The Impacts Of Heterogeneous Microenvironment On Cellular Senescence Of Subpopulations Derived From H-DFSCs

Objectives:Stem cells have been wildly used in cell therapy and tissue regeneration because of their self-renewal ability and multilineage differentiation potential.However,the internal composition of stem cells is not homogeneous,but composed of different subpopulations with diverse phenotypes and functions,leading to instability effects in practical applications.Conversely,subpopulations within the seeding cells showed advantaged differentiation towards specified lineage due to their relatively univocal phenotype,which drives them more ideal seeding cells.It’s still worth noting that separation of subpopulations means the destruction of the original heterogeneous microenvironment,whereas heterogeneity of stem cells is of great significance to the functional activities of cells and tissues,with loss of the heterogeneity often accompanied by tissue aging.Whether subpopulations of stem cells are susceptible to cellular senescence after separation operations needs to be considered before their application as seeding cells.In this study,human dental follicle stem cells(H-DFSCs)were isolated as experimental subjects,and subclones were screened by disturbing heterogeneous microenvironment.By contrasting the aging process among heterogeneous H-DFSCs and subclones,the effects of changed heterogeneous microenvironment on cellular senescence of subclones could be studied,so as to provide a new idea for the long-term application of subpopulations derived from heterogeneous stem cells.Methods:This topic cultured H-DFSCs with enzyme digestion method and explanted tissue culture method.The surface molecules of stem cells were identified by flow cytometry,proliferation ability was measured by plate clonality assays and multilineage differentiation potential was speculated by osteogenic differentiation and adipogenic differentiation.Then ALP staining was used to test their heterogeneity followed by subclones screening through limited dilution method.CCK-8 method and plate clonality assay was used to assay cellular proliferation potential.Then we researched cell cycle distribution by flow cytometry,and counted cells stained with senescence associated β-galactosidase.TP53,CDKN1 A,CDKN2A,MAD2L1 and BUB1 B m RNA expression levels were detected by q RT-PCR.All above senescent-related detections were compared among subclones and the heterogeneity H-DFSCs obtained in the same passage and the same culture duration.Results:1.This experiment cultured H-DFSCs using both enzyme digestion method and explanted tissue culture method.Flow cytometry detection showed their positive expression of CD90 and CD146(mesenchymal stem cell marker),and negative expression of CD34 and CD45(hematopoietic stem cell marker).Plate clonality assays showed strong ability for colony-forming.H-DFSCs owned capacity of osteogenic differentiation and adipogenic differentiation.2.ALP staining combined with plate clonality assays showed positive,weakly positive and negative expression of H-DFSCs.Then subclones were screened by limited dilution method and three subclones DF1,DF2 and DF3 which had proliferated for 11 generations were harvested,showing spindle shape,strip shape and polygon shape respectively.3.CCK8 results showed that,compared with the heterogeneous H-DFSCs at the same passage 11(P11),DF1 showed stronger proliferation capacity,while DF2 and DF3 showed weaker proliferation capacity and DF3 was the worst.The plate clonality assay showed that colony formation ability of DF2 and DF3 is extremely lower than P11,DF1 was similar to P11 but slightly higher than H-DFSCs at passage 16(P16),while H-DFSCs at passage 21(P21)had the highest colony formation efficiency.4.Cell cycle results showed that the proportion of G1 phase cells in DF1 was similar to P11,DF2 and P21 showed less cells in G1 phase and more cells in S phase,while DF3 and P16 had more cells in G1 phase,among which DF3 had the most cells in G1 phase.5.Senescence associated β-galactosidase staining showed the fewest senescent cells in P11,then the numbers began to increase in P16 and accumulated more in P21,but the positive rate of DF1,DF2 and DF3 was still higher than that of P21.6.qRT-PCR results showed that,compared with P11,the expression of genes TP53,CDKN1 A and CDKN2 A decreased in P21 after reaching the peak in P16,while the expression of MAD2L1 increased in P16 and maintained until P21,and no significant change was observed in the expression of BUB1 B gene.As for subclones DF1,DF2 and DF3,the expressions of TP53,CDKN1 A and CDKN2 A were significantly higher than P11,and the expressions of MAD2L1 and BUB1 B were significantly lower than P11.Conclusions:In this experiment,we isolated H-DFSCs with good proliferation ability and multilineage differentiation potential.Three subclones were successfully separated by limited dilution method,among which showing different biological behaviors.Compared with the heterogeneous H-DFSCs,all the subclones showed cellular senescence earlier and exhibited lower repair capacity after senescence.It is therefore suggested that heterogeneous microenvironment plays an important role in maintaining the normal biological behavior of subpopulations,and destruction of heterogeneous environment can significantly affect the biological potential of subpopulations,which need to be considered before application.