Therapeutic Effects of Gingival Mesenchymal Stem Cells and Their Exosomes in a Chimeric Model of Rheumatoid Arthritis (original) (raw)
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Formation of cartilage and synovial tissue by human gingival stem cells
2014
Human gingival stem cells (HGSCs) can be easily isolated and manipulated in culture to investigate their multipotency. Osteogenic differentiation of bone-marrow-derived mesenchymal stem/stromal cells has been well documented. HGSCs derive from neural crests, however, and their differentiation capacity has not been fully established. The aim of the present report was to investigate whether HGSCs can be induced to differentiate to osteoblasts and chondrocytes. HGSCs were cultured either in a classical monolayer culture or in threedimensional floating micromass pellet cultures in specific differentiation media. HGSC differentiation to osteogenic and chondrogenic lineages was determined by protein and gene expression analyses, and also by specific staining of cells and tissue pellets. HGSCs cultured in osteogenic differentiation medium showed induction of Runx2, alkaline phosphatase (ALPL), and osterix expression, and subsequently formed mineralized nodules consistent with osteogenic differentiation. Interestingly, HGSC micromass cultures maintained in chondrogenic differentiation medium showed SOX9-dependent differentiation to both chondrocyte and synoviocyte lineages. Chondrocytes at different stages of differentiation were identified by gene expression profiles and by histochemical and immunohistochemical staining. In 3-week-old cultures, peripheral cells in the micromass cultures organized in layers of cuboidal cells with villous structures facing the medium. These cells were strongly positive for cadherin-11, a marker of synoviocytes. In summary, the findings indicate that HGSCs have the capacity to differentiate to osteogenic, chondrogenic, and synoviocyte lineages. Therefore, HGSCs could serve as an alternative source for stem cell therapies in regenerative medicine for patients with cartilage and joint destructions, such as observed in rheumatoid arthritis.
Arthritis Research & Therapy - ARTHRITIS RES THER, 2001
There is increasing interest in adeno-associated virus (AAV) vectors for a wide variety of gene therapy applications. AAV is a nonpathogenic human parvovirus that can mediate long-term transduction of a number of cell types without provoking a significant immune response. These properties make AAV especially attractive for use in gene therapy of rheumatoid arthritis (RA), a chronic inflammatory disease. To investigate the potential of AAV in gene therapy of arthritis, the ability of AAV to infect synovium in vitro and in vivo was tested. Three human RA synovial fibroblast cell lines and two murine (one DBA/1J and one DBA1J×C3H F1) synovial fibroblast cell lines were used to test AAV transduction in vitro. The cell lines (2 × 10 5 cells) were infected with 10 4 particles/cell of a murine IL-10-encoding vector (AAV-mIL-10) alone or with the addition of a low titer (100 particles/cell) of an E1-, E3-deleted recombinant adenovirus to provide E4orf6 activity to enhance second-strand synthesis. The supernatants were harvested from the wells at various time points and assayed for mIL-10 expression by ELISA. Both human synovial cell lines infected with AAV alone demonstrated low-level transgene expression throughout the course of the study. However, by day 10, all human cultures coinfected with adenovirus showed a 16-to 56-fold increase in mIL-10 compared to cultures infected with AAV-mIL10 alone. By day 30, a 31-to 135-fold increase was observed. No such increase was observed in any of the mouse cell lines. To determine the AAV transduction efficiency for synovium in vivo, human RA synovial tissues obtained from patients undergoing joint-replacement surgery were implanted subcutaneously on the backs of NOD.CB17-Prkdc SCID mice. After allowing a 2-week period for engraftment, tissues were injected with 3.4 × 10 11 particles of AAV-luciferase alone or in combination with 1.0 × 10 11 particles of adenovirus. Two weeks following AAV administration, the tissues were homogenized and assayed for expression of luciferase. Only the tissues coinfected with adenovirus had luciferase levels above background. A similar experiment with AAV-LacZ demonstrated X-gal staining only of synovial tissues coinfected with adenovirus. These findings demonstrate a preferential ability of AAV to transduce human, compared to mouse, synovial tissue and suggest that second strand synthesis may be a limiting factor in gene transduction. Further studies to elucidate the mechanisms limiting gene transduction in human synovium may allow optimization of this vector for the treatment of arthritis. P2 Delivery of antisense constructs and ribozymes to inhibit cartilage destruction in the SCID mouse model of RA
