Journal: Advanced Science

Article Title: Discovery and Application of Postnatal Nucleus Pulposus Progenitors Essential for Intervertebral Disc Homeostasis and Degeneration

doi: 10.1002/advs.202104888

Figure Lengend Snippet: RegNPs were a metabolically active and mechanically sensitive population, while HomNPs were sensitive to hypoxia with “degenerative” potential. A) t‐SNE plots and representative violin plots showing the expression of Unc5c, Runx3, Bmp7, Wnt4, Tgfb2, CNTFR, Matn3, Grb10, Fgfr3, Epyc, Ptch1, and Pth1r on the t‐SNE map. B) Representation analysis of GO categories showing different functions for RegNPs. C) Heatmap revealing metabolic‐related functions and pathways for RegNPs. D) Representative images of lumbar spine sections from 4‐week‐old wild‐type mice stained for BMP7. Scale bars, 100 µm ( n = 3 mice per group). E) t‐SNE plots and representative violin plots showing the expression of Gdf5, Agt, Eln, Grem1, Mmp3, and Plau on the t‐SNE map. F) Representative analysis of GO categories showing different functions for HomNPs. G) Representative images of lumbar spine sections from 4‐week‐old WT mice stained for GDF5. The lower image shows a high‐magnification view of the indicated area from the upper image. Scale bars, 100 µm ( n = 3 mice per group).

Article Snippet: Dissociated single cells were then stained with AF488 anti‐mouse UTS2R (R&D Systems, FAB9245G‐100UG), APC anti‐mouse GDF5 (ASSAYPRO, 32579‐05161), anti‐mouse CNTFRa (Santa Cruz Biotechnology, SC9993), BB700 anti‐mouse CD146 (BD Biosciences, 742 280), APC anti‐mouse CD44 (BioLegend, 559 250), BV421 anti‐mouse CD73 (BioLegend, 127 217), BV605 anti‐mouse CD90 (BioLegend, 140 317), APC anti‐mouse PDGFa/CD140a (BioLegend, 135 907), FITC anti‐mouse Sca‐1 (BioLegend, 108 106), PerCP/Cy5.5 anti‐CD31 (BioLegend, 102 420), PerCP/Cy5.5 anti‐CD45 (BioLegend, 103 132), PerCP/Cy5.5 anti‐mouse TER‐119 (BioLegend, 116 228), FITC anti‐mouse 6C3/Ly‐51 (BioLegend, 108 305), Brilliant Violet 605 anti‐mouse CD90.2 (BioLegend, 140 317), PE/Cy7 anti‐mouse CD105 (BioLegend, 120 409), APC anti‐mouse CD200 (BioLegend, 123 809), and anti‐mouse Alexa Fluor 488 (Molecular Probes, A21202, 1:1000).

Techniques: Metabolic Labelling, Expressing, Staining

Journal: Advanced Science

Article Title: Discovery and Application of Postnatal Nucleus Pulposus Progenitors Essential for Intervertebral Disc Homeostasis and Degeneration

doi: 10.1002/advs.202104888

Figure Lengend Snippet: Lineage tracing of UTS2R + NP cells. A) Dot plot showing the expression of Uts2r on the t‐SNE map. B) Representative immunofluorescence imaging of UTS2R (green) in postnatal 1‐month‐old WT mice. The right image shows high magnification of the indicated area from the left image ( n = 3 mice per group). Scale bars, 100 µm. C) Construction strategy of Uts2r‐CreER transgenic mice using the CRISPR/Cas9 System. D) Diagram showing postnatal day 1 (P1) Uts2r‐CreER;Ai9 /+ mice administered with one dosage tamoxifen and sacrificed at postnatal day 3 (P3), 1 month (P1M), or 2 months (P2M). E) Representative immunofluorescence imaging of Uts2r‐CreER;Ai9 + cells (red). The right images show high magnification of the indicated area from the left image ( n = 3 mice per group). Scale bars, 100 µm. F,G) Representative immunofluorescence imaging of Uts2r‐CreER;Ai9 + cells (red), BMP7 (green) (F) or GDF5 (green) (G). The right images show high magnification of the indicated area from the left image ( n = 3 mice per group). Scale bars, 100 µm.

Article Snippet: Dissociated single cells were then stained with AF488 anti‐mouse UTS2R (R&D Systems, FAB9245G‐100UG), APC anti‐mouse GDF5 (ASSAYPRO, 32579‐05161), anti‐mouse CNTFRa (Santa Cruz Biotechnology, SC9993), BB700 anti‐mouse CD146 (BD Biosciences, 742 280), APC anti‐mouse CD44 (BioLegend, 559 250), BV421 anti‐mouse CD73 (BioLegend, 127 217), BV605 anti‐mouse CD90 (BioLegend, 140 317), APC anti‐mouse PDGFa/CD140a (BioLegend, 135 907), FITC anti‐mouse Sca‐1 (BioLegend, 108 106), PerCP/Cy5.5 anti‐CD31 (BioLegend, 102 420), PerCP/Cy5.5 anti‐CD45 (BioLegend, 103 132), PerCP/Cy5.5 anti‐mouse TER‐119 (BioLegend, 116 228), FITC anti‐mouse 6C3/Ly‐51 (BioLegend, 108 305), Brilliant Violet 605 anti‐mouse CD90.2 (BioLegend, 140 317), PE/Cy7 anti‐mouse CD105 (BioLegend, 120 409), APC anti‐mouse CD200 (BioLegend, 123 809), and anti‐mouse Alexa Fluor 488 (Molecular Probes, A21202, 1:1000).

