Immunoselected STRO-3+ mesenchymal precursor cells reduce inflammation and improve clinical outcomes in a large animal model of monoarthritis

Abstract

Background: The purpose of this study was to investigate the therapeutic efficacy of intravenously administered immunoselected STRO-3 + mesenchymal precursor cells (MPCs) on clinical scores, joint pathology and cytokine production in an ovine model of monoarthritis.

Methods: Monoarthritis was established in 16 adult merino sheep by administration of bovine type II collagen into the left hock joint following initial sensitization to this antigen. After 24 h, sheep were administered either 150 million allogeneic ovine MPCs (n = 8) or saline (n = 8) intravenously (IV). Lameness, joint swelling and pain were monitored and blood samples for leukocytes and cytokine levels were collected at intervals following arthritis induction. Animals were necropsied 14 days after arthritis induction and gross and histopathological evaluations were undertaken on tissues from the arthritic (left) and contralateral (right) joints.

Results: MPC-treated sheep demonstrated significantly reduced clinical signs of lameness, joint pain and swelling compared with saline controls. They also showed decreased cartilage erosions, synovial stromal cell activation and angiogenesis. This was accompanied by decreased infiltration of the synovial tissues by CD4⁺ lymphocytes and CD14⁺ monocytes/macrophages. Over the 3 days following joint arthropathy induction, the numbers of neutrophils circulating in the blood and plasma concentrations of activin A were significantly reduced in animals administered MPCs.

Conclusions: The results of this study have demonstrated the capacity of IV-administered MPCs to mitigate the clinical signs and some of the inflammatory mediators responsible for joint tissue destruction in a large animal model of monoarthritis.

Keywords: Animal model; Collagen-induced arthritis; Mesenchymal stem cells; Neutrophils.

Fig. 1

Effect of MPC administration on lameness (A), pain on joint flexion (B) and joint swelling (C) in sheep with CIA in the left hock. Intra-articular injection of BCII was performed on day 28 and MPC administration on day 29. There was a significant reduction in all measured parameters and the aggregate score (D) 2 days after MPC treatment. Values were analysed using two-way ANOVA with Sidak’s multiple comparison tests and each point represents the mean ± SEM of seven to eight sheep with ^*, ^**, ^***and ^**** representing p ≤ 0.05, 0.01, 0.001 and 0.0001 respectively. MPC mesenchymal precursor cells

Fig. 2

Effect of MPC administration on total blood leukocyte (A), neutrophil (B), lymphocyte (C) and monocyte (D) counts. Values were analysed using two-way ANOVA with Sidak’s multiple comparison tests and each point represents the mean ± SEM of seven to eight sheep with ^*, ^** and ^*** representing p ≤ 0.05, 0.01 and 0.001, respectively. MPC mesenchymal precursor cells

Fig. 3

Effect of MPC administration on plasma cytokines in sheep with CIA. Plasma concentrations of activin A (A), and IL-17A (B) were compared between the MPC-treated and control group using two-way ANOVA with Sidak’s multiple comparison tests. The AUC_I for the maximal responses between days 29 and 36 of activin A (A) and IL-17A (B) were compared using Mann-Whitney tests. Each point represents the mean ± SEM for concentrations or mean ± SD for AUC of seven to eight sheep with ^*and ^** representing p ≤ 0.05 and 0.01, respectively). MPC mesenchymal precursor cells

Fig. 4

Effect of MPC administration on SF cytokines in sheep with CIA. Concentrations of activin A (A), IL-17A (B), IFN-γ (C) and IL-10 (D), were compared between the MPC-treated and the control group using paired Wilcoxon (left versus right) and unpaired Mann-Whitney (saline versus MPC treatment) tests. Lines represent mean ± SEM of seven to eight sheep and ^* and ^** represent p ≤ 0.05 and 0.01, respectively. IFN interferon, IL interleukin, MPC mesenchymal precursor cells

Fig. 5

Effect of MPC treatment on gross joint pathology in the sheep CIA model. Representative photographs of joint swelling and gross appearance of articular cartilage from the left hock joints of control (A and C) and MPC-treated (B and D) sheep 14 days after the induction of arthritis, the arthritic left hock joint from the control animal shows more severe erosion of the cartilage on the articular surface of the talus bone (panel C; arrow) and on the articular surface of the distal tibia, compared to the MPC-treated sheep (panel D). MPC mesenchymal precursor cells

Fig. 6

Effect of MPC treatment on cartilage erosion in the sheep CIA model. The cartilage on the surface of the talus was assessed macroscopically using a 5-point scale based on the OARSI recommendations for macroscopic scoring of cartilage pathology, as described in the Methods section. Values were compared between the MPC-treated and the control group using paired Wilcoxon (left and right) and unpaired Mann-Whitney (saline and MPC treatment) tests. Lines represent mean ± SEM of seven to eight sheep. ^** represents significant difference; p ≤ 0.01. MPC mesenchymal precursor cells

Fig. 7

A Effect of MPC treatment on synovial histopathology in the sheep CIA model. Synovial histopathology scores were determined for samples from the left hock joints of control and MPC-treated sheep 14 days after the induction of arthritis. The parameters included stromal cell activation (panel a), intimal hyperplasia (panel b) and inflammatory cell infiltration (panel c), plus a combined total score (panel d). Values were compared between the MPC-treated and the control group using paired Wilcoxon (left and right) and unpaired Mann-Whitney (saline and MPC treatment) tests. Lines represent mean ± SEM of seven to eight sheep. ^* and ^** represent significant difference; p ≤ 0.05 and p ≤ 0.01, respectively. B Representative histopathological images of synovial membranes from arthritic hock joints following saline (a - c) or MPC (d - f) treatment. Paraffin-embedded tissues were stained with haematoxylin and eosin. The bars represent 250 μm. MPC mesenchymal precursor cells

Fig. 8

A Effect of MPC treatment on the number of inflammatory cells in SMs from sheep with CIA. Lines represent median and interquartile ranges of seven to eight sheep. Frozen sections were immunostained for CD4 (a), CD8 (b), γδ TCR (c), CD79a (d), CD14 (e) and Ki-67 (f). Blood vessels were identified by vWF expression (g). The data were compared using paired Wilcoxon (left and right) and unpaired Mann-Whitney (saline and MPC treatment) tests. Lines represent mean ± SEM of six to eight sheep with ^* and ^** representing p ≤ 0.05 and p ≤ 0.01, respectively. B Immunohistology of synovial membranes from arthritic hock joints following saline (left column) or MPC (right column) treatment. The frozen sections were stained with antibodies to CD4 (a , b), CD8 (c , d), γδTCR (e , f), CD79a for B cells (g , h) and CD14 (i , j). The bars represent 25 μm. MPC mesenchymal precursor cells