Cyprus Journal of Medical Sciences
BACKGROUND/AIMS: Dental mesenchymal stem cells are easily accessible sources for mesenchymal stem cells (MSCs) and can rapidly proliferate in culture conditions. Rheumatoid arthritis (RA) is a chronic and multi-systemic autoimmune inflammatory disease that results in cartilage damage. The present study aimed to investigate the regenerative potential of dental MSCs in the synovial fluid microenvironment of patients with RA. MATERIALS AND METHODS: Synovial fluid samples (8-10 mL/patient) were collected from patients with RA (age; 48−67 years). Dental pulp (DP) and periodontal ligament (PL) tissues were collected from healthy individuals, and the tissues were enzymatically digested in 3 mg/mL collagenase type I. MSCs were cultured in Dulbecco's Modified Eagle Medium (DMEM). The cells were cultured in the presence and absence of the synovial fluid samples of the patients with RA and subjected to flow cytometry analysis for cell surface expressions of positive markers for chondrogenesis (CD49e) and an osteogenic marker (alkaline phosphatase; ALP). The differentiation capacity of MSCs was evaluated with osteogenic or chondrogenic stimulation media and analyzed by staining the cells with Alizarin Red or Alcian Blue, respectively. RESULTS: The cytokines interleukin (IL)-1β (61.1±9.8) and IL-6 (2386.7±397.4) were significantly higher in the end-stage RA-SF samples, compared to the early-stage RA-SF samples (IL-1β:35.2±4.8, IL-6:561.3±197.6) (p<0.05, p<0.001, respectively). DP-MSCs were significantly differentiated to osteocytes and formed calcium deposits cultured with end-stage RA-SF samples, whereas PL-MSCs were differentiated to osteocytes in limited levels, and low concentrations of calcium deposits were observed. Chondrocytes were observed in DP and PL-MSCs, and cartilage formation was observed only in DP-MSCs when cultured with end-stage RA-SF samples. The neutralization of IL-1β or IL-6 tended to decrease osteogenic marker expressions of DP-MSCs cultured in the presence of end-stage RA-SF samples. CONCLUSION: The present study showed the differentiation potential of DP-MSCs into osteogenic and chondrogenic lineage in the inflammatory microenvironment of SF. DP-MSCs may be candidates for tissue regeneration, especially in patients with RA with bone or cartilage erosions.
Journal of Translational Medicine, 2014
Background: Rheumatoid arthritis (RA) is a debilitating and painful disease leading to increased morbidity and mortality and novel therapeutic approaches are needed. The purpose of this study was to elucidate if mesenchymal stem cells (MSCs) injected in the joints of mice with arthritis are therapeutic, reducing joint swelling and cartilage destruction. Methods: Murine mesenchymal stem cells (mMSCs) were isolated from bone marrow of C57Bl/6 mice and expanded in culture. Cells were tested for immunophenotype and their ability to form colonies and to differentiate into chondrocytes, osteocytes and adipocytes. Antigen-induced arthritis (AIA) was induced by intra-articular injection of methylated bovine serum albumin into the knee joints of preimmunized C57Bl/6 mice. After one day, when peak swelling occurs, 500,000 mMSCs labelled with red fluorescent cell tracker CM-DiI were injected intra-articularly in the right knee joint. Left knee joints were treated as controls by receiving PBS injections. Differences between groups were calculated by Mann Whitney U test or unpaired t tests using GraphPad Prism software version 5. Results: Knee joint diameter (swelling) was measured as a clinical indication of joint inflammation and this parameter was significantly less in MSC-treated mice compared to control-treated animals 48 hours after arthritis induction. This difference continued for~7 days. CM-DiI-labelled MSCs were clearly visualised in the lining and sublining layers of synovium, in the region of the patella and femoral and tibial surfaces. By day 3, parameters indicative of disease severity, including cartilage depletion, inflammatory exudate and arthritic index were shown to be significantly reduced in MSC-treated animals. This difference continued for 7 days and was further confirmed by histological analysis. The serum concentration of tumour necrosis factor α was significantly decreased following MSC administration. Conclusions: Our results reveal that MSCs injected in the joints of mice with AIA are therapeutic, reducing inflammation, joint swelling and cartilage destruction. These cells also integrate into the synovium in AIA.
