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The finding of stem/progenitor cells in postnatal bone marrow and umbilical cord blood, opens up a possibility of using stem cells to treat neurologic diseases. There is a controversy, whether intravenously administered human umbilical cord blood cells (HUCBC) migrate to the brain, differentiate and improve recovery after ischemia. In this study, 13 ×106 cells from non-cultured (non-committed) mononuclear HUCBC fraction were intravenously infused 1, 2, 3 or 7 days after a transient middle cerebral artery occlusion (MCAo) in adult rats. We found few human cells only in the ischemic area, localized mostly around blood vessels with few positive cells in the brain parenchyma. Timing of HUCBC delivery after ischemia or injection of Cyclosporin A at the time of delivery, had no effect on the number of human cells detected in the ischemic brain. Infusion of HUCBC did not reduce infarct volume and did not improve neurologic deficits after MCAo, suggesting that HUCBC failed to migrate/survive in the ischemic brain and did not provide significant neurological benefits.
Human umbilical cord blood is frequently used as a source of transplantable hematopoietic cells and more recently as a target of gene therapy - a new approach for treatment of various disorders. The aim of our study was optimisation of the transfection conditions of cord blood-derived CD34+ hematopoietic cells. Mononuclear cells fraction was isolated from cord blood samples by density gradient centrifugation. Subsequently, CD34+ hematopoietic cells were separated on immuno- magnetic MiniMACS columns. Pure population of CD34+ cells was incubated in a se­rum free medium supplemented with thrombopoietin, stem cell factor and Flt-3 ligand for 48 h and then transfected with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene. We studied the influence of vari­ous pulse settings and DNA concentrations on the transfection efficiency, measured by flow cytometry as the fluorescence of target cells due to the expression of EGFP. The optimal settings were as follows: 4 mm cuvette, 1600 iF, 550 V/cm, and 10 ug of DNA per 500 ul. With these settings we obtained a high transfection frequency (41.2%) without a marked decrease of cell viability. An increase of the pulse capaci­tance and/or of DNA concentration resulted in a greater electroporation efficiency, but also in a decrease of cell viability. In conclusion, the results described here allow one to recommend electroporation as an efficient method of gene delivery into CD34+ hematopoietic cells derived from human umbilical cord blood.
We compared three methods usually applied in biological dosimetry for estimation of radiation-induced DNA damage in human T and B lymphocytes: alkaline comet assay, micronucleus (MN) test and formation of histone gamma-H2AX foci. Human peripheral blood lymphocytes were fractionated using T cells and B cells isolation kits. Cells were irradiated with doses in the range of 0-1 Gy of X-rays. Induction of DNA damage was assessed by the standard alkaline comet assay, MN test and histone gammaH2AX foci immunofluorescence assay. Notwithstanding different end-points measured by the applied methods, all tests revealed a similar induction of DNA damage in B lymphocytes as compared with T lymphocytes. The results indicated that all three tests detect DNA damage with similar sensitivity, the lowest dose being approximately 0.3 Gy. The difference between irradiated and control cells was expressed as the ratio of the value obtained for irradiated cells (1 Gy) to that for control cells. The highest ratio was obtained for formation of gammaH2AX foci and was 6.2 for T and 13.8 for B lymphocytes, whereas those for comet assay and micronucleus test were 3.5; 3.6 and 5.6; 4.8, respectively.
Tissue formation and maintenance is regulated by various factors, including biological, physiological and physical signals transmitted between cells as well as originating from cell-substrate interactions. In our study, the osteogenic potential of mesenchymal stromal/stem cells isolated from umbilical cord Wharton's jelly (UC-MSCs) was investigated in relation to the substrate rigidity on polyacrylamide hydrogel (PAAM). Osteogenic differentiation of UC-MSCs was enhanced on stiff substrate compared to soft substrates, illustrating that the mechanical environment can play a role in differentiation of this type of cells. These results show that substrate stiffness can regulate UC-MSCs differentiation, and hence may have significant implications for design of biomaterials with appropriate mechanical properties for regenerative medicine.
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