Objective To compare the characterization and myocardial differentiation capacity of arnniotic fluid-derived mesenchymal stromal cells （AF MSCs） and umbilical cord Wharton＇s Jelly-derived mesenchymal stromal cells ...Objective To compare the characterization and myocardial differentiation capacity of arnniotic fluid-derived mesenchymal stromal cells （AF MSCs） and umbilical cord Wharton＇s Jelly-derived mesenchymal stromal cells （WJ MSCs）. Methods The human AF MSCs were cultured from amniotic fluid samples obtained by amniocentesis. The umbilical cord WJ MSCs were obtained from Wharton＇s Jelly of umbilical cords of infants delivered full-term by normal labor. The morphology, growth curves, and analyses by flow cytometry of cell surface markers were compared between the two types of cells. Myocardial genes （GATA-4, c-TnT, a-actin, and Cx43） were detected by real-time PCR and the corresponding protein expressions were detected by Western blot analysis after myocardial induced in AF MSCs and WJ MSCs. Results Our findings revealed AF MSCs and WJ MSCs shared similar morphological characteristics of the fibroblastoid shape. The AF MSCs were easily obtained than the WJ MSCs and had a shorter time to reach adherence of 2.7 ± 1.6 days to WJ MSCs of 6.5 ± 1.8 days. The growth curves by MTT cytotoxic assay showed the AF MSCs had a similar proliferative capacity at passage 5 and passage 10. However, the proliferative capacities ofWJ MSCs were decreased at 5 passage relative to 10 passage. Both AF stem cells and WJ stem cells had the characteristics of mesenchymal stromal cells with some characteristics of embryonic stem cells. They express CD29 and CD105, but not CD34. They were positive for Class I major histocompatibility （MHC I） antigens （HLA-ABC）, and were negative, or mildly positive, for MHC Class II （HLA-DR） antigen. Oct-4 was positive in all the two cells types. Both AF MSCs and WJ MSCs could differentiate along myocardium. The differentiation capacities were detected by the expression of GATA-4, c-TnT, a-actin, Cx43 after myocardial induction. Conclusions Both AF MSCs and WJ MSCs have the potential clinical application for myogenesis in cardiac regenerative therapy.展开更多
In this study, Computational Fluid Dynamics(CFD) is used to investigate and compare the impact of bioreactor parameters(such as its geometry, medium flow-rate, scaffold configuration) on the local transport phenomena ...In this study, Computational Fluid Dynamics(CFD) is used to investigate and compare the impact of bioreactor parameters(such as its geometry, medium flow-rate, scaffold configuration) on the local transport phenomena and, hence, their impact on human mesenchymal stem cell(hM SC) expansion. The geometric characteristics of the TissueFlex174;(Zyoxel Limited, Oxford, UK) microbioreactor were considered to set up a virtual bioreactor containing alginate(in both slab and bead configuration) scaffolds. The bioreactor and scaffolds were seeded with cells that were modelled as glucose consuming entities. The widely used glucose medium, Dulbecco's Modified Eagle Medium(DMEM), supplied at two inlet flow rates of 25 and 100 μl·h^(-1), was modelled as the fluid phase inside the bioreactors. The investigation, based on applying dimensional analysis to this problem, as well as on detailed three-dimensional transient CFD results, revealed that the default bioreactor design and boundary conditions led to internal and external glucose transport, as well as shear stresses, that are conducive to h MSC growth and expansion. Furthermore, results indicated that the ‘top-inout' design(as opposed to its symmetric counterpart) led to higher shear stress for the same media inlet rate(25 μl·h^(-1)), a feature that can be easily exploited to induce shear-dependent differentiation. These findings further confirm the suitability of CFD as a robust design tool.展开更多
Objective: To investigate the effects of porous poly lactide-co-glycolide (PLGA) modified by type I collagen on the adhesion, proliferation, and differentiation of rabbit marrow-derived mesenchymal stem cells (MSCs). ...Objective: To investigate the effects of porous poly lactide-co-glycolide (PLGA) modified by type I collagen on the adhesion, proliferation, and differentiation of rabbit marrow-derived mesenchymal stem cells (MSCs). Methods: The third generation MSCs isolated from mature rabbits by density gradient centrifugation were cultured at different initial concentrations on 0.3 cm×1.2 cm×2.0 cm 3-D porous PLGA coated by type I collagen in RPMI 1640 containing 10% fetal calf serum, while cultured on PLGA without type I collagen as control. The cells adhesive and proliferative behavior at 7, 14, and 21 days after inoculation was assessed by determining the incorporation rate of [3H]-TdR. In order to examine MSCs differentiation, the expression of osteoblasts marker genes, osteocalcin (OCN), alkaline phosphatase (ALP), osteopontin (OPN) mRNA, were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), and further more, the cell morphology at 21 days was also observed by scanning electron microscope (SEM). Results: Type I collagen promoted cell adhesion on PLGA. The valve was significantly higher than controls (6 h, 2144 cpm±141cpm vs. 1797 cpm±118 cpm, P=0.017; 8 h, 2311 cpm±113 cpm vs. 1891 cpm±103 cpm, P=0.01). The cells which cultured on PLGA coated with type I collagen showed significantly higher cell proliferation than controls on the 7th day (1021 cpm±159 cpm vs. 451 cpm±67 cpm, P=0.002), the 14th day (1472 cpm±82 cpm vs. 583 cpm±67 cpm, P<0.001) and 21th day (1728 cpm±78 cpm vs. 632 cpm±55 cpm, P<0.001). Osteoblasts markers, OCN, ALP, OPN mRNA, were all detected on PLGA coated by type I collagen on the 21th day, but OCN, OPN mRNA could not be found in controls. Spindle and polygonal cells well distributed on the polymer coated by type I collagen while cylindric or round cells in controls. Conclusions: Type I collagen is effective in promoting the adhesion, proliferation and differentiation of MSCs on PLGA.展开更多
