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Unidirectional rotating molecular motors dynamically interact with adsorbed proteins to direct the fate of mesenchymal stem cells

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Science Advances  29 Jan 2020:
Vol. 6, no. 5, eaay2756
DOI: 10.1126/sciadv.aay2756
  • Fig. 1 Molecular motion–induced restructuring of the adsorbed protein cell adhesion layer affects stem cell differentiation.

    (A) Schematic illustration of the molecular motion directing the fate of hBM-MSCs. The molecular motion originating from unidirectional rotating molecular motors mediates the initial protein adsorption behavior that affects the fibronectin (Fn) adsorption, which subsequently regulates the FA cytoskeleton actin transduction pathway to govern the gene and protein expressions of hBM-MSCs. (B) Structural details of motor-modified (dynamic layer), amine-modified, and stator-modified (static control layer) surfaces.

  • Fig. 2 Cell adhesion on the molecular motor and amine surfaces.

    (A) Fluorescent images of hBM-MSCs cultured on the molecular motor and amine surfaces for 12 hours. Red is F-actin, visualized by tetramethyl rhodamine isothiocyanate (TRITC)–phalloidin staining, and blue is nucleus, stained by 4′,6-diamidino-2-phenylindole (DAPI); green staining is vinculin. (B to E) Cell density, area per cell, F-actin per cell, and cell elongation on the molecular motor and amine surfaces (n = 3). Data reported as means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001. A.U., arbitrary units.

  • Fig. 3 Subcellular characteristics on the molecular motor and amine surfaces.

    (A) Fluorescent images of a single hBM-MSC cultured on molecular motor and amine surfaces under different conditions for 12 hours. Red staining is F-actin, visualized by TRITC-phalloidin staining, and blue staining is nucleus, stained by DAPI; green staining is vinculin. Scale bars, 40 μm. (B and C) FA area per cell and filopodia number per cell on molecular motor and amine surfaces under different conditions (n = 3), respectively. Data reported as means ± SD (***P < 0.001).

  • Fig. 4 Biocompatibility on the molecular motor surfaces.

    (A) The percentage of living cells and (B) metabolic activity per cell on molecular motor surfaces under different conditions for 12 hours and 4 days (n = 3). (C) Fluorescent images of hBM-MSCs cultured on molecular motor surfaces under different conditions. Scale bars, 75 μm. (D to G) Area per cell, F-actin per cell, cell elongation, and FA area per cell on molecular motor surfaces under different conditions after 4 days (n = 3). Data reported as means ± SD (*P < 0.05, **P < 0.01, ***P < 0.001).

  • Fig. 5 The osteogenic differentiation and stemness maintenance of hBM-MSCs on the molecular motor surfaces.

    (A) Fluorescent images of hBM-MSCs cultured on molecular motor surfaces with different cell media for 14 days. Scale bars, 50 μm. Red, OPN; blue, nucleus. (B) Protein level: Quantification of the expression of OPN normalized by the cell number over 14 days of culture (n = 3). (C) Gene level: Real-time PCR (RT-PCR) analysis of relative mRNA expression levels of OPN genes in different media over 14 days of culture (n = 3). (C and D) RT-PCR analysis of relative mRNA expression level of Nanog and OCT4 genes at different time points (n = 3; *P < 0.05).

  • Fig. 6 The adsorbed protein behaviors on molecular motor and amine surfaces.

    (A) Protein adsorption on the molecular motor surface under different conditions measured by QCM-D. (B) Serum protein mass on the samples quantified by QCM-D (n = 3 to 4). (C) Fn mass quantified from QCM-D curves (n = 3 to 4). (D) Circular dichroism (CD) spectra of protein from BSA adsorbed on the molecular motor surface under different conditions as indicated. Data reported as means ± SD (*P < 0.05, **P < 0.01).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/5/eaay2756/DC1

    Fig. S1. Altered cell behavior induced by molecular motion–mediated protein layer restructuring.

    Fig. S2. Proliferation capacity of MSCs on molecular motor–functionalized surfaces in static and rotating mode upon protein adhesion.

    Fig. S3. Immunofluorescence analysis of cell morphology during osteogenic differentiation of MSCs on dynamic and static molecular motor–functionalized surfaces.

    Fig. S4. Protein adhesion and structural integrity mediated by molecular motion and UV irradiation.

    Fig. S5. MD simulations of protein–molecular motor interactions and motion-induced structural deformation.

    Fig. S6. AFM analysis of motion-induced alterations to structural alterations of protein adhesion layers.

    Fig. S7. Physicochemical influences on the morphology of protein adhesion layers.

    Table S1. Primer sequences of human-specific genes used for qRT-PCR.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Altered cell behavior induced by molecular motion–mediated protein layer restructuring.
    • Fig. S2. Proliferation capacity of MSCs on molecular motor–functionalized surfaces in static and rotating mode upon protein adhesion.
    • Fig. S3. Immunofluorescence analysis of cell morphology during osteogenic differentiation of MSCs on dynamic and static molecular motor–functionalized surfaces.
    • Fig. S4. Protein adhesion and structural integrity mediated by molecular motion and UV irradiation.
    • Fig. S5. MD simulations of protein–molecular motor interactions and motion-induced structural deformation.
    • Fig. S6. AFM analysis of motion-induced alterations to structural alterations of protein adhesion layers.
    • Fig. S7. Physicochemical influences on the morphology of protein adhesion layers.
    • Table S1. Primer sequences of human-specific genes used for qRT-PCR.

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