Identification of a sub-population of synovial mesenchymal stem cells with enhanced treatment efficacy in a rat model of osteoarthritis
- McCaig Institute for Bone and Joint Health, University of Calgary, Canada
- University of Calgary, Biomedical Engineering Graduate Program, Canada
- Department of Physiology and Pharmacology, University of Calgary, Canada
- Department of Surgery, University of Calgary, Canada
- Department of Anatomy and Cell Biology, University of Calgary, Canada
Figures
Figure 1 with 3 supplements
In vitro differentiation potential of clones derived from normal individuals and patients with osteoarthritis (OA).
Representative histological staining of Alizarin Red, Safranin O, and Oil Red O from four normal (A) and four OA clones (E). Quantitative PCR (QPCR) data (n=3 biological replicates and n=3 technical replicates, each point is the mean of the technical replicates) is presented for each of the clones for osteogenic (B, F), chondrogenic (C, G), and adipogenic (D, H) markers. Signifiance determined by t-Test. *p<0.05, **p<0.01. Scale bars equal 200 µm.
Figure 1—figure supplement 1
Association between population doublings, sex, and age.
A significant negative association was observed between age and population doublings in clones derived from normal and osteoarthritis (OA) synovium. There was no association between sex and population doubling determined by Spearman Correlation, p<0.05.
Figure 1—figure supplement 2
In situ and in vitro cell surface protein expression on clones derived from normal individuals and patients with osteoarthritis (OA).
Representative histograms from 4 clones from normal (A) and OA (B) synovium are shown. The in situ expression of each CD marker is represented by the vertical dashed black bar. The in vitro expression of each marker in the clonal-derived cell population is represented by the red histogram. The isotype/negative control for each CD marker is represented by the blue histogram.
Figure 1—figure supplement 3
Overview of the experimental design employed in the current study.
Figure 2 with 1 supplement
Osteoarthritis (OA) scoring with and without cell injection in a rat DMM model.
Representative images of rats with sham surgery (A, A’), DMM injury with injection of saline (B, B’), DMM injury with injection of mesenchymal stem cells (MSCs) (C, C’) or non-MSCs (D, D’). The Osteoarthritis Research Society International (OARSI) and Krenn scoring was quantified in each group (E). Scale bars equal 200 µm in A, B, C, D and 20 µm in A’, B’, C’, D’. *p<0.05. Sample size as follows: control (sham) n=6, DMM (saline injected) n=6, DMM (MSC injected) n=3 rats per cell line, eight cell lines used (normal MSC x2 and non-MSC x2, OA MSC x2 and non-MSC x2). Normal and OA MSC groups and non-MSC groups added together. Signifiance determined by 2 way ANOVA. p<0.05.
Figure 2—figure supplement 1
Safranin O staining sections of rat synovium with or without cell delivery.
Representative images of synovium tissue from all four treatment groups (A–D). The summed Krenn score is shown along with the individual categories, including synovial lining, cellular density, and infiltration (E). Scale bars equal 40 µm. Signifiance determined by 1 way ANOVA. p<0.05.
Figure 3 with 2 supplements
Exogenous cell contribution to cartilage repair in a rat DMM model.
Representative images of rats with sham surgery (A) without cell injection (no TdTomato – B) and endogenous PRG4/Lubricin staining (C). In rats injected with saline (D), no TdTomato (E) and disrupted PRG4/Lubricin (F) staining was observed. When normal (G) or osteoarthritis (OA) (M), mesenchymal stem cells (MSCs) were injected into injured rats, TdTomato expression (H.N) was observed. When normal (J) or OA (P) non-MSCs were injected, no TdTomato (K, Q) and disrupted PRG4/Lubricin (L, R) staining was observed. Scale bars equal 30 µm. Tissue cytometry was employed to quantify the number of tdTomato-positive, PRG4-positive, and double-positive cells within the articular cartilage (S). Signifiance determined by 2 way ANOVA. *p<0.05, **p<0.01. *=p<0.05, N.S.=not significant . Sample size as follows: control (sham) n=6, DMM (saline injected) n=6, DMM (MSC injected) n=3 rats per cell line, eight cell lines used (normal MSC x2 and non-MSC x2, OA MSC x2 and non-MSC x2).
Figure 3—figure supplement 1
Representative tissue cytometry gates.
Examples of gating strategy for each marker (DAPI, tdTomato, PRG4, tdTomato plus PRG4) in each experimental group.
Figure 3—figure supplement 2
Mesenchymal stem cells (MSCs) within the cartilage produce collagen 2.
Representative images of a rat with DMM surgery (A) with MSC injection (TdTomato – B) and collagen 2 staining (C). Scale bars equal 20 µm.
Figure 4
Contribution of transplanted cells to inflammatory signaling.
