Different strategies of mixed chimerism induction may determine stem/progenitor cell populations in recipient mice.

Mixed chimerism has been suggested to induce tolerance to transplanted alloantigens. As the precise influence of mixed chimerism induction on the host organism has still not been fully elucidated, the aim of the present study was to explore this phenomenon in relation to the stem cell compartment. The experiment was performed on B6.SJL-Ptprc(a)Pep3(b) mice. Mixed chimerism induction protocols involved 3 Gy TBI (Day -1 of the experiment), injection of 20-30 × 10(6) Balb C bone marrow cells (Day 0), and administration of blocking antibodies against CD40L (Day 0 and Day 4), anti-CD8 (Day -2) with/without anti-NK1.1 (Day -3). Selected groups of mice were also treated with cyclophosphamid (175 mg/kg) on Day 2. The presence of mixed chimerism was assessed in peripheral blood, bone marrow, and spleen, as well as in various subpopulations of leukocytes (CD4(+), CD8(+), CD45/B220(+), Gr-1(+), lin(-)/Sca-1(+)/c-kit(-), lin(-)/Sca-1(+)/c-kit(+), lin(-)/Sca-1(-)/c-kit(+)). Furthermore, the percentage of stem/progenitor cells (lin(-)/Sca-1(+)/c-kit(-), lin(-)/Sca-1(+)/c-kit(+), lin(-)/Sca-1(-)/c-kit(+), VSEL, HSC) was analysed for the first time in bone marrow and peripheral blood of chimeric mice. The range of mixed chimerism differed significantly among various cell populations: it was lowest in CD8-positive cells and lin(-)/Sca-1(+)/c-kit(-) cells, and highest in granulocytes. The induction of mixed chimerism revealed a significant impact on the stem/progenitor cell frequency in recipient mice, providing potential therapeutic insights into the long-term immunologic tolerance observed in chimeric mice. Collectively, these findings contribute to further optimization of mixed chimerism induction protocols and might help in the introduction of this phenomenon into clinical practice.

Bone marrow of multiorgan donors underutilized: implications for improvement of accessibility of hematopoietic cells for transplantations.

BACKGROUND: The demand for human hematopoietic stem and progenitor cells (HSPCs) for transplantation is increasing. Thus, effective alternative sources of HSPCs are required. Consequently, we sought to expand the accessibility of hematopoietic cells for clinical purposes by the investigation of hematopoietic reconstitution after transplantation of human HSPCs harvested from the bone marrow (BM) of heparinized deceased organ donors (HDODs). METHODS: For multipart research comparison, human BM HDODs-, healthy donor-derived, umbilical cord blood nuclear cells, or CD34(+) cells were transplanted into sublethally irradiated NOD/SCID mice. Twenty-eight days after transplantation nuclear cells were isolated from the murine BM, spleen, and peripheral blood and were used to quantitatively detect human CD45 antigen by quantitative real-time reverse transcriptase-polymerase chain reaction and flow cytometry. The clonogenic growth of human colony-forming units was also investigated. RESULTS: We found that umbilical cord blood-derived HSPCs showed the greatest transplantation potential in our in vivo model. Interestingly, the transplantation potential of HSPCs collected from the BM of HDODs was of the same quality as cells obtained from healthy BM donors. CONCLUSION: Based on these results, we conclude that HDODs are a strongly underappreciated source of HSPCs that are ready to use for clinical purposes.

Different populations of circulating endothelial cells in patients with age-related macular degeneration: a novel insight into pathogenesis.

PURPOSE: Circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) may serve as novel markers of endothelial dysfunction. The presence and clinical implications of CECs and the expression of endothelin (ET)-1, one of the most potent vasoconstrictors, have not been evaluated in patients with the neovascular form of age-related macular degeneration (AMD). This study was conducted to determine the different populations of endothelial cells (ECs) in the peripheral blood of AMD patients and to correlate these findings with the expression of ET-1 and the cytokines and growth factors responsible for EC migration and function. METHODS: Peripheral blood samples were collected from 29 patients with diagnosed neovascular AMD and from 38 healthy control subjects. CD133(-)CD144(+) CECs and CD34(+)CD133(+)CD144(+) EPCs were counted and analyzed by flow cytometry. The intracellular expression of ET-1 in peripheral blood nuclear cells (PBNCs) was studied by using qRT-PCR, Western blot, and immunocytofluorescence assays, and ET-1, IGF-1, VEGF, SDF-1, and HGF plasma concentrations were measured in enzyme-linked immunosorbent assays. RESULTS: Increased CECs and EPCs were found in the AMD patients compared with the counts in healthy individuals. The expression of intracellular ET-1 was significantly elevated in PBNCs from the AMD patients compared with the control subjects. In addition a significantly higher plasma concentration of IGF-1 was observed, but a lower SDF-1 level in the group of AMD patients. CONCLUSIONS: These findings suggest that circulating endothelial cells, together with high ET-1 content, may contribute to the development of AMD. Further prospective investigations on the mechanism involved may be relevant to the potential treatment of this disease.