Research ArticleStem Cells

Thy-1 (CD90) promotes bone formation and protects against obesity

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Science Translational Medicine  08 Aug 2018:
Vol. 10, Issue 453, eaao6806
DOI: 10.1126/scitranslmed.aao6806
  • Fig. 1 Thy-1 supports osteogenic differentiation of MSC while concurrently inhibiting adipogenic differentiation.

    MSCs were isolated from WT and Thy-1–deficient mice (KO). (A) Calcification was detected 14 days after initiating osteogenic differentiation. Alizarin red staining (red, mineralized bone matrix) and ALP activity (purple) by Fast blue RR solution (representative images; scale bars, 25 μm). (B) Gene expression of osteogenesis-related genes detected after 3 (Runx2) or 14 days (Oc, Tnalp1, and Osx) in culture by reverse transcription polymerase chain reaction (RT-PCR). mRNA values were normalized to the reference gene Rpl26, and arbitrary units (AUs) are indicated. (C) Oil red staining of lipid accumulation visualized 14 days after initiation of adipogenic differentiation (representative images; scale bars,100 μm). (D) Solubilization of Oil red absorption measured by enzyme-linked immunosorbent assay (ELISA). (E) Adipogenesis-related genes detected after 3 (Pparγ and Cebpα) or 14 days (AdipoQ and Fabp4) by RT-PCR. The mRNA values were normalized to the reference gene Rpl26, and AUs are indicated. (F and G) WT and KO MSC were differentiated into (F) osteoblasts (representative images; scale bars, 25 μm) and (G) adipocytes in the presence of function-blocking antibody against integrin β3 (aCD61) or integrin β1 (aCD29). Osteoblast differentiation was detected after 14 days by Alizarin red staining and adipocyte differentiation by Oil red staining and subsequent solubilization. (H and I) WT and KO MSC seeded on immobilized rThy-1 or control protein (Fc) and differentiated into osteoblasts or adipocytes. Osteoblast differentiation was detected after 14 days by Alizarin red staining (H) and adipocyte differentiation by Oil red staining (I) and subsequent solubilization. Data represent means ± SD of MSCs from at least four different mice per genotype. *P < 0.05, **P < 0.01, ***P < 0.001 [Student’s t test (B and E) and one-way analysis of variance (ANOVA) (G to I)].

  • Fig. 2 Thy-1 decreases body fat mass.

    (A) Body weights of male and female WT and Thy-1–deficient (KO) mice and ratio of subcutaneous (sc) AT and epigonadal fat to body weight of WT and KO mice. Fat volume per bone volume (FV/BV) in male mice was detected by osmium tetroxide staining (representative images, right). (B) Body weight, percentage of fat mass, and ratio of epigonadal and subcutaneous fat weight to body weight for male WT and KO mice fed an HFD for 12 weeks. (C) Gene expression of AT-related genes in epigonodal AT in (B) analyzed by RT-PCR. mRNA values were normalized to the reference gene Rpl26, and AUs are indicated. (D) Skin of WT and KO mice in (B) stained by hematoxylin and eosin (representative images; scale bars, 100 μm). Thickness of white dermal AT (WdAT) and size of adipocytes were measured using Keyence Analyzer software. Each point represents one mouse. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test).

  • Fig. 3 Thy-1 increases bone mass and stability.

    (A to G) Bone volume per total volume (BV/TV) of the trabecular (A) and cortical (B) bone compartments, trabecular (Tb.Th; C) and cortical (Ct.Th; D) thickness, cortical BMD and TMD (E and F), and cortical porosity (Ct.Po; G) detected by microcomputed tomography (μCT) in femora from WT and Thy-1–deficient mice (KO) under standard control diet. (H) Fourier transform infrared (FTIR) spectroscopy evaluation of crystallinity of the tibia cortical bone compartment. (I and J) Representative images of trabecular (I) and cortical (J) bone areas by μCT analysis. (K) Maximum force (Fmax) and elasticity modulus (Emod) analyzed using three-point bending test. (L) First cycle indentation distance (CID 1st) and total indentation distance (TID) evaluated by reference-point analysis. Each point represents one mouse. *P < 0.05 and **P < 0.01 (Student’s t test).

  • Fig. 4 Thy-1 positively influences bone formation.

