Dataset Information

SIRT3 Enhances Mesenchymal Stem Cell Longevity and Differentiation.

ABSTRACT: Mesenchymal stem cells (MSCs) are multipotent cells that are currently being investigated in a wide variety of clinical trials for their anti-inflammatory and immunomodulatory properties as well as their osteogenic and chondrogenic capabilities. However, there are considerable interdonor variability and heterogeneity of MSC populations, making it challenging to compare different products. Furthermore, proliferation, differentiation, and immunomodulation of MSCs decrease with aging and ex vivo expansion. The sirtuins have emerged as a class of protein deacylases involved in aging, oxidative stress, and metabolism. Sirtuin 3 (SIRT3) is the major mitochondrial deacetylase involved in reducing oxidative stress while preserving oxidative metabolism, and its levels have been shown to decrease with age. This study investigated the role of SIRT3 in MSC differentiation and aging. As MSCs were expanded ex vivo, SIRT3 levels decreased. In addition, SIRT3 depletion reduced MSC differentiation into adipocytes and osteoblasts. Furthermore, overexpression of SIRT3 in later-passage MSCs reduced aging-related senescence, reduced oxidative stress, and enhanced their ability to differentiate. These data suggest that overexpressing SIRT3 might represent a strategy to increase the quality and quantity of MSCs utilized for clinical applications.

SUBMITTER: Denu RA

PROVIDER: S-EPMC5499245 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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SIRT3 Enhances Mesenchymal Stem Cell Longevity and Differentiation.

Denu Ryan A RA

Oxidative medicine and cellular longevity 20170621

Mesenchymal stem cells (MSCs) are multipotent cells that are currently being investigated in a wide variety of clinical trials for their anti-inflammatory and immunomodulatory properties as well as their osteogenic and chondrogenic capabilities. However, there are considerable interdonor variability and heterogeneity of MSC populations, making it challenging to compare different products. Furthermore, proliferation, differentiation, and immunomodulation of MSCs decrease with aging and ex vivo ex ...[more]

PMID: 28717408

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Project description:Skeletal tissue regeneration following traumatic injury involves a complex cascade of growth factor signals that direct the differentiation of mesenchymal stem cells (MSCs) within the fracture. The necessity for controlled and localized expression of these factors has highlighted the role gene therapy may play as a promising treatment option for bone repair. However, the design of nanocarrier systems that negotiate efficient intracellular trafficking and nuclear delivery represents a significant challenge. Recent investigations have highlighted the roles histone tail sequences play in directing nuclear delivery and activating DNA transcription. We previously established the ability to recapitulate these natural histone tail activities within non-viral nanocarriers, improving gene transfer and expression by enabling effective navigation to the nucleus via retrograde vesicular trafficking. Herein, we demonstrate that histone-targeting leads to ∼4-fold enhancements in osteogenic bone morphogenetic protein-2 (BMP-2) expression by MSCs over 6 days, as compared with standard polymeric transfection reagents. This improved expression augmented chondrogenesis, an essential first step in fracture healing. Importantly, significant enhancements of cartilage-specific protein expression were triggered by histone-targeted gene transfer, as compared with the response to treatment with equivalent amounts of recombinant BMP-2 protein. In fact, an ∼100-fold increase in recombinant BMP-2 was required to achieve similar levels of chondrogenic gene and protein expression. The enhancements in differentiation achieved using histone-targeting were in part enabled by an increase in transcription factor expression, which functioned to drive MSC chondrogenesis. These novel findings demonstrate the utility of histone-targeted gene transfer strategies to enable substantial reductions in BMP-2 dosing for bone regenerative applications.Statement of significanceThis contribution addresses significant limitations in non-viral gene transfer for bone regenerative applications by exploiting a novel histone-targeting approach for cell-triggered delivery that induces osteogenic BMP-2 expression coincident with the initiation of bone repair. During repair, proliferating MSCs respond to a complex series of growth factor signals that direct their differentiation along cellular lineages essential to mature bone formation. Although these MSCs are ideal targets for enhanced transfection during cellular mitosis, few non-viral delivery approaches exist to enable maximization of this effect. Accordingly, this contribution seeks to utilize our histone-targeted nanocarrier design strategy to stimulate BMP-2 gene transfer in dividing MSCs. This gene-based approach leads to significantly augmented MSC chondrogenesis, an essential first step in bone tissue repair.

Project description:BackgroundMesenchymal stem cells (MSCs) have been recognized as a promising tool for the treatment of various inflammatory disorders and autoimmune diseases. Stress conditions affect immune-mediated treatment and activate autophagy in MSCs. However, whether autophagy affects the MSC-mediated recruitment and differentiation of CD4+ T cells remains elusive.MethodsMSCs were pretreated with 3-methyladenine (3-MA) and rapamycin to regulate autophagy, and then co-cultured with CD4+ T cells. CD4+ T cell migration and differentiation were detected by flow cytometry. Further, gene expression levels of well-known chemokines were analyzed by quantitative real-time PCR. Enzyme-linked immunosorbent assays and western blot analysis were performed to detect C-X-C motif chemokine ligand 8 (CXCL8) and transforming growth factor (TGF)-β1 protein levels. An exogenous antibody and short hairpin RNA were used to regulate CXCL8 and TGF-β1 levels, which enabled us to evaluate how autophagy affected MSC-mediated CD4+ T cell migration and differentiation.Results3-MA inhibited autophagy in MSCs, which was activated by rapamycin. Rapamycin increased the migration of CD4+ T cells, whereas 3-MA decreased their migration. Mechanistically, we found that autophagy strengthened CXCL8 secretion, and the addition of exogenous CXCL8 and an anti-CXCL8 antibody eliminated the difference of CD4+ T cell migration among groups. Further, the ratio of regulatory T (Treg) cells was increased in rapamycin-pretreated MSCs, but the ratio of T helper 1 (Th1) cells was decreased, while pretreatment of MSCs with 3-MA induced the opposite effect compared with the control group. TGF-β1 overexpression and knockdown using lentiviruses rectified the differences in the ratios of Treg and Th1 cells among the groups.ConclusionThis study demonstrates that autophagy of mesenchymal stem cells mediates CD4+ T cell migration and differentiation through CXCL8 and TGF-β1, respectively. These results provide a potential new strategy for improving MSC-mediated therapy.

Dataset Information

SIRT3 Enhances Mesenchymal Stem Cell Longevity and Differentiation.

Publications

SIRT3 Enhances Mesenchymal Stem Cell Longevity and Differentiation.

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