| Part 1 Comparison of biological characteristics of human umbilical cord mesenchymal stem cells isolated by two methods in different culture dimensions Objective: To compare the biological characteristics of human umbilical cord mesenchymal stem cells(hUC-MSCs)isolated by enzymatic digestion and tissue adherence under two-dimensional(2D)and three-dimensional(3D)culture.Methods: hUC-MSCs were isolated by collagenase type I digestion and adherent extraction from tissue blocks.After being expanded to the P3 generation,they were grouped into 2D and 3D culture.Cell spheroids were observed by scanning electron microscope.Dead and alive staining was used to record the survival status of cell spheroids.Alamar blue was selected to detect the proliferation ability and growth curve.The expression of immunophenotype CD34,CD44,CD45,CD90 and CD105 was detected by flow cytometry.Immunofluorescence and western blot were used to detect the expression of pluripotency transcription factors Sox2,Nanog and Oct4.Results: Average on the 3rd day of enzymatic digestion,it was found that the cells adhered and began to grow,while the cells of the tissue adherent crawled out from the edge of the tissue block after 7th day.Tight junctions between cells in spheroid were observed by scanning electron microscope.Dead and alive staining showed that cell spheroids were in good survival condition after 3 days culture,and a few cells dead in the center of spheroids.The survival curve drawn through Alamar Blue showed that,under 2D culture,the logarithmic growth phase of the digestion method was 4-6 days,and the adherent method was 2-3 days;cell spheroids proliferation was very slow under3 D culture.Flow cytometry results showed that under 2D culture the cells isolated by enzyme digestion under 2D culture strongly expressed CD44(99.83±0.06%),CD90(99.50±0.46%)and CD105(89.03±0.12%),and negatively expressed CD34 and CD45;cells isolated by tissue adherence method were also strongly expressed CD44(99.87±0.06%),CD90(98.70±0.17%),CD105(71.04±0.64%),but negatively expressed CD34 and CD45.In 2D culture,the expression of CD105 was higher in enzymatic digestion method(P<0.001),and there was no difference in the expression of CD44 and CD90.Under 3D culture,the cells extracted by enzyme digestion strongly expressed CD44(98.73±0.38%)and CD90(94.50±0.36%),lowly expressed CD105(16.37±0.63%),and did not express CD34 and CD45;the cells extracted by the adherent method also strongly expressed CD44(97.73±0.76%),CD90(84.23±0.56%),low expression of CD105(6.66±1.63%),and no expression of CD34 and CD45.In 3D culture,the expression of CD90 and CD105 of enzymatic digestion method was higher than tissue block adherence method(P < 0.01),and there was no difference in the expression of CD44.The expression of the transcription factors Sox2,Nanog and Oct4 in 2D cultured cells isolated by enzymatic digestion method was higher than that of adherent method(P < 0.01);under 3D culture conditions,the expression of Sox2 was higher in tissue adherence method(P < 0.01);and the expressions of Nanog and Oct4 were not different between the two methods.Conclusion: The enzymatic digestion method takes less time to isolate original hUCMSCs,but the time required for cell passaging is shorter with the adherent method in the expansion stage.Whether in 2D or 3D culture conditions,the enzymatic digestion method expresses a higher immunophenotype.In 2D culture,the enzyme digestion method expressed higher levels of pluripotency transcription factors Sox2,Nanog,and Oct4,but this trend was reversed in 3D culture.Part 2 Effects of culture dimension conversion on biological characteristics and induced differentiation of human mesenchymal stem cells Objective: To compare the biological characteristics and the osteogenic/ adipogenic differentiation function indexes of human mesenchymal stem cells in 2D,3D culture and after dimensional conversion into 2D to 2D(2-2D),2D to 3D(2-3D),3D to 2D(3-2D),and 3D to 3D(3-3D).Methods: hUC-MSCs were isolated by collagenase type I digestion method,then expanded to P3 generation and cultured in 2D and 3D for 5 days,after that dimension conversion into 2-2D,2-3D,3-2D,and 3-3D.The expressions of pluripotency transcription factors Sox2,Nanog and Oct4 were detected by IF,Western Blot and RTqPCR,and the expressions of immunophenotypes CD34,CD44,CD90,CD105 and HLA-DR were detected