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SCIENCE CHINA Life Sciences, https://doi.org/10.1007/s11427-021-1933-7

Engineering osteoarthritic cartilage model through differentiating senescent human mesenchymal stem cells for testing disease-modifying drugs

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  • ReceivedMar 2, 2021
  • PublishedJun 4, 2021

Abstract


Funded by

Department of Orthopaedic Surgery at the University of Pittsburgh and the Albert B. Ferguson

Jr.

M.D. Orthopaedic Fund of The Pittsburgh Foundation.


Acknowledgment

This work was supported by Department of Orthopaedic Surgery at the University of Pittsburgh and the Albert B. Ferguson, Jr., M.D. Orthopaedic Fund of The Pittsburgh Foundation. Ning Wang is a medical student at the University of Pittsburgh School of Medicine supported by the Central South University Xiangya School of Medicine.


Interest statement

The author(s) declare that they have no conflict of interest. Life Length SL did not provide any financial support and did not influence the results reported in this study. With the approval from the Institutional Review Boards (University of Pittsburgh and University of Washington), the cartilage tissues and bone marrows were collected from the patients who underwent total knee joint replacement, which were used to isolate chondrocytes and mesenchymal stem cells, respectively.


Supplement

SUPPORTING INFORMATION

The supporting information is available online at https://doi.org/10.1007/s11427-021-1933-7. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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  • Figure 1

    Assessment of cartilage harvested from the P-C and D-C areas in OA knee joint. A, The cartilage explants used to collect P-C and D-C were harvested from the same human donor. The black arrows indicated the locations of the extraction sites of the P-C and D-C tissues. B–E, (B) Safranin O/Fast green staining, (C) MMP13, (D) p16INK4a immunohistochemistry (IHC), and (E) SA-β-gal staining were used to assess cartilage quality. Red arrows indicated positive staining. Bar=50 µm. F–H, The relative expression levels of (F) MMP13, (G) p16INK4a, and (H) SA-β-gal were assessed with ImageJ based on the images shown in C–E (N=3). All data was normalized to that in P-C group (set as 1). **, P<0.01; ***, P<0.001.

  • Figure 2

    Characterization of chondrocytes (CHs), isolated from P-C (P-CHs) and D-C (D-CHs), and MSCs at passage 4 (P4-MSCs) and 10 (P10-MSCs). A, SA-β-gal staining and p16INK4a immunofluorescence (IF) were used to examine cellular senescence. Bar=20 µm in SA-β-gal staining and 50 µm in p16INK4 IF. B, The relative expression levels of p16INK4a were assessed using ImageJ based on the images in A. Data was normalized to that from P-CHs group (set as 1). C, Ratios of senescent cells in different cell populations. D, Immunofluorescent images to detect the cellular nuclei (blue) and telomeres (green). Red arrows indicated the positive staining of the telomere. Bar=20 µm. E, MTS assay was used to assess cell proliferation potential. F, Median telomere length (left) and cell percentile with telomere length <3 kb (right); (N=5). G, In this study, we hypothesized that cartilage generated from the senescent MSCs displayed a similar phenotype to damaged cartilage in OA. *, P<0.05; **, P<0.01; ****, P<0.0001.

  • Figure 3

    Examination of CHs and MSCs chondrogenic potential. Expression levels of (A and C) chondrogenesis, (E and H) degradation, (F and I) fibrosis, and (G and J) inflammation-relevant genes in cartilage tissues created by CHs (A, E–G) and MSCs (C, H–J). The data was normalized to that in P-CHs or P4-MSCs group (set as 1). B and D, The GAG/DNA in cartilage generated by CHs (B) and MSCs (D); N=3. K, Safranin O/Fast green staining and COL2 IF were conducted to assess cartilage matrix content. Bar=50 µm.*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

  • Figure 4

    Assessment of senescence and OA characteristics in cartilage tissues derived from CHs and MSCs. A, The expression levels of p16INK4a, p21, and p53 in D-CHs and P10-MSCs derived cartilage were examined with qRT-PCR, which were normalized to respective control from P-CHs or P4-MSCs. N=3. B, Western blot for SOX9, MMP13, p21, p53, and p16INK4a. C, Based on the result shown in B, relative protein levels between D- and P-CHs groups, and between P4- and P10-MSCs groups were shown through a heatmap. D–F, (D) SA-β-gal staining, (E) p16INK4a, and (F) MMP13 IHC were used to assess the senescence level. Red arrows indicated positive staining. Bar=50 µm. G–I, The relative protein levels of (G) SA-β-gal, (H) p16INK4a, and (I) MMP13 were quantitated with ImageJ based on the images shown in D–F; (N=3). Data was normalized to that from P-CHs group (set as 1). **, P<0.01; ***, P<0.001; ****, P<0.0001.

  • Figure 5

    RNA sequencing analysis for CHs and MSCs-derived cartilage tissues. A, PCA for four subgroups. B, Volcano plot of differential expressed genes in human OA cartilage (E-MTAB-4304, (Dunn et al., 2016)), MSCs, and CHs derived cartilage. Top common DEGs were labeled throughout all three comparisons. C, Protein-protein interaction (PPI) network for common DEGs shooting to OA pathway. Each node represented different DEGs, and the colors of nodes represent the relative gene expression level. Colors of connecting lines imply the correlation strength between different DEGs. D, Bar graph showed −log10(P-value) of the 28 common pathways that were identified in all three comparisons. Pathways that are related to OA pathogenesis were marked with red frames. E, Bubble graph for OA- and senescence-related pathways that were identified in both comparisons between P-CHs and D-CHs groups, and between P4-MSCs and P10-MSCs groups. F, Heatmap showed senescence-related DEGs and their corresponding pathways.

  • Figure 6

    Assessment of the therapeutic potential of DMOADs and senolytics on cartilage derived from CHs and MSCs. A, Potential utility of P4- and P10-MSCs-derived cartilage in screening DMOADs, which can predict the influence of drugs on both preserved and damaged cartilage in OA knee joint. B, The agents were tested in this study. Their known functions were also listed. Engineered cartilage derived from CHs and MSCs were treated with different compounds for 7 d. C, Safranin O/Fast green staining was used to assess the GAG content after different treatments; Bar=100 µm. D, Expression levels of 17 selective genes were measured by qRT-PCR. A heatmap was used to qualitatively display the difference among groups.

  • Figure 7

    Influence of potential DMOADs and senolytics on cellular senescence in engineered cartilage tissues. A, IHC assay for p16INK4a; Bar=100 and 20 µm in top and bottom panels. B, Levels of p16INK4a protein were quantitated with ImageJ based on the images shown in A; (N=3). Data was normalized to that from the respective P10-MSCs or D-CHs control group (set as 1). C–G, Expression levels of p16INK4a and SASP-relevant factors in engineered cartilage tissues after different treatments. Data was normalized to that from the respective P10-MSCs or D-CHs control group (set as 1). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

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