Techniques: Expressing, Immunofluorescence, Imaging, Transgenic Assay, CRISPR

Journal: PLoS ONE

Article Title: Human Chondrocytes Respond Discordantly to the Protein Encoded by the Osteoarthritis Susceptibility Gene GDF5

doi: 10.1371/journal.pone.0086590

Figure Lengend Snippet: The amino acid substitutions of GDF5 variants A and B.

Article Snippet: The cells were stimulated with four different recombinant GDF5 proteins (mouse GDF5, R&D Systems; human wild type GDF5 and two variants A and B, Biopharm GmbH) at 10 ng/ml, 30 ng/ml, 100 ng/ml and 300 ng/ml.

Techniques: Variant Assay

Journal: PLoS ONE

Article Title: Human Chondrocytes Respond Discordantly to the Protein Encoded by the Osteoarthritis Susceptibility Gene GDF5

doi: 10.1371/journal.pone.0086590

Figure Lengend Snippet: Y-axis represents the luciferase activity readings generated in SW1353 cells in response to exogenous growth factor. Cells were stimulated for 6, 12, 24 and 48 hours with wildtype mouse GDF5 (A), wildtype human GDF5 (B), human GDF5 variant A (C) and human GDF5 variant B (D). BMP2 and TGF-β1 stimulations were used as positive controls. Error bars represent the standard error of the mean. GDF5 10, 10 ng/ml; GDF 30, 30 ng/ml; GDF5 100, 100 ng/ml; GDF5 300, 300 ng/ml. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, *****P<0.00001, two-tailed Student’s t-test.

Article Snippet: The cells were stimulated with four different recombinant GDF5 proteins (mouse GDF5, R&D Systems; human wild type GDF5 and two variants A and B, Biopharm GmbH) at 10 ng/ml, 30 ng/ml, 100 ng/ml and 300 ng/ml.

Techniques: Luciferase, Activity Assay, Generated, Variant Assay, Two Tailed Test

Journal: PLoS ONE

Article Title: Human Chondrocytes Respond Discordantly to the Protein Encoded by the Osteoarthritis Susceptibility Gene GDF5

doi: 10.1371/journal.pone.0086590

Figure Lengend Snippet: Chondrocytes were cultured in monolayer and stimulated with each of the four GDF5 proteins for 15 minutes, 30 minutes, 1-β1 stimulation for 1 hour was used as a positive control. Protein was extracted and subjected to western blot analysis using an antibody against Smad 1/5/8 (anti-Smad1/5/8), with anti-β-actin antibody used as a loading control. This data comes from one OA patient. Identical data was obtained for a second OA patient (data not shown).

Article Snippet: The cells were stimulated with four different recombinant GDF5 proteins (mouse GDF5, R&D Systems; human wild type GDF5 and two variants A and B, Biopharm GmbH) at 10 ng/ml, 30 ng/ml, 100 ng/ml and 300 ng/ml.

Techniques: Cell Culture, Positive Control, Western Blot, Control

Journal: PLoS ONE

Article Title: Human Chondrocytes Respond Discordantly to the Protein Encoded by the Osteoarthritis Susceptibility Gene GDF5

doi: 10.1371/journal.pone.0086590

Figure Lengend Snippet: Chondrocytes were stimulated for 6, 12, 24 and 48 hours with wildtype mouse GDF5 (A), wildtype human GDF5 (B), human GDF5 variant A (C), human GDF5 variant B (D) and TGF-β1 (E). Each cross represents a significant (P<0.05, two-tailed Student’s t-test) up/down regulation of gene expression relative to untreated cells in one patient. Twelve patients were studied for each of the GDF5 growth factor treatments and ten patients for the TGF-β1 treatments.

Article Snippet: The cells were stimulated with four different recombinant GDF5 proteins (mouse GDF5, R&D Systems; human wild type GDF5 and two variants A and B, Biopharm GmbH) at 10 ng/ml, 30 ng/ml, 100 ng/ml and 300 ng/ml.

Techniques: Variant Assay, Two Tailed Test, Gene Expression

Journal: PLoS ONE

Article Title: Human Chondrocytes Respond Discordantly to the Protein Encoded by the Osteoarthritis Susceptibility Gene GDF5

doi: 10.1371/journal.pone.0086590

Figure Lengend Snippet: Chondrocytes were stimulated for 5 days with wildtype mouse GDF5 (A), wildtype human GDF5 (B), human GDF5 variant A (C), human GDF5 variant B (D) and TGF-β1 (E). Each cross represents a significant (P<0.05, two-tailed Student’s t-test) up/down regulation of gene expression relative to untreated cells in one patient. Ten patients were studied for each of the growth factor treatments.

Article Snippet: The cells were stimulated with four different recombinant GDF5 proteins (mouse GDF5, R&D Systems; human wild type GDF5 and two variants A and B, Biopharm GmbH) at 10 ng/ml, 30 ng/ml, 100 ng/ml and 300 ng/ml.

Techniques: Variant Assay, Two Tailed Test, Gene Expression