The Study Of The Regulation Of Senescence In Bone Marrow-derived Mesenchymal Stem Cells And The Asymmetric Division Model Of Single Cell

Bone marrow-derived mesenchymal stem cells(bMSCs) are autologous cell resource employed in many clilnical cell therapy trials for tissue and organs repairing. However, in vitro cultured bMSCs will exhibit replicative senescence phenomenon. This kind of senescence complied with loss of proliferation ability and differentiation potency. Meanwhile, bMSCs are highly heterogeneous, asymmetric divided after mitosis. The heterogeneous and unpredictable properties such as senescence hamper the studies of the potency optimizing of bMSCs.This study, on the one hand, is aim to find genes that play critical roles in senescence regulation of bMSCs through gene expression profiling. On the other hand, we wish to establish a asymmetric division model for bMSCs research via single cell suspension culture.By taking advantage of single-cell sequencing, eventually we will find new marker genes that regulate senescence and(or) asymmetric division.Part1: The study of senescence regulation genes in bMSCsMethods: Through depicting growth curve and cell morphology, performing EdU incorporation and β-Gal staining, comparing expression levels of cell cycle negative regulators, detecting H2 A.X Ser139 phosphorylation level, we compared the difference of proliferation potency between early and later passages of bMSCs, defined the phenotype of senescence. Meanwhile, gene microarray were performed for discoving the genes expression in MAPCs, combining the GEO database, we compared the difference of gene expression in ESCs/MAPCs/bMSCs, found specific genes expressed in MAPCs. qPCR were employed for screening out the senescence regulation relative candidate gene DDR2 in bMSCs aging process. siRNA specific target DDR2 were transfected in bMSCs, cell morphology, EdU incorporation and CCK8 proliferative test revealed proliferation ability of bMSCs after DDR2 were inhibited. β-Gal staining, p16 and hTERT level and DNA comets array revealed the phenotype of senescence and DNA damage. CARM1 specific siRNA were transfected in b MSCs,same methods were perfomed for same purpose. Ectopic expressing CARM1/DDR2 and rescure experiments were used for confirming wether CARM1 regulate DDR2 and up-regulate bMSCs growth. After all, we employed CARM1 inhibitor and ChIP for discovering wether CARM1 could modify H3R17 methylation on DDR2 promoters. Results: Later passages of bMSCs growth slowly than early passages, showing no colony formation, hypertrophy, more senescence phenotype and DNA damage. DDR2 were selected as an candidate gene specific expression in MAPCs, inhibited DDR2 expression induced cell senescence, decreased cell proliferation, up regulated H2 A.X Ser139 phosphorylation, DNA damage and p16 levels, down regulated hTERT level. Iinhibited CARM1 expression induced similar results and down regulated DDR2. Ectopic expressed CARM1 up regulated DDR2, ectopic expressed CARM1/DDR2 displayed no effects on promoting cell proliferation. CARM1 inhibitor down regulated DDR2 and promoted cell senescence. CARM1 enriched on DDR2 promoters and catalyzed H3R17 methylation. Conclusions: Specific gene expression profile was found in MAPCs. Later passages of bMSCs exhibit cell senescence and lower proliferation potency, DDR2 plays a critical role in regulating senescence, CARM1 regulate senescence via activate DDR2 transcription. Ectopic express CARM1/DDR2 is not an ideal way of promoting proliferation. Single-cell study will be performed for researching cell asymmetric division and senescence.Part II: The study of single bMSC asymmetric division modelMethods: bMSCs were separated from bone marrow and passaged, by using CFSE cell tracker we lablled P3 cells and disgested them to single bMSC(S-b MSC). Cell were cultured in adherent and suspension medium respectively. Labbled S-bMSC were cocultured with uMSCs in suspension medium. Unlabbled S-bMSC were cultured in suspension medium alone. Daughter cells of divided S-bMSC were separated and stored in lysis solution for single-cell transcriptome amplification, cDNA library construting and sequencing. Results: CFSE labbed S-bMSC are not able to divided alone, but divided after coculture with uMSCs, and formed clusters, but not appropriate for follow-up works. Unlabbled S-bMSC divided into 3,5,6,7 stages besides 2,4,8 stages.Daughter cells from 2,3,4 cell stages were separated and stored successfully. Conclusions: S-bMSC exhibits multi-stages during mitosis under suspension medium condition.. The difference among daught cells from same stage provid a proper model for asymmetric division study, the difference among daught cells from different stages provid a model for proliferation and senescence study.Part III: S-bMSC transcriptome amplification, library construction and surface markers analysisMethods: Daughter cells from 2,3,4 stages were lysised, their mRNA were reverse transcripted by 3’poly(A) tailing, then amplified by two rounds of PCR and purification. bMSCs surface markers of 20 daughter cells were detected by qPCR using amplified cDNA to identify daughter cells, analysis heterogeneity and different potency. Results: Daughter cells’ trancriptomes from each stages were amplified successfully, every cell expressed CD90 positive marker,and non negative markers were found. CD105 positive marker expressed differently among cells in same stages.CD73 positive marker detected only in 4 cell stage daughter cells. S-bMSC trancriptomes were successfully amplified, libraries were qualified. Conclusions: Asmmetric division happens in early proliferation stage of S-bMSC and throughout later stages. bMSCs surface markers expression are time-space specificity in S-bMSC asymmetric division model. The follow up work on single-cell sequencing will reveal much more genes plays vital roles in asymmetric division and senescence process.