SATB2-Nanog Axis Links Age-related Intrinsic Changes Of Mesenchymal Stem Cells From Human Craniofacial Bone

Bone mesenchymal stem cells (BMSCs) senescence contributes to age-related bone loss. The alveolar bone in jaws originates from neural crest cells and possesses significant site- and age-related properties. However, such intrinsic characteristics of BMSCs from alveolar bone (AB-BMSCs) and the underlying regulatory mechanisms still remain incompletely know. Here, we found that the expression of special AT-rich binding protein 2 (SATB2) in AB-BMSCs significantly decreased with aging. SATB2 knockdown on AB-BMSCs from young donors displayed these aging-related phenotypes in vitro. Meanwhile, enforced SATB2 overexpression could rejuvenate AB-BMSCs from older donors. Mechanistically, the sternness regulator Nanog was identified as the direct transcriptional target of SATB2 in BMSCs and functioned as a downstream mediator of SATB2. Collectively, our data reveal that SATB2 in AB-BMSCs associates with their age-related properties, and prevents AB-BMSCs senescence via maintaining Nanog expression. These findings highlight the translational potential of transcriptional factor-based cellular reprogramming for anti-aging therapy.Part I Biological characterization in AB-BMSCs Declines with AgeObjective:To isolate and culture bone mesenchymal stem cells from alveolar bone in vitro and to study the in vitro biologic characters of AB-BMSCs.Methods:1).AB-BMSCs were isolated and cultured by whole bone marrow adherent culture and the nonadherent cells were removed by changing the culture medium. Cells were passaged when the monoclonal colony formed and attached each others; 2).Real-time PCR and Western bolts for SATB2 expression in AB-BMSCs.3). Proliferating function of BMSCs was identified by colony formation assays.4).CCK8 assays for cell proliferation ability.5).Western bolts and Immunofluorescence for sternness associated genes expression in AB-BMSCs.6).Real-time PCR, Western bolts, Oil red stain and alizarin redstain were used to detected the multi-differentiation ability.7) Cell Senescence-associated P-galactosidase Staining and Western bolts for senescence associated genes expression in AB-BMSCs.Results:(1) SATB2 mRNA levels in BMSC from older donors (O) were significantly lower as compared to middle (M) and young counterparts (Y). (2) The proliferation rates of AB-BMSCs gradually decreased with aging. The expression of three master sternness factors Nanog, SOX2 and OCT4 was remarkably reduced with aging. (3)Osteogenic potential of AB-BMSCs gradually decreased with aging; adipogenic potential enhanced with aging;We found that both BMSCs from M and O groups exhibited higher M-CSF and lower OPG compared to Y group The RANKL expression and the ratios of RANKL/RANK, RANKL/OPG were increased in M group compared to Y group, but then decreased in O group. (4) More SA-β-Gal positive cells were observed in M and O groups than Y group. Senescence-related factors P16, P21 and P53 showed markedly higher expression in M and O groups compared to Y group.Conclusions:The results indicate that SATB2 expression in AB-BMSCs is decreased with aging as well as stemness and osteogenetic ability and senescence ability increased.Part Ⅱ Study the effects of SATB2-modified BMSCs in stemness and senescenceObjective:To explore the roles and associated regulatory mechanisms of SATB2 in stemness and senescence regulation of AB-BMSCs by SATB2 overexpression or knockdown.Methods:1)AB-BMSCs from older subjects were transfected by exogenous SATB2 by lentiviral vector and endogenous SATB2 in AB-BMSCs from young donors was silenced by shRNA-mediated gene knockdown.CCK8 assays for cell proliferation ability; 2) Western bolts for sternness associated genes and senescence associated genes expression.3) β-galactosidase staining for aging-related phenotypes of AB-BMSCs.4) Alizarin redstain and CPC were used to detected the osteogenic ability.5) Western bolts for RUNX2, OSX and OPN expression.6) Real-time PCR for Leptin, PPAR-y and C/EBP-a mRNA expressionResults:(1) SATB2 overexpression significantly enhanced pluripotency markers expression, promoted osteogenic differentiation of older AB-BMSCs and inhibited their adipogenic differentiation, reduced cellular senescenceas reflected by reduced SA-β-Gal-positive cells and senescence marker expression. (2)The cellular proliferation rate significantly decreased after SATB2 knockdown; SATB2 knockdown reduced the expression abundance of Nanog, SOX2 and OCT4, and increased cell senescence as shown by induced SA-β-Gal-positive cells and enhanced senescence markers expression, inhibited osteogenic differentiation and promoted adipogenic differentiation of BMSCs.Conclusions:Our results from both gain-and loss-of function experiments strongly support the idea that SATB2 is a critical regulator responsible for age-associated propertiesof AB-BMSCs.P art Ⅲ Roles of SATB2-Nanog axis in BMSCs senescence and aging-related bone lossObjective:To analysis the influence of SATB2-Nanog axis in BMSCs senescence and aging-related bone loss and to study the mechanism of senescence.Methods:1) we knocked down Nanog in SATB2 stable overexpressing older AB-BMSCs, Western bolts and Real-time PCR for Nanog, SOX2 and OCT4 expression;Immunofluorescence for Nanog expression; Western bolts for P16, P21 and P53 expression; Alizarin redstain and Western bolts were used to detected the osteogenic ability.2) The enrichment of SATB2 in Nanog promoter region was assessed by Chromatin Immunoprecipitation(ChIP).3) Dual Luciferase Reporter Assay for putative binding region of SATB2 in human Nanog promoter.Results:(1) SATB2 knockdown reduced the expression abundance of Nanog, SOX2 and OCT4, and increased cell senescence as shown by induced SA-β-Gal-positive cells and enhanced senescence markers expression, inhibited osteogenic differentiation of BMSCs. (2) Significant enrichment of SATB2 was identified in four putative binding sites in human Nanog promoter region. (3) The luciferase activity was significantly increased in reporter containing the binding site 10.Conclusions:There existed a SATB2-Nanog axis regulating AB-BMSCs senescence and differentiation.