[Comparative characterization of mesenchymal stem cells from human umbilical cord tissue and bone marrow].

This study was purposed to compare the biological characteristics of umbilical cord-derived mesenchymal stem cells (UC-MSCs) and bone marrow-derived mesenchymal stem cells (BM-MSCs). The frequency of successful isolation, cell yield, colony-forming units-fibroblastics (CFU-F), proliferation capacity, immunophenotype and multi-differentiation potentials of UC-MSCs and BM-MSCs were determined by limiting dilution assay, flow cytometry, invert microscopy, RT-PCR and so on, the determined results were compared. The results showed that MSCs were successfully isolated from all the 36 portion of UC tissue and 8 portion of BM. Although the mean number of nucleated cells isolated from UC tissue was significantly lower than that from BMs (1 x 10(6)/cm vs 5.5 x 10(7)/ml) (p=0.0002), no significant differences of the yield of adherent cells were observed (8.6 x 10(5)/cm vs 8.4 x 10(5)/ml) (p>0.05). UC-MSCs shared the most of the characteristic of BM-MSC, including fibroblastic-like morphology, typical immunophenotype, cell cycle status, adipogenic and osteogenic differentiation potentials. However, the CFU-F frequency was higher in UC (1:1609+/-0.18) than that in BM (1:35700+/-0.01) (p<0.05). Furthermore, the proliferation capacity of UC-MSCs was higher than that of BM-MSCs; the expressions of CD106 and HLA-class I in UC-MSCs were lower than those in BM-MSCs (p<0.05). It is concluded that the cell yield and most biological characteristics of UC-MSCs are similar to BM-MSCs, but UC-MSCs possess the higher proliferation capacity, and the lower expression of HLA-class I and HLA-DR as compared with BM-MSCs, therefore the human umbilical cord tissue may be considered as a promising alternative to bone marrow as a source of MSCs.

Comparative characterization of mesenchymal stem cells from human umbilical cord tissue and bone marrow / 中国实验血液学杂志

This study was purposed to compare the biological characteristics of umbilical cord-derived mesenchymal stem cells (UC-MSCs) and bone marrow-derived mesenchymal stem cells (BM-MSCs). The frequency of successful isolation, cell yield, colony-forming units-fibroblastics (CFU-F), proliferation capacity, immunophenotype and multi-differentiation potentials of UC-MSCs and BM-MSCs were determined by limiting dilution assay, flow cytometry, invert microscopy, RT-PCR and so on, the determined results were compared. The results showed that MSCs were successfully isolated from all the 36 portion of UC tissue and 8 portion of BM. Although the mean number of nucleated cells isolated from UC tissue was significantly lower than that from BMs (1 x 10(6)/cm vs 5.5 x 10(7)/ml) (p=0.0002), no significant differences of the yield of adherent cells were observed (8.6 x 10(5)/cm vs 8.4 x 10(5)/ml) (p>0.05). UC-MSCs shared the most of the characteristic of BM-MSC, including fibroblastic-like morphology, typical immunophenotype, cell cycle status, adipogenic and osteogenic differentiation potentials. However, the CFU-F frequency was higher in UC (1:1609+/-0.18) than that in BM (1:35700+/-0.01) (p<0.05). Furthermore, the proliferation capacity of UC-MSCs was higher than that of BM-MSCs; the expressions of CD106 and HLA-class I in UC-MSCs were lower than those in BM-MSCs (p<0.05). It is concluded that the cell yield and most biological characteristics of UC-MSCs are similar to BM-MSCs, but UC-MSCs possess the higher proliferation capacity, and the lower expression of HLA-class I and HLA-DR as compared with BM-MSCs, therefore the human umbilical cord tissue may be considered as a promising alternative to bone marrow as a source of MSCs.

[Construction of recombinant adenovirus vector containing human survivin gene and its expression in dendritic cells].