by flow cytometry after dimension conversion.In 2D and 3D culture,hUC-MSCs were osteogenic induced for 14 days,and then the cells after induced were converted into 2-2D,2-3D,3-2D,and 3-3D cultured for 3 days.Alizarin red S staining was used to observe the deposition of calcified matrix,and RT-qPCR was used to detect osteogenesis-related marker genes Runx2,BMP2,and OC.Human adipose-derived mesenchymal stem cells(hAD-MSCs)were isolated by collagenase type I digestion method,and then expanded for 14 days adipogenic induced under 2D and 3D culture.The cells after induced were converted into 2-2D,2-3D,3-2D,3-3D and cultured for 3 days.Oil red O staining was used to observe the accumulation of lipid droplets,and RT-qPCR was used to detect the adipogenic-related marker genes PPARγ,FABP4,and LPL.Results: The expression of pluripotency transcription factors Sox2,Nanog and Oct4 in hUC-MSCs was higher in 3D culture than in 2D culture(P < 0.05).After dimension conversion,the levels of Sox2,Nanog and Oct4 in 2-3D group were higher than those in 2-2D group(P < 0.001);2-3D group was higher than 3-3D group(P < 0.001);3-2D group was higher than 2-2D group(P < 0.05);3-2D group had no difference in Sox2 and Nanog expression compared with 3-3D group,but Oct4 expression in 3-2D group was higher than 3-3D group(P < 0.001).After dimension conversion,the flow cytometry results showed that all four groups did not expressed CD34 and HLA-DR,but strongly expressed CD44 and CD90,and there is no difference between groups;as for the expression of CD105,2-3D group and 3-3D group were very low and there was no difference between,while 2-2D group was higher than 2-3D group(P < 0.001),2-2D group was higher than 3-2D group(P < 0.001),3-2D group was higher than 3-3D group(P < 0.001).After 2D and 3D osteogenic induction for 14 days,alizarin red S staining observed red calcified matrix in hUC-MSCs.RT-qPCR results of the Runx2,BMP2 and OC expressions showed that 2D induction group was higher than 2D control group(P < 0.01),3D induction group was higher than 3D control group(P < 0.01),and simultaneously 3D induction group was higher than 2D induction group(P < 0.001).Alizarin red S staining also observed red calcified matrix after dimension conversion.RT-qPCR detection of Runx2,BMP2,and OC expression showed that 2-3D group was higher than the 2-2D group(P < 0.05);2-3D group was lower than 3-3D group(P <0.001);the 3-2D group was lower than the 3-3D group(P < 0.001);3-2D group and 2-2D group had no difference in the mRNA levels of Runx2 and OC,but the expression of BMP2 in 3-2D group was higher than that in 2-2D group(P < 0.01).After 2D and3 D adipogenic induction for 14 days,oil red O staining observed red lipid droplets in hAD-MSCs.RT-qPCR results of PPARγ,FABP4 and LPL expressions showed that 2D induction group was higher than 2D control group(P < 0.001),3D induction group was higher than 3D control group(P < 0.001),and simultaneously 2D induction group was higher than 3D induction group(P < 0.001).Red lipid droplets were still visible after dimension conversion.RT-qPCR detection showed that the transcription levels of PPARγ and FABP4 in 2-3D group were higher than those in 2-2D group(P < 0.001);2-3D group was higher than 3-3D group(P < 0.001);3-2D group was lower than 3-3D group(P < 0.01);3-2D group was lower than 2-2D group(P < 0.01).While the transcription level of LPL had no difference between 3-2D group and the 2-2D group,the comparison results between the other groups were the same as PPARγ and FABP4.Conclusion: Compared with maintaining a fixed culture dimension,the conversion of culture dimension will promote the expression of pluripotency transcription factors,and 2-3D is better than 3-2D.The expression of CD105 in mesenchymal stem cells was significantly decreased under 3D culture,and the dimensional transition of 2-3D decreased the expression of CD105,while the expression of CD105 was restored in 3-2D.3D induction was better than 2D induction for the expression of osteogenesisrelated genes,while 2D induction was better than 3D induction for the expression of adipogenesis-related genes.The dimensional conversion of 2-3D after induction promoted the expression of osteogenesis and adipogenesis-related genes,while 3-2D decreased the expression of osteogenesis and adipogenesis-related genes. |
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