Representative images of mesenchymal stem cells (MSCs) (A) and non-MSCs (B) in the synovium of rats post-DMM. In rats injected with MSCs, only minimal CCL2 (A”) expression is observed and/or colocalized with tdTomato staining (A’). In rats injected with non-MSCs, intense CCL2 staining (B”) was observed that colocalized with tdTomato staining (B’). This was quantified using tissue cytometry (C). In rats injected with MSCs, little Ki67 staining was observed in the cartilage (D-D”). In rats injected with non-MSCs, nearly every transplanted cell in the synovium was also positive for Ki67 (E-E”). This was quantified using tissue cytometry (F). Signifiance determined by t-Test. p<0.05. Scale bars equal 75 µm (A-B), 30 µm (D-E).
Figure 5
Proteomics analysis of mesenchymal stem cell (MSC) vs. non-MSC populations.
Schematic representation of the quantitative shotgun proteomics workflow (A). Volcano plot demonstrating the distribution of statistically changing proteins identified in MSCs (red) and Non-MSCs (blue) (C). Metascape analysis and heatmap showing pathways regulated by differentially expressed proteins in MSCs vs. non-MSCs (D). STRING-db analysis of protein-protein interaction networks with elevated proteins in MSCs (D).
Figure 6 with 1 supplement
Analysis of synovial cells expressing CD47.
The gating strategy to identify and sort CD47Hi vs. CD47Lo cell populations (A–C). CD47Hi vs. CD47Lo cells were identified and isolated from normal and osteoarthritis (OA) synovial tissue (B). Chondrogenic differentiation of CD47Hi vs. CD47Lo cells using pellet culture and stained with Alcian Blue (D). GAG quantification of the pellets (E). The pellets were also stained with Collagen 2 (Col2) as a marker of mature cartilage ECM (E, G). Signifiance determined by1 way ANOVA. p<0.05. Scale bars equal 100 µm.
Figure 6—figure supplement 1
Analysis of synovial cells expressing CD47.
CD47 cells from normal and osteoarthritis (OA) synovium were assayed for the expression of ITAG5 and DPP4/CD26 by flow cytometry. The isotype control shows little to no reactivity. CD47Hi cells co-expressed ITAG5 and DPP4/CD26, whereas CD47Lo cells did not.
Figure 7
CD47Hi vs. CD47Lo cell contribution to cartilage repair in a rat DMM model.
The CD47Hi vs. CD47Lo cells were injected into rats that underwent DMM injury (A), and it was observed that rats which received the CD47Hi cells presented with significantly lower OARSI scores than rats receiving CD47Lo cells (B) with representative images presented of joints receiving CD47Hi (C) and CD47Lo (D) cells. Immunofluorescence analysis demonstrated that the transplanted human CD47Hi cells (TdTomato) integrated into the rat articular cartilage and expressed the chondrocyte markers Prg4 and Sox9 (H–J). This was not observed in the cartilage of rats that received the CD47Lo cells (E–G). Scale bars equal 50 µm. Signifiance determined by1 way ANOVA. p<0.05.
Additional files
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Supplementary file 1
Summary of the participants in the study.
There was no difference in the ages between the normal and OA group (P=0.13).
- https://cdn.elifesciences.org/articles/103332/elife-103332-supp1-v1.docx
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Supplementary file 2
Summary of all clonal lines derived from all patients included in the study.
Gray highlight: Patient sample did not yield any cell growth following single cell sorting. Blue highlight: Clonal cell line yielded enough number of cells for full characterization.
- https://cdn.elifesciences.org/articles/103332/elife-103332-supp2-v1.docx
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Supplementary file 3
Summary of the self-renewal capacity (population doublings) from all clonal lines derived in the study.
- https://cdn.elifesciences.org/articles/103332/elife-103332-supp3-v1.docx
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Supplementary file 4
Summary of the cell surface marker expression (in situ and in vitro) and differentiation potential of clones derived from normal individuals.
- https://cdn.elifesciences.org/articles/103332/elife-103332-supp4-v1.docx
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Supplementary file 5
Summary of the cell surface marker expression (in situ and in vitro) and differentiation potential of clones derived from OA patients.
- https://cdn.elifesciences.org/articles/103332/elife-103332-supp5-v1.docx
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Supplementary file 6
Summary of marker expression and differentiation potential from CD90+CD44+CD73+ cells derived from normal and OA synovium.
- https://cdn.elifesciences.org/articles/103332/elife-103332-supp6-v1.docx
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Supplementary file 7
Shotgun proteomics of proteins from mesenchymal stem cells and non-mesenchymal stem cells identified without constraint by enzyme specificity rules during spectrum-to-sequence matching.
- https://cdn.elifesciences.org/articles/103332/elife-103332-supp7-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/103332/elife-103332-mdarchecklist1-v1.docx
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Identification of a sub-population of synovial mesenchymal stem cells with enhanced treatment efficacy in a rat model of osteoarthritis
eLife 14:RP103332.
https://doi.org/10.7554/eLife.103332.3