The study was aimed to construct the recombinant adenovirus vectors containing human survivin gene, and to investigate their expression in transfected dendritic cells. Full length cDNA encoding survivin was obtained by PCR amplification from plasmid pcDNA3.0-survivin. The PCR product was restricted, and then inserted into pShuttle-CMV. The plasmids of pShuttle-CMV-survivin were linearized with PmeI, and the fragment containing survivin was ligated with pShuttle-CMV and transfected into E. coli BJ5183. After homologous recombination in bacteria, the extracted plasmid from the positive bacteria were linearized with PacI, transfected into HEK293 cells with liposome Lipofectamine 2000. Then, the harvested adenovirus supernatants were transfected into dendritic cells. The results showed that the recombinant adenovirus-survivin was constructed successfully and its titer was about 2.65 x 10(9) pfu/ml. The expression of survivin in transfected dendritic cells was confirmed by Western blot analysis. It is concluded that the recombinant adenovirus vector containing human survivin was constructed successfully, which may provide preliminary laboratory evidence for anti-leukemia immunotherapy.

Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials.

BACKGROUND AND OBJECTIVES: Adult bone marrow (BM) is the major source of mesenchymal stem cells (MSC) for cell therapy. However, aspiration of BM involves invasive procedures. We isolated MSC from human full term umbilical cord tissues (UC). The biological characteristics of MSC derived from UC (UC-MSC) were further determined and compared with normal adult bone marrow-derived MSC (BM-MSC). DESIGN AND METHODS: MSC were isolated from UC by enzyme digestion and cultured in appropriate growth medium. The isolation efficiency, cell yield, colony-forming unit-fibroblast (CFU-F) frequency, growth kinetics, phenotypic characteristics, multi-lineage differentiation capacity, cytokine spectrum as well as hematopoiesis-supportive function of UC-MSC were determined and compared with those of BM-MSC. RESULTS: MSC were successfully isolated from all 36 UC and six BM samples we collected for this study. The mean number of nucleated cells isolated from UC was 1yen106/cm and the yield of adherent cells was 8.6yen105/cm. UC-MSC shared most of the characteristic of BM-MSC, including fibroblastic-like morphology, immunophenotype, cell cycle status, adipogenic and osteogenic differentiation potentials, and hematopoiesis-supportive function. The CFU-F frequency was higher in UC nucleated cells (1:1609 +/- 0.18) than in BM nucleated cells (1:35700 +/- 0.01) (p < 0.05). Furthermore, in comparison with BM-MSC, the UC-MSC had a higher proliferation capacity and lower levels of expression of CD106 and HLA-ABC (p < 0.05). Immunofluoresent and western blot assays revealed that UC-MSC had a higher percentage of neuron specific enolase-positive cells than had BM-MSC after neuronal induction. Finally, reverse transcriptase polymerase chain reaction analysis showed that UC-MSC had a cytokine spectrum very similar to that of BM-MSC, including expression of the mRNA of stem cell factor, leukemia inhibitor factor, macrophage-colony stimulating factor, Flt3-ligand, interleukin-6, vascular endothelial growth factor and stromal-derived factor-1, but UC-MCS additionally expressed mRNA of granulocyte macrophage and granulocyte colony-stimulating factors. After co-culture with CD34+ cord blood cells for 5 weeks, no significant difference in colony-forming cells was observed between the CD34+ cells/UC-MSC and CD34+ cells/BM-MSC co-cultures (p > 0.05). INTERPRETATION AND CONCLUSIONS: We have established a protocol to isolate abundant MSC from human umbilical cords with a 100% success rate. The comparative study indicates that UC is an excellent alternative to BM as a source of MSC for cell therapies.

Construction of recombinant adenovirus vector containing human survivin gene and its expression in dendritic cells / 中国实验血液学杂志

The study was aimed to construct the recombinant adenovirus vectors containing human survivin gene, and to investigate their expression in transfected dendritic cells. Full length cDNA encoding survivin was obtained by PCR amplification from plasmid pcDNA3.0-survivin. The PCR product was restricted, and then inserted into pShuttle-CMV. The plasmids of pShuttle-CMV-survivin were linearized with PmeI, and the fragment containing survivin was ligated with pShuttle-CMV and transfected into E. coli BJ5183. After homologous recombination in bacteria, the extracted plasmid from the positive bacteria were linearized with PacI, transfected into HEK293 cells with liposome Lipofectamine 2000. Then, the harvested adenovirus supernatants were transfected into dendritic cells. The results showed that the recombinant adenovirus-survivin was constructed successfully and its titer was about 2.65 x 10(9) pfu/ml. The expression of survivin in transfected dendritic cells was confirmed by Western blot analysis. It is concluded that the recombinant adenovirus vector containing human survivin was constructed successfully, which may provide preliminary laboratory evidence for anti-leukemia immunotherapy.