    Histological and histomorphometrical analysis of tibiae from male WT and Thy-1–deficient mice (KO). (A) Bone volume per total volume (BV/TV) and (B) osteoid surface per bone perimeter (Osteoid.Srf/B.Pm) by (C) von Kossa staining and van Giesson counterstaining (black, mineralized bone matrix; red, unmineralized osteoid/collagen; scale bars, 50 μm). (D and E) Bone formation rate per bone surface (BFR/BS; D) and mineral surface per bone surface (MS/BS; E) analyzed by double calcein labeling [green, calcein labels (F); scale bars, 50 μm]. (G to I) Number of osteoclasts per bone perimeter (N.Oc/B.Pm; G) and osteoclast surface per bone surface (OC.S/BS; H) detected via staining for tartrate-resistant acid phosphatase [TRAP; red, osteoclasts (I); scale bars, 50 μm]. (J and K) Serum concentrations of P1NP and CTX detected in WT and KO mice by ELISA. (L to N) WT and KO MSCs encapsulated in alginate beads and subcutaneously transplanted into WT mice. (L) Calcification (bone/total volume, BV/TV) detected after 3 weeks by μCT analysis. (M) Representative images of WT and KO MSCs in alginate beads 3 weeks after implantation. (N) Gene expression of Tnalp1 and Oc detected by RT-PCR. The mRNA values were normalized to the reference gene Rpl26, and AUs are indicated. Each point represents one mouse. *P < 0.05, **P < 0.01, and ***P < 0.0001 for effect of Thy-1 deficiency (Student’s t test).

  • Fig. 5 Thy-1 deficiency is associated with altered Wnt pathway.

    (A and B) Gene expression of Wnt ligands (A) and Wnt inhibitors Sost and Dkk-1 (B) in bone detected by RT-PCR. The mRNA values were normalized to the reference gene Gapdh and x-fold expression in Thy-1–deficient mice (KO) compared to WT mice. nd, not detectable. (C and D) Serum concentrations of Wnt inhibitors Dkk-1 (C) and sclerostin (D) were detected by ELISA. Each point represents one mouse. (E) MSCs were isolated from WT and Thy-1–deficient (KO) mice, and gene expression of Wnt ligands was detected by RT-PCR. The mRNA values were normalized to the reference gene Rpl26. X-fold expression in KO mice compared to WT mice is indicated (MSCs of ≥11 different mice per genotype). (F) KO-MSCs cultured for 24 hours on immobilized rThy-1 (KO-rThy-1) or control protein (Fc). Thy-1/β3 integrin interaction was blocked by a function-blocking antibody against integrin β3 (aCD61), and expression of Wnt5a and Wnt16 was detected by RT-PCR. The mRNA values were normalized to the reference gene Rpl26, and x-fold expression is indicated (MSCs of six different mice per genotype). (G) WT and KO MSCs were stimulated with Wnt3a (50 ng/ml) or vehicle for 24 hours. Gene expression of Cyclin D1 and Lef1 was detected by RT-PCR. The mRNA values were normalized to the reference gene Rpl26, and AUs are indicated (MSCs of three different mice per genotype). *P < 0.05, **P < 0.01, ***P < 0.001 [Student’s t test (A to E) and one-way ANOVA (F and G)].

  • Fig. 6 Disturbed bone formation and AT accumulation in obesity are associated with decreased Thy-1 expression.

    (A to C) Body weight (A), percentage of fat mass (B), and fat volume per bone volume (FV/BV; C) in male WT mice fed control chow or HFD for 12 weeks. FV/BV detected by osmium tetroxide staining (C; representative images, right). (D) Bone volume per total volume (BV/TV) analyzed by μCT (representative images, right). (E) Bone formation rate per bone surface (BFR/BS) analyzed by double calcein labeling. (F) Gene expression of Osx, Oc, and Runx2 in bone of control and HFD-fed mice. (G) The number of CFU per 100,000 cells detected from digested bone from chow and HFD mice. (H) Number of CD45/Sca-1+/CD73+ cells per 1 × 106 living cells in bone marrow of WT mice under chow and HFD analyzed by flow cytometry. (I and J) Expression of Pdgfrα (I) and Thy-1 (J) in bone detected by RT-PCR. The mRNA values were normalized to the reference gene Gapdh and AUs are indicated. (K) Expression of Thy-1 on CD45/Sca-1+ cells in bone detected by flow cytometry. Dead cells were excluded by Zombie staining. Mean fluorescence intensity of Thy-1 was detected on CD45/Sca-1+ cells of chow- and HFD-fed mice. Each point represents one mouse. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test).