TURKISH JOURNAL OF MEDICAL SCIENCES, 2019
Background/aim: Multipotent mesenchymal stem cells (MSCs) have been investigated in autoimmune diseases such as rheumatoid arthritis (RA) due to their immunomodulatory and regenerative properties. In this study, their immunosuppressive effects on peripheral blood mononuclear cells (PBMC) of RA patients were studied. Materials and methods: Dental follicle stem cells (DFSCs) were isolated from follicle tissue in the orofacial region. Characterization and multipotency analyses were performed. Lymphocytes were isolated from peripheral venous blood of RA patients (n = 5) and healthy individuals (n = 5). DFSCs were preincubated with IFN-γ for 48 h. PBMCs of RA patients and healthy individuals were separately cultured with or without DFSCs for 72 h. After culture period, lymphocyte proliferation and viability, the frequency of CD4 + CD25 + FoxP3 + T regulatory cells, IL-10 and TNF-α levels in the culture supernatants were measured via flow cytometry. Results: Our results demonstrated that DFSCs suppressed proliferation of T lymphocytes by increasing the number of FoxP3 expressing CD4 + CD25 + T regulatory cells and suppressed lymphocyte apoptosis in RA patients. Also, DFSCs reduced TNF-α cytokine secretion and upregulated IL-10 secreting cells. Conclusions: Such cells could potentially be a source for future immunomodulatory treatments of RA patients.
Bone stem cell mediated gene therapy and tissue engineering
Arthritis Research & Therapy, 2001
There is increasing interest in adeno-associated virus (AAV) vectors for a wide variety of gene therapy applications. AAV is a nonpathogenic human parvovirus that can mediate long-term transduction of a number of cell types without provoking a significant immune response. These properties make AAV especially attractive for use in gene therapy of rheumatoid arthritis (RA), a chronic inflammatory disease. To investigate the potential of AAV in gene therapy of arthritis, the ability of AAV to infect synovium in vitro and in vivo was tested. Three human RA synovial fibroblast cell lines and two murine (one DBA/1J and one DBA1J×C3H F1) synovial fibroblast cell lines were used to test AAV transduction in vitro. The cell lines (2 × 10 5 cells) were infected with 10 4 particles/cell of a murine IL-10-encoding vector (AAV-mIL-10) alone or with the addition of a low titer (100 particles/cell) of an E1-, E3-deleted recombinant adenovirus to provide E4orf6 activity to enhance second-strand synthesis. The supernatants were harvested from the wells at various time points and assayed for mIL-10 expression by ELISA. Both human synovial cell lines infected with AAV alone demonstrated low-level transgene expression throughout the course of the study. However, by day 10, all human cultures coinfected with adenovirus showed a 16-to 56-fold increase in mIL-10 compared to cultures infected with AAV-mIL10 alone. By day 30, a 31-to 135-fold increase was observed. No such increase was observed in any of the mouse cell lines. To determine the AAV transduction efficiency for synovium in vivo, human RA synovial tissues obtained from patients undergoing joint-replacement surgery were implanted subcutaneously on the backs of NOD.CB17-Prkdc SCID mice. After allowing a 2-week period for engraftment, tissues were injected with 3.4 × 10 11 particles of AAV-luciferase alone or in combination with 1.0 × 10 11 particles of adenovirus. Two weeks following AAV administration, the tissues were homogenized and assayed for expression of luciferase. Only the tissues coinfected with adenovirus had luciferase levels above background. A similar experiment with AAV-LacZ demonstrated X-gal staining only of synovial tissues coinfected with adenovirus. These findings demonstrate a preferential ability of AAV to transduce human, compared to mouse, synovial tissue and suggest that second strand synthesis may be a limiting factor in gene transduction. Further studies to elucidate the mechanisms limiting gene transduction in human synovium may allow optimization of this vector for the treatment of arthritis. P2 Delivery of antisense constructs and ribozymes to inhibit cartilage destruction in the SCID mouse model of RA
Stem Cell Therapy as a Treatment Method for Rheumatoid Arthritis
Stem Cell and Regenerative Medicine, 2020
Rheumatoid Arthritis (RA) is a progressive, autoimmune disorder that causes joint deterioration in the body. RA begins with swelling and inflammation in the joints, and eventually progresses to cause cartilage and bone deterioration at the site of the joint. Early stages of RA typically affect smaller joints, and the impact spreads to larger joints as the disease continues to advance within an individual. There are various treatment methods that are currently used by RA patients, which aim to mitigate RA symptoms and induce periods of remission. However, stem cell therapies using BM-MSCs, hASCs, UC-MSCs, and hUCB-MSCs are currently being studied as potential treatment methods for RA. Many scientists and researchers believe that the characteristics of stem cells will allow stem cell therapy to be an extremely beneficial treatment method. Pre-clinical trials and clinical trials using various stem cell types have been completed, but further testing is necessary to determine the most safe and effective method to treat RA in humans.
