Research ArticleDEVELOPMENTAL BIOLOGY

Synthetic presentation of noncanonical Wnt5a motif promotes mechanosensing-dependent differentiation of stem cells and regeneration

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Science Advances  16 Oct 2019:
Vol. 5, no. 10, eaaw3896
DOI: 10.1126/sciadv.aaw3896
  • Fig. 1 Summary of the experimental procedures and the characterization of the hMSC-embedded, porous RGD-Foxy5 MeHA hydrogels.

    (A) Porous, Foxy5 + RGD peptide–conjugated MeHA hydrogels were developed by conjugating cysteine-containing functional peptides to MeHA molecules. (B) The porous scaffolds were used to copresent the adhesive ligand (RGD) and noncanonical Wnt5a–activating ligand (Foxy5) to synergistically induce the osteogenic lineage commitment of stem cells both in vitro and in vivo. (C) Rat MSC (rMSC)–seeded hydrogels were used to fill calvarial defects for regeneration. (D) Micrographs of the 3D porous hydrogels with Ø 200-μm pores. The inset shows the microstructure of the MeHA porous hydrogel; scale bar, 50 μm. (E) The Young modulus of the DTT-crosslinked MeHA hydrogels was verified using the Mach-1 mechanical tester. (F) The live/dead staining of the hMSCs seeded in the porous MeHA hydrogels showed the uniform distribution of the cells in the hydrogels. (G) Viable cell metabolic activity in the RGD, Foxy5 + RGD, and Scram + RGD hydrogels on day 7 of culture was characterized by alamarBlue assay. Data are shown as the means ± SD (n = 3).

  • Fig. 2 Foxy5 peptide–conjugated hydrogel scaffolds activate noncanonical Wnt signaling to trigger RhoA signaling and elevate intracellular calcium.

    (A) Schematic illustration of the seeding of hMSCs on the Foxy5/Scram + RGD peptide–functionalized 2D hydrogel substrate. (B) Gene expression level of the RhoA signaling cascade (Wnt5a coreceptor Dvl2, RhoA, ROCK), downstream mechano-effector (NMII), and major focal adhesion adaptor protein (vinculin) in hMSCs in 3D porous hydrogels conjugated with RGD peptide alone (RGD), Foxy5 and RGD peptide (Foxy5 + RGD), or scrambled Foxy5 peptide and RGD peptide (Scram + RGD), respectively, after 7 days of osteogenic culture (n = 9). (C) Representative micrographs of fluorescence staining for F-actin (red), nuclei (blue), and RhoA (green) in hMSCs cultured on the 2D RGD, Foxy5 + RGD, and Scram + RGD hydrogels. Quantification showed a significantly higher RhoA staining intensity in the Foxy5 + RGD group than in the RGD and Scram + RGD groups (n = 20). a.u., arbitrary units. (D) Western blot bands and quantification of the expression level of mechano-responsive kinases ROCK2 and p-FAK (phosphorylated at the Ser722 sites) in each group (RGD, Foxy5 + RGD, Scram + RGD). (E) Representative merged fluorescence and bright-field micrographs of intracellular calcium in hMSCs cultured on RGD, Foxy5 + RGD, and Scram + RGD 2D hydrogels (stained with Fura-AM). (F) Quantification showed a significantly higher intracellular calcium level in the Foxy5 + RGD group than in the RGD and Scram + RGD groups. Scale bars, 50 μm. Data are shown as the means ± SD (n = 9). Statistical significance: *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 3 Noncanonical Wnt5a mimetic peptide (Foxy5) conjugated to 2D hydrogel substrates enhances the mechanosensing and osteogenesis of hMSCs.

    (A) Fluorescence micrographs of hMSCs stained for F-actin (red), nuclei (blue), and the mechanosensing marker YAP (green) or the osteogenic marker RUNX2 (green) (B) and ALP (blue in bright-field) (C), cultured on the RGD, Foxy5 + RGD, and Scram + RGD hydrogels. (D) Analysis of the nuclear localization of YAP determined by the nuclear-to-cytoplasmic fluorescence intensity ratio (N/C ratio) (n = 20) and (E) RUNX2 nuclear localization (n = 20) and (F) ALP expression of representative cells cultured on 2D hydrogels in the different experimental groups (n = 9). Scale bars represent 50 μm in the fluorescence micrographs and 200 μm in the bright-field images. Data are shown as the means ± SD. Statistical significance: *P < 0.05, **P < 0.01, and ***P < 0.001 significant difference.

  • Fig. 4 Conjugating Wnt5a mimetic peptide (Foxy5) to a 3D porous hydrogel scaffold promotes hMSC osteogenic gene expression and osteogenic matrix synthesis.

    (A) Schematic illustration of the seeding of hMSCs in the Foxy5/Scram + RGD peptide–functionalized 3D hydrogel scaffolds. (B) Quantitative gene expression of osteogenic markers (type I collagen, RUNX2, ALP, and OPN) in hMSCs seeded in porous hydrogels conjugated with RGD peptide alone (RGD), Foxy5 and RGD peptides (Foxy5 + RGD), or Scram and RGD peptides (Scram + RGD) in osteogenic culture. (C) von Kossa staining, Alizarin Red S staining, and immunohistochemistry staining of type I collagen and (D) quantification of the staining intensities after 14 days of osteogenic culture. Scale bars, 50 μm. Data are shown as the means ± SD (n = 9). Statistical significance: * P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 5 Functionalization of biomaterial scaffolds with the Wnt5a mimetic peptide substantially enhances the in situ regeneration of integrated and mature bone tissues.

