| Background and objectives: Allogeneic hematopoietic stem cell transplantation is an effective therapy for hematopoietic malignancies, nonmalignancies dieases, congenital disorders,and some solid tumors. Successful allogeneic stem cell transplantation requires that hematopoietic precursor cells from normal donors be able to home in the recipients' bone marrow microenvironment and attain rapid and persistent hematoimmunologic reconstitution. Graft-versus-leukemia (GVL), which accompanies with transplantation, could improve our ability to treat refractory malignant disorders. Homing, immune reconstitution, graft-versus-host disease (GVHD) and GVL are important pathophysiologic processes during allogeneic hematopoietic stem cell transplantation (HSCT), and their occurrence correlates directly with the cellular makeup of the donor grafts. It has been reported that the process of homing and mobilization may be determined by the selective migration of hematopoietic stem/progenitor cells (HSPC). Recent research shows that stromal cell derived factor l(SDF-l) and its receptor CXCR4 may be critical in homing and mobilization process. Furthermore, the accessory cells in allografts may be required to facilitate engraftment of stem cells across allogeneic barriers. GVHD is the major complication of allogeneic stem cell transplantation. Recognition of host antigens by donor T cells, and the subsequent T cell activation and cytokine production, play an important role in the induction and progression of GVHD. Thedelayed immune reconstitution may result in infections and secondary tumors post transplantation. GVL is the major mechanism to eliminate hematological malignant cells. Adoptive allogeneic immunotherapy is an effective means to treat mini-residual disease (MRD) and converse relapse. All these pathophysiologic processes depend on the number and functions of the cellular components in allografts. Normal human bone marrow (BM), cord blood (CB) and mobilized peripheral blood (MPB) are the most commonly used sources for allogeneic stem cell transplantation. Stem cells from different sources have different characteristics during transplantation. We obtained purified CD34+cells from different stem cell sources by the CD34 MultiSort Kit immunomagnetic bead system and assayed the expression of CXCR4 on CD34+ cells by flow cytometry. Moreover, we detected lymphocyte subsets in each type of allograft by three-color flow cytometry. The aim of this study was to investigate the expression of CXCR4 in CD34+ cells from different allografts, and to investigate the mechanism of hematoimmunologic reconstitution, GVHD, GVL, and peripheral blood precursor cell mobilization.Methods: CD34+ hematopoietic cells were separated from BM, CB and MPB by the CD34 MultiSort Kit immunomagnetic bead system. The expression of CXCR4 in CD34+cells was assayed by flow cytometry.The lymphocyte subsets in each type of allograft were detected by three-color flow cytometry. In brief, normal human bone marrow samples were obtained from health donors in bone marrow transplantation (n=10). CB samples were obtained from full-term normal pregnant women who underwent caesarean section (n=10). Peripheral blood progenitor cells were obtained from normal donors after mobilization (n=10). Peripheral blood samples were obtained from the same peripheral blood progenitor cells donors before mobilization (n=10). First, red blood was eliminated from CB and BM samples by HESPAN. Second, mononuclear cells (MNC) were enriched by density gradient centrifugation over Ficoll-paque. Third, blocking reagent and the antibody reagent from the CD34 Isolation Kit (MACS) were added simultaneously to the MNC suspension.After incubation at 4 , the cells were washed and submicroscopic magnetic beads were added. A separation column was placed in the magnetic field of the MACS Separator, and then the magnetically labeled cells were applied to the column. The column was then washed with buffer, and CD34+cells were eluted by removing column from the magnetic field and flu...
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