Stem Cell Research & Therapy, 2016
Background: Mesenchymal stem cells (MSCs) have been isolated from a variety of tissues, including bone marrow, adipose, and mucosa. MSCs have the capacity for self-renewal and differentiation. Reports have been published on the systemic administration of MSCs leading to functional improvements by engraftment and differentiation, thus providing a new strategy to regenerate damaged tissues. Recently, it has become clear that MSCs possess immunomodulatory properties and can therefore be used to treat diseases. However, the therapeutic effect mechanisms of MSCs are yet to be determined. Here, we investigated these mechanisms using a medicationrelated osteonecrosis of the jaw (MRONJ)-like mouse model. Methods: To generate MRONJ-like characteristics, mice received intravenous zoledronate and dexamethasone two times a week. At 1 week after intravenous injection, maxillary first molars were extracted, and at 1 week after tooth extraction, MSCs were isolated from the bone marrow of the mice femurs and tibias. To compare "diseased MSCs" from MRONJ-like mice (d-MSCs) with "control MSCs" from untreated mice (c-MSCs), the isolated MSCs were analyzed by differentiation and colony-forming unit-fibroblast (CFU-F) assays and systemic transplantation of either d-MSCs or c-MSCs into MRONJ-like mice. Furthermore, we observed the exchange of cell contents among d-MSCs and c-MSCs during coculture with all combinations of each MSC type. Results: d-MSCs were inferior to c-MSCs in differentiation and CFU-F assays. Moreover, the d-MSC-treated group did not show earlier healing in MRONJ-like mice. In cocultures with any combination, MSC pairs formed cell-cell contacts and exchanged cell contents. Interestingly, the exchange among c-MSCs and d-MSCs was more frequently observed than other pairs, and d-MSCs were distinguishable from c-MSCs. Conclusions: The interaction of c-MSCs and d-MSCs, including exchange of cell contents, contributes to the treatment potential of d-MSCs. This cellular behavior might be one therapeutic mechanism used by MSCs for MRONJ.
Mesenchymal stem cells are conditionally therapeutic in preclinical models of rheumatoid arthritis
Annals of the Rheumatic Diseases, 2012
Objective The role of mesenchymal stem cells (MSC) in experimental arthritis is undoubtedly confl icting. This study explored the effect of bone marrow-derived MSC in previously untested and pathogenetically different models of rheumatoid arthritis (RA). Methods MSC were tested both in an induced (adjuvant-induced) and a spontaneous (K/BxN) arthritis model. Arthritis was assessed clinically and histologically. The proliferation of splenocytes and fi broblast-like synoviocytes (FLS) in the presence of MSC was measured by radioactivity incorporation. Toll-like receptor (TLR) expression was measured by real-time PCR. T-regulatory cell (Treg) frequency, T-cell apoptosis and cytokine secretion were monitored by fl ow cytometry. Results MSC, in vitro, strongly inhibited critical cell populations; splenocytes and FLS. In contrast, MSC proved ineffective in vivo, unless they were administered before disease onset, an effect implying that the infl ammatory arthritic milieu potentially abrogates MSC immunomodulatory properties. In order to alleviate infl ammation before MSