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Thawed cryopreserved synovial mesenchymal stem cells show comparable effects to cultured cells in the inhibition of osteoarthritis progression in rats.

ABSTRACT: Intra-articular injections of mesenchymal stem cells (MSCs) can inhibit the progression of osteoarthritis (OA). Previous reports have used cultured MSCs, but the ability to use thawed cryopreserved MSC stocks would be highly advantageous. Our purpose was to elucidate whether thawed cryopreserved MSCs show comparable inhibitory effects on OA progression in rats to those obtained with cultured MSCs. Cultured rat synovial MSCs or thawed MSCs were compared for in vitro viability and properties. The inhibitory effect of thawed MSCs on OA progression was evaluated by injecting cryopreservation fluid and thawed MSCs in meniscectomized rats. Cartilage degeneration was assessed using gross finding and histological scores. Cultured MSCs were then injected into one knee and thawed MSCs into the contralateral knee of the same individual to compare their effects. Cultured MSCs and MSCs thawed after cryopreservation had comparable in vitro colony formation and chondrogenic potentials. In the rat OA model, the gross finding and histological scores were significantly lower in the thawed MSC group than in the cryopreservation fluid group at 8 weeks. Finally, cartilage degeneration did not differ significantly after injection of cultured and thawed MSCs. In conclusion, thawed MSCs showed comparable inhibitory effects on OA progression to cultured MSCs.

SUBMITTER: Horiuchi K

PROVIDER: S-EPMC8102597 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Thawed cryopreserved synovial mesenchymal stem cells show comparable effects to cultured cells in the inhibition of osteoarthritis progression in rats.

Horiuchi Kiyotaka K Ozeki Nobutake N Endo Kentaro K Mizuno Mitsuru M Katano Hisako H Akiyama Masako M Tsuji Kunikazu K Koga Hideyuki H Sekiya Ichiro I

Scientific reports 20210506 1

Intra-articular injections of mesenchymal stem cells (MSCs) can inhibit the progression of osteoarthritis (OA). Previous reports have used cultured MSCs, but the ability to use thawed cryopreserved MSC stocks would be highly advantageous. Our purpose was to elucidate whether thawed cryopreserved MSCs show comparable inhibitory effects on OA progression in rats to those obtained with cultured MSCs. Cultured rat synovial MSCs or thawed MSCs were compared for in vitro viability and properties. The ...[more]

PMID: 33958682

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Project description:BackgroundVariations in bone morphology in patients with hip osteoarthritis (HOA) can be broadly categorized into three types: atrophic, normotrophic, and hypertrophic. Despite the investigations examining clinical elements, such as bone morphology, pain, and range of motion, our understanding of the pathogenesis of HOA remains limited. Previous studies have suggested that osteophytes typically originate at the interface of the joint cartilage, periosteum, and synovium, potentially implicating synovial mesenchymal stem cells (SMSCs) in the process. This study aimed to investigate the potential factors that drive the development of bone morphological features in HOA by investigating the characteristics of the synovium, differentiation potential of SMSCs, and composition of synovial fluid in different types of HOA.MethodsSynovial tissue and fluid were collected from 30 patients who underwent total hip arthroplasty (THA) with the variable bone morphology of HOA patients. RNA sequencing analysis and quantitative reverse transcription-polymerase chain reaction (RT-qPCR) were performed to analyse the genes in the normotrophic and hypertrophic synovial tissue. SMSCs were isolated and cultured from the normotrophic and hypertrophic synovial tissues of each hip joint in accordance with the variable bone morphology of HOA patients. Cell differentiation potential was compared using differentiation and colony-forming unit assays. Cytokine array was performed to analyse the protein expression in the synovial fluid.ResultsIn the RNA sequencing analysis, 103 differentially expressed genes (DEGs) were identified, predominantly related to the interleukin 17 (IL-17) signalling pathway. Using a protein-protein interaction (PPI) network, 20 hub genes were identified, including MYC, CXCL8, ATF3, NR4A1, ZC3H12A, NR4A2, FOSB, and FOSL1. Among these hub genes, four belonged to the AP-1 family. There were no significant differences in the tri-lineage differentiation potential and colony-forming capacity of SMSCs. However, RT-qPCR revealed elevated SOX9 expression levels in synovial tissues from the hypertrophic group. The cytokine array demonstrated significantly higher levels of CXCL8, MMP9, and VEGF in the synovial fluid of the hypertrophic group than in the normotrophic group, with CXCL8 and MMP9 being significantly expressed in the hypertrophic synovium.ConclusionUpregulation of AP-1 family genes in the synovium and increased concentrations of CXCL8, MMP9, and VEGF were detected in the synovial fluid of the hypertrophic group of HOA patients, potentially stimulating the differentiation of SMSCs towards the cartilage and thereby contributing to severe osteophyte formation.

Project description:UnlabelledRecent data suggest that freshly thawed previously frozen mesenchymal stromal cells (MSCs) may not have the same effectiveness or breadth of anti-inflammatory activities as do continuously cultured MSCs. This has significant implications for clinical use, in which many infusion schemes use frozen cells thawed at the bedside for administration. The available data, however, predominantly evaluate in vitro MSC properties, and so far there has been limited in vivo analysis. To further assess this issue, we compared freshly thawed (thawed) versus continuously cultured (fresh) human bone marrow-derived MSC (hMSC) administration in a mouse model of mixed Th2/Th17 allergic airway inflammation induced by Aspergillus hyphal extract (AHE) exposures in immunocompetent C57Bl/6 mice. Control cell populations included fresh versus thawed murine bone marrow-derived MSCs (mMSCs) and human lung fibroblasts (HLFs). Systemic administration of both thawed and fresh hMSCs and mMSCs, but not HLFs, at the onset of antigen challenge in previously sensitized mice significantly ameliorated the AHE-provoked increases in airway hyper-reactivity, lung inflammation, and antigen-specific CD4 T-cell Th2 and Th17 phenotype. Notably, there was no difference in effects of fresh versus thawed hMSCs or mMSCs on any outcome measured except for some variability in the effects on the bronchoalveolar lavage fluid composition. These results demonstrated potent xenogeneic effects of human MSCs in an immunocompetent mouse model of allergic airways inflammation and that thawed MSCs are as effective as fresh MSCs. The question of fresh versus thawed MSC effectiveness needs to be investigated carefully and may differ in different in vivo disease-specific models.SignificanceThis study addressed whether freshly thawed mesenchymal stromal cells (MSCs) are as effective in in vivo settings as those that have been continuously cultured. It also provided further data demonstrating that xenogeneic use of MSCs in immunocompetent mice is as effective as murine MSCs. This information provides further support and direction for potential clinical use of MSCs in patients with severe asthma.

Project description:BackgroundDetailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples.MethodsMultiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq.ResultsUpon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 μg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified.ConclusionsWe have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.

Dataset Information

Thawed cryopreserved synovial mesenchymal stem cells show comparable effects to cultured cells in the inhibition of osteoarthritis progression in rats.

Publications

Thawed cryopreserved synovial mesenchymal stem cells show comparable effects to cultured cells in the inhibition of osteoarthritis progression in rats.

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