablated baboons resulted in delayed short-term engraftment (recovery of normal neutrophil and platelet counts) compared to freshly isolated BM CD34+ cells . Autologous transplantation of irradiated cats with cultured bone marrow cells resulted in an engraftment rate of 40% compared with 100% for non-cultured cells . Xenotransplantation of cultured human cord blood (CB) cells in SCID or NOD/SCID mice have been reported to result in delayed and significantly reduced engraftment compared with fresh CD34+ cells [27C33]. When fresh and expanded human CB CD34+ cells were transplanted together in a competitive repopulation assay, the fresh CD34+ cells significantly out-competed the expanded cells . An important finding from these xenogeneic studies is that the reduced in vivo repopulating capacity was evident as early as 24 to 48 hours.