文摘Objective To compare the characterization and myocardial differentiation capacity of arnniotic fluid-derived mesenchymal stromal cells （AF MSCs） and umbilical cord Wharton＇s Jelly-derived mesenchymal stromal cells （WJ MSCs）. Methods The human AF MSCs were cultured from amniotic fluid samples obtained by amniocentesis. The umbilical cord WJ MSCs were obtained from Wharton＇s Jelly of umbilical cords of infants delivered full-term by normal labor. The morphology, growth curves, and analyses by flow cytometry of cell surface markers were compared between the two types of cells. Myocardial genes （GATA-4, c-TnT, a-actin, and Cx43） were detected by real-time PCR and the corresponding protein expressions were detected by Western blot analysis after myocardial induced in AF MSCs and WJ MSCs. Results Our findings revealed AF MSCs and WJ MSCs shared similar morphological characteristics of the fibroblastoid shape. The AF MSCs were easily obtained than the WJ MSCs and had a shorter time to reach adherence of 2.7 ± 1.6 days to WJ MSCs of 6.5 ± 1.8 days. The growth curves by MTT cytotoxic assay showed the AF MSCs had a similar proliferative capacity at passage 5 and passage 10. However, the proliferative capacities ofWJ MSCs were decreased at 5 passage relative to 10 passage. Both AF stem cells and WJ stem cells had the characteristics of mesenchymal stromal cells with some characteristics of embryonic stem cells. They express CD29 and CD105, but not CD34. They were positive for Class I major histocompatibility （MHC I） antigens （HLA-ABC）, and were negative, or mildly positive, for MHC Class II （HLA-DR） antigen. Oct-4 was positive in all the two cells types. Both AF MSCs and WJ MSCs could differentiate along myocardium. The differentiation capacities were detected by the expression of GATA-4, c-TnT, a-actin, Cx43 after myocardial induction. Conclusions Both AF MSCs and WJ MSCs have the potential clinical application for myogenesis in cardiac regenerative therapy.
基金Department of Engineering Science, University of Oxford, Scholarship
文摘In this study, Computational Fluid Dynamics(CFD) is used to investigate and compare the impact of bioreactor parameters(such as its geometry, medium flow-rate, scaffold configuration) on the local transport phenomena and, hence, their impact on human mesenchymal stem cell(hM SC) expansion. The geometric characteristics of the TissueFlex174;(Zyoxel Limited, Oxford, UK) microbioreactor were considered to set up a virtual bioreactor containing alginate(in both slab and bead configuration) scaffolds. The bioreactor and scaffolds were seeded with cells that were modelled as glucose consuming entities. The widely used glucose medium, Dulbecco's Modified Eagle Medium(DMEM), supplied at two inlet flow rates of 25 and 100 μl·h^(-1), was modelled as the fluid phase inside the bioreactors. The investigation, based on applying dimensional analysis to this problem, as well as on detailed three-dimensional transient CFD results, revealed that the default bioreactor design and boundary conditions led to internal and external glucose transport, as well as shear stresses, that are conducive to h MSC growth and expansion. Furthermore, results indicated that the ‘top-inout' design(as opposed to its symmetric counterpart) led to higher shear stress for the same media inlet rate(25 μl·h^(-1)), a feature that can be easily exploited to induce shear-dependent differentiation. These findings further confirm the suitability of CFD as a robust design tool.
文摘Objective: To investigate the effects of porous poly lactide-co-glycolide (PLGA) modified by type I collagen on the adhesion, proliferation, and differentiation of rabbit marrow-derived mesenchymal stem cells (MSCs). Methods: The third generation MSCs isolated from mature rabbits by density gradient centrifugation were cultured at different initial concentrations on 0.3 cm×1.2 cm×2.0 cm 3-D porous PLGA coated by type I collagen in RPMI 1640 containing 10% fetal calf serum, while cultured on PLGA without type I collagen as control. The cells adhesive and proliferative behavior at 7, 14, and 21 days after inoculation was assessed by determining the incorporation rate of [3H]-TdR. In order to examine MSCs differentiation, the expression of osteoblasts marker genes, osteocalcin (OCN), alkaline phosphatase (ALP), osteopontin (OPN) mRNA, were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), and further more, the cell morphology at 21 days was also observed by scanning electron microscope (SEM). Results: Type I collagen promoted cell adhesion on PLGA. The valve was significantly higher than controls (6 h, 2144 cpm±141cpm vs. 1797 cpm±118 cpm, P=0.017; 8 h, 2311 cpm±113 cpm vs. 1891 cpm±103 cpm, P=0.01). The cells which cultured on PLGA coated with type I collagen showed significantly higher cell proliferation than controls on the 7th day (1021 cpm±159 cpm vs. 451 cpm±67 cpm, P=0.002), the 14th day (1472 cpm±82 cpm vs. 583 cpm±67 cpm, P<0.001) and 21th day (1728 cpm±78 cpm vs. 632 cpm±55 cpm, P<0.001). Osteoblasts markers, OCN, ALP, OPN mRNA, were all detected on PLGA coated by type I collagen on the 21th day, but OCN, OPN mRNA could not be found in controls. Spindle and polygonal cells well distributed on the polymer coated by type I collagen while cylindric or round cells in controls. Conclusions: Type I collagen is effective in promoting the adhesion, proliferation and differentiation of MSCs on PLGA.