  • Fig. 7 Regulation of Thy-1 in vitro and in vivo.

    (A to F) Regulation of Thy-1 in MSCs in vitro. (A) Thy-1 expression detected by RT-PCR (means ± SD of MSCs from ≥3 different mice) and x-fold expression of murine WT MSCs stimulated with indicated cytokines compared to untreated cells. (B) Representative Western blot of Thy-1 protein expression (n = 4). GAPDH, glyceraldehyde phosphate dehydrogenase. (C and D) Thy-1 gene and protein expression detected by RT-PCR (C) and Western blot (D) from human MSCs stimulated with TNFα or IL-1β. Means ± SD of MSCs from three different donors performed in duplicate. X-fold expression compared to untreated cells is indicated. (E) Gene expression of Tnfα and Il-1β in bone of chow and HFD-fed mice detected by RT-PCR. The mRNA values were normalized to the reference gene Gapdh, and AUs are indicated. Each point represents one mouse. ***P < 0.001 (Student’s t test). (F) Correlation analysis of Tnfα and Thy-1 expression in bone of chow- and HFD-fed mice (Spearman correlation analysis). (G to K) Detection of human sThy-1 by ELISA. (G) Serum sThy-1 in female and male subjects with BMI of <26 and age of <50 or >60. (H) Serum sThy-1 in individuals with normal weight (BMI, <25 kg/m2), overweight (BMI, 25 to 30 kg/m2), and obese subjects (BMI, >30 kg/m2). (I) Serum sThy-1 in healthy (T-score between −1 and +1) and osteoporotic (T-score, ≤2.5) nonobese (BMI, <30) women. (J) Spearman correlation analysis of P1NP concentration and sThy-1 in serum of healthy and osteoporotic nonobese (BMI, <30) women. (K) Serum sThy-1 in patients with polymyalgia rheumatica. Individuals were divided into patient subgroups without (T-score, ≥1) and with osteoporosis (T-score, ≤2.5). Each point represents one patient. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/453/eaao6806/DC1

    Fig. S1. Characterization of MSCs in Thy-1–deficient and WT mice.

    Fig. S2. Thy-1 does not affect adhesion of WT and Thy-1–deficient MSCs.

    Fig. S3. Food intake and activity do not differ between WT and Thy-1–deficient mice.

    Fig. S4. Blood glucose metabolism, food intake, and activity of WT and Thy-1–deficient mice under HFD.

    Fig. S5. Thy-1 increases bone mass in female mice.

    Fig. S6. Thy-1 deficiency affects femur size but does not alter femur geometry.

    Fig. S7. Osteoclasts in WT and Thy-1–deficient mice.

    Fig. S8. Thy-1–deficient MSCs exhibited higher cell growth and proliferation in parallel to a decreased apoptosis rate.

    Fig. S9. Thy-1 supports osteogenesis and bone formation while inhibiting adipogenesis.

    Table S1. Murine primer sequences for RT-PCR.

    Table S2. Antibodies.

    Table S3. Characterization of healthy and osteoporosis subjects (cohort 2).

    Table S4. Characterization of patients with polymyalgia rheumatica (cohort 3).

    Table S5. Individual subject-level data (Excel file).

  • The PDF file includes:

    • Fig. S1. Characterization of MSCs in Thy-1–deficient and WT mice.
    • Fig. S2. Thy-1 does not affect adhesion of WT and Thy-1–deficient MSCs.
    • Fig. S3. Food intake and activity do not differ between WT and Thy-1–deficient mice.
    • Fig. S4. Blood glucose metabolism, food intake, and activity of WT and Thy-1–deficient mice under HFD.
    • Fig. S5. Thy-1 increases bone mass in female mice.
    • Fig. S6. Thy-1 deficiency affects femur size but does not alter femur geometry.
    • Fig. S7. Osteoclasts in WT and Thy-1–deficient mice.
    • Fig. S8. Thy-1–deficient MSCs exhibited higher cell growth and proliferation in parallel to a decreased apoptosis rate.
    • Fig. S9. Thy-1 supports osteogenesis and bone formation while inhibiting adipogenesis.
    • Table S1. Murine primer sequences for RT-PCR.
    • Table S2. Antibodies.
    • Table S3. Characterization of healthy and osteoporosis subjects (cohort 2).
    • Table S4. Characterization of patients with polymyalgia rheumatica (cohort 3).

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S5. Individual subject-level data (Excel file).

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