ABSTRACT
BACKGROUND: Perivascular cells have been shown to be the precursor cells of mesenchymal stem cells, which regulate the behavior of hematopoietic stem cells and support hematopoiesis through cell-to-cell contact or paracrine effects. Hematopoietic support of human skeletal muscle-derived pericytes/perivascular cells (hMD-PCs) remains to be studied. OBJECTIVE: To identify the biological characteristics of hMD-PCs isolated from human skeletal muscle and to study their supporting effect on umbilical cord blood CD34+ cells in vitro. METHODS: (1) hMD-PCs with phenotype CD146+ CD56-CD34-CD144-CD45- were sorted from human skeletal muscle by enzymatic digestion and multiparameter fluorescence-activated cell sorting, and their biological characteristics were identified. (2) The in vitro culture system of umbilical cord blood CD34+ cells co-cultured with human CD146+ hMD-PCs (experimental group) or with human bone marrow mesenchymal stem cells (positive control group) was established. After 1, 2 and 4 weeks of co-culture, the number of cells, the colony formation ability and immunophenotype were measured and statistically analyzed. RESULTS AND CONCLUSION: (1) CD146+ hMD-PCs were sorted by multiparameter fluorescence-activated cell sorting and the purity was (91.5±1.85)% (n=5). CD146+ hMD-PCs expressed mesenchymal surface markers CD73, CD90, CD105, CD44, and did not express hematopoietic cell and endothelial cell markers CD45, CD34, and CD31. After induced culture, CD146+ hMD-PCs could differentiate into osteoblasts, chondrogenesis, adipocytes and myoblasts. (2) There were no significant differences in the cell number, colony f ormation ability or immunophenotype (CD45+, CD34+ CD33-, CD14+, CD10+/CD19+) between experimental and positive control groups (P > 0.05, n=6). The number of cells in the blank control group without feeder was significantly decreased at 1 week of culture, and there was almost no cell survival at 2 weeks of culture. (3) In summary, CD146+ hMD-PCs, like human bone marrow mesenchymal stem cells, have hematopoietic support capacity in vitro.
ABSTRACT
Objective To investigate the effect of transplantation of human umbilical cord blood CD34+cells on spinal cord injury. Methods CD34+cells were separated from fresh human umbilical cord blood by magnetic cell sorting. Ninety-six female Wistar rats were injured at T10 by IMPACTOR MODEL-Ⅱ, and then randomly assigned to three groups:Cyclo?sporin A (CsA)+Dexamethasone (Dex) treated group (Ⅰ, n=32), local transplantation of cells+CsA+Dex treated group (Ⅱ) at the first day after operation (DAO 1, n=32), local transplantation of cells+CsA+Dex treated group (Ⅲ) at DAO 6 (n=32). BBB locomotor scoring system was used to assess the recovery of the lower limbs. The survival and neural differentiation of transplanted cells at the injury site were observed by double immunofluorescence. The tissue vitality at the injury site was ob?served by 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) staining, and the blood vessel density was observed by infusing mixture of Chinese ink and glutin followed by HE staining. Results BBB score at DAO 8-56 was significantly higher inⅡgroup than that of other two groups (P<0.05). TTC staining showed that the proportion of decreased vitality area was signifi?cantly smaller inⅡgroup than that of other two groups (P<0.01). The result of gelatin ink perfusion showed that the blood vessel density at the injury site was significantly bigger inⅡgroup than that of other two groups (P<0.01). There were more survival transplanted cells inⅡgroup than those of III group (per visual field, 7.51 ± 1.00 vs 5.51 ± 0.89,t=6.051, P<0.01). All the transplanted cells didn’t differentiate into neural cells. Conclusion Human umbilical cord blood CD34+cells can promote the recovery of the lower limbs after spinal cord injury by repairing blood vessels to increase tissue vitality at the in?jury site in rats.
ABSTRACT
BACKGROUND: In vitro generated cells from cord blood (CB) CD34+ cells increase cell dose and may reduce the severity and the duration of pancytopenia after transplantation. But safe engraftment for adolescents and adults is still not predictable and standardized. We attempted to establish a clinically application of in vitro generated cells by investigating the use of cytokines for the culture of CB CD34+ cells. METHODS: CD34+ cells, purified from four separate human CB units by magnetic bead selection, were cultured in IMDM with several cytokines. Differentiation of the in vitro generated cells has been confirmed by flowcytometry with specific erythroid, myeloid and megakaryocytic lineage surface markers. And apoptosis of these cells also was analysed with annexin V staining and morphologic analysis under the electron microscopic examination was done, simultaneously. RESULTS: Purified CB CD34+ cells were expanded with differentiation from CD38- to CD38+ expression with time. CB CD34+ cells could be terminally differentiated into erythroid (GPA+) and myeloid (CD33+/CD15+) lineage cells without apoptosis (annexin-V - ) in the presence of EPO and G-CSF, respectively. Megakaryocytic differentiation was partially arrested in early stage due to apoptosis in the presence of TPO. Morphological examination using electron microscope revealed all stages of erythroid and myeloid lineage cells without apoptosis, and apoptotic megakaryocytic lineage cells of early stage. But we could observe the small number of fully maturated platelets. CONCLUSION: CB CD34+ cells were terminally differentiated to erythroid, myeloid, and megakaryocytic lineage cells with or without apoptosis by several cytokines.