    (A) Schematic illustration of the implantation of rMSC-seeded and peptide-functionalized porous hydrogels in rat calvarial defects. (B) H&E staining and immunohistochemical staining of the native healthy bone tissue and the calvarial defects treated with the RGD hydrogels, Foxy5 + RGD hydrogels, Scram + RGD hydrogels, and no hydrogels (blank) 8 weeks after implantation (n = 3). High-magnification images showing the defect/native bone boundaries highlighted in yellow and red boxes and defect center areas in blue boxes in the low-magnification images of H&E-stained sections. The dotted lines indicate the boundary between the defect and native bone. The newly formed bone was seamlessly integrated with the neighboring native bone in the Foxy5 + RGD group. Scale bars, 50 μm. (C) Top view of 3D micro-CT images showing calvarial bone defects after 8 weeks in all groups (n = 3). (D) Bone volume (normalized to total tissue volume, BV/TV) in the calvarial defects in all groups after 8 weeks (n = 3). The bone volume of healthy rat calvarial bone is shown as the benchmark. Quantification of the immunohistochemical staining intensity of the osteogenic markers, including osteocalcin and type I collagen, showing the higher intensity in the Foxy5 + RGD group compared with those of the RGD and Scram + RGD control groups. Data are shown as the means ± SD (n = 9). Statistical significance: *P < 0.05, **P < 0.01, and ***P < 0.001 significant difference.

  • Fig. 6 Schematic illustration of the Wnt5a mimetic peptide–conjugated, porous HA hydrogels promoting the osteogenesis of seeded hMSCs by activating RhoA signaling and elevating the intracellular calcium level.

    Presentation of the Wnt5a mimetic Foxy5 peptide by the hydrogel scaffold to Wnt receptors (Frizzled) and coreceptors on the membrane of seeded MSCs activates noncanonical Wnt signaling, leading to the activation of Dvl2 and downstream RhoA-ROCK2 signaling. The activation of RhoA signaling leads to enhanced cytoskeletal stability and actomyosin contractility (increased NMII) and therefore boosts mechanotransduction and the formation of focal adhesions (elevated vinculin expression). Meanwhile, the presentation of Foxy5 peptide mediates elevated intracellular calcium levels, contributing to the amplification of mechanosensing and osteogenic lineage commitment. The amplified mechanosensing of hMSCs residing in Foxy5 peptide–functionalized hydrogels is demonstrated by the increased YAP nuclear localization and up-regulated RhoA and ROCK2 expression, and these events promote the commitment of MSCs to the osteogenic lineage in the synthetic osteogenic niche microenvironment.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/10/eaaw3896/DC1

    Fig. S1. Characterization of the methacrylated hyaluronic acid (MeHA) macromers.

    Fig. S2. The mechanical characterizations of the MeHA hydrogels conjugated with various groups of peptide.

    Fig. S3. Foxy5 peptide-conjugated hydrogels upregulate the expression of mechanotransduction signaling molecules in the seeded hMSCs.

    Fig. S4. The promoting effect of conjugated Foxy5 peptide on the osteogenesis of hMSCs is dependent on ROCK and non-muscle myosin II activities.

    Fig. S5. Conjugated Foxy5 peptides enhance the expression of canonical Wnt-related genes of hMSCs seeded in the 3D hydrogel.

    Fig. S6. The hydrogel-conjugated Foxy5 peptide promotes the osteogenesis in a wide range of hydrogel substrate stiffness.

    Fig. S7. The conjugated Foxy5 peptide promotes the osteogenesis of the hMSCs from multiple donors.

    Fig. S8. The conjugated Foxy5 peptide promotes the mechanotransduction and osteogenesis of the seeded rMSCs.

    Table S1. The sequence of the primers and probes used for real-time PCR are listed.

    Table S2. The donor information of the hMSCs used for the in vitro experiments is listed.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Characterization of the methacrylated hyaluronic acid (MeHA) macromers.
    • Fig. S2. The mechanical characterizations of the MeHA hydrogels conjugated with various groups of peptide.
    • Fig. S3. Foxy5 peptide-conjugated hydrogels upregulate the expression of mechanotransduction signaling molecules in the seeded hMSCs.
    • Fig. S4. The promoting effect of conjugated Foxy5 peptide on the osteogenesis of hMSCs is dependent on ROCK and non-muscle myosin II activities.
    • Fig. S5. Conjugated Foxy5 peptides enhance the expression of canonical Wnt-related genes of hMSCs seeded in the 3D hydrogel.
    • Fig. S6. The hydrogel-conjugated Foxy5 peptide promotes the osteogenesis in a wide range of hydrogel substrate stiffness.
    • Fig. S7. The conjugated Foxy5 peptide promotes the osteogenesis of the hMSCs from multiple donors.
    • Fig. S8. The conjugated Foxy5 peptide promotes the mechanotransduction and osteogenesis of the seeded rMSCs.
    • Table S1. The sequence of the primers and probes used for real-time PCR are listed.
    • Table S2. The donor information of the hMSCs used for the in vitro experiments is listed.

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