| Background:Mesenchymal stem cells (MSCs) are widespread in adult organisms and involved in tissue maintenance and repair as well as in the regulation of hematopoiesis and immunologic responses. It represents a promising cell-based therapy for a number of degenerative conditions and inflammatory diseases. To thoroughly understand the surface markers of MSCs is of theoretical and practical significance for separation, purification, identification, functional studies and eventually application of specific MSCs in clinical settings. Although CD73, CD90, CD105 are recognized as MSCs surface markers, they do not reflect the "stemness" characteristics of MSCs, which means self-renewing, multipotent differentiation and micro-environmental sensitivity. In previous studies, we found CD49f (Integrin α6) was differential expressed in the fetal and adult bone marrow-derived MSCs (BMSCs). Being an important class of cell surface proteins, adhesion molecules mediate the interactions between stem cells and their niches and thus play key roles in maintaining the sternness of stem cells. CD49f is recognized as a stemness marker for a variety of stem cells, such as embryonic stem cells, neural stem cells, hematopoietic stem cells. By binding to ECM component laminin, CD49f also plays an important role in the interactions between stem cell and the microenvironment. Currently, there is only limited information about the roles of CD49f in MSCs, further studies are needed to prove whether CD49f is a sternness marker for BMSCs. Meanwhile, being an important part of the microenvironment, inflammatory cytokines communicate physiological or pathological signaling to the stem cells via adhesion molecules, whether CD49f play a role in BMSCs sensing microenvironment inflammatory signaling and further modulating their biological characteristics to meet the body’s requirement is still need further study.Objective:1. To compare the self-renew and differentiation potential of CD49f+ and CD49f-BMSC, and to explore the possibility of CD49f being a sternness surface marker for BMSCs.2. To investigate the influence of inflammatory cytokines on CD49f and the role of CD49f in BMSCs’ sensing of inflammatory signaling and further modulating the biological behaviors in accordance with the body’s requirement.3. To further elucidate the regulatory mechanisms of TNF-a on CD49f in BMSCs.Methods and Results:1. Evaluation of CD49f as a stemness surface maker for BMSCs.Methods:Flow cytometry and Real-Time PCR were applied for detection of CD49f expression in different sources of stem cells/cells with varied differentiation potential; fetal BMSCs were verified to be mesenchymal stem cell in terms of morphology, surface markers and differentiation capacity; serial passage of fetal BMSCs was performed to observe the dynamic change of CD49f; CD49f-and CD49f+ cell population were sorted with flow cytometry and CFU-F and osteogenic or adipogenic induction were used to compare their self-renewal and differentiation capacity.Results:CD49+cell proportion and mRNA levels in fetal BMSCs and skin fibroblasts were much higher than in the adult corresponding cells, and almost all human embryonic stem cells (hESCs) and amniotic epithelial cells expressed CD49f; the morphology and surface markers of fetal BMSCs were consistent with adult BMSCs and met the standard of MSCs defined by International Society for Cell therapy; CD49f+ cells was decreased during in vitro passage; CD49f+ cell showed more enhanced ability of colony formation and differentiation than CD49f-cells.2. The role of CD49f in the regulation of BMSCs’biological behaviors under inflammatory conditions.Methods:Different types and concentrations of inflammatory cytokines were added to the culture medium of BMSCs, then CD49f expression was checked with flow cytometry, Real-Time PCR and Westent Blot; CD49f was knockdowned and overexpressed in order to verify its role in maintaining the BMSCs differentiation potential; Adhesion assays were applied to detect the effects of inflammatory cytokines on the adhesion of BMSCs on laminins, and the role of CD49f in the process was also verified by functional blocking antibody; The binding proteins of CD49f was screened by co-immunoprecipitation, and their relationship with adhesion and migration of BMSCs under flammatory environment was checked.Results:After TNF-α treatment, CD106 on BMSCs was up-regulated and CD49f isoform A was down-regulated, while isoform B experienced no significant change; TGF-β1 treatment down-regulated CD 106 expression and showed no effects on CD49f; CD49f play important roles in TNF-α induced impairement of osteogenic and adipogenic differentiation capacity as the overexpression or knockdown of CD49f alone could promote or inhibit the differentiation of BMSCs; however, isoform A showed much more potential in enhancing the differentiation capacity of BMSCs; CD49f mediated the adhesion of BMSCs on laminins, and TNF-a decreased the adhesion; migration of BMSCs increased after TNF-a treatment, TG2 was specifically precipitated by CD49f isoform A and B, and TG2 expression was increased after TNF-a treatment.3. Study of the Mechanism by which TNF-a regulates CD49f expression.Methods:TNF-a was added to the culture medium of BMSCs for certain time points, then the cells were collected and proteins were extracted. Western blot was applied to detect the activation of NF-κB and mTOR pathways, immunofluorescence method was also used for detection of nuclear translocation of NF-κB P65; for inhibitor assays, BMSCs were pretreated with NF-κB inhibitor SN50 and mTOR inhibitor rapamycin and PP242, respectively, then stimulated with TNF-α, the activation state of NF-κB and mTOR pathways were check as above, and meanwile, CD49f expressions in inhibitor pretreated BMSCs then stimulated with TNF-α or normal culture passages were checked with flow cytometry.Results:NF-κB and mTOR pathway in BMSCs were quickly activated by TNF-α; SN50 can specifically and efficiently inhibit TNF-α-induced NF-κB p65 nuclear translocation, and rapamycin and PP242 inhibited mTOR and its downstream effector S6K1 and AKT activation; however, SN50 showed no effects on TNF-α induced decline of CD49f; only rapamycin and PP242 can inhibit the decline of CD49f caused by both TNF-a stimulation or in vitro passaging.Conclusion:1. CD49f is preferentially expressed in fetal BMSCs than adults BMSCs; CD49f is sensitive to environmental change and can be as a sternness surface marker to enrich CD49f+ cell with higher colony formation and differentiation ability than CD49f-cells.2. TNF-a downregulates CD49f expression in BMSCs in a concentration dependent manner, and the downregulation of CD49f plays important role in TNF-α-induced impairement of differentiation capacity of BMSCs. CD49f mediates the binding of BMSCs to laniinins, and TG2 can specifically bind to CD49f; The downregulated expression of CD49f together with the upregulation of TG2 may contribute to the decreased adhesion of BMSCs to laminins and increased migration induced by TNF-α.3. TNF-α induces the activation of NF-κB and mTOR signal transduction pathways; SN50, rapamycin or PP242 significantly inhibite TNF-a-induced NF-κB and mTOR activation in BMSCs. mTOR inhibitors rapamycin and PP242 antaganize the TNF-α or in vitro passags-induced decline of CD49f, which indicates mTOR play an important role in the regulation of CD49f.4. The establishment of CD49f as a stemness surface marker for BMSCs provides a new marker for the sorting of MSCs in clinical studies, and the important roles of CD49f in the modulation of biological behaviors induced by TNF-α provide a new mechanism to clarify the impact of inflammation on stem cells. Furthermore, mTOR inhibitors may be applied to prevent the exhaustion of stem cells both in vivo caused by inflammation and in vitro by long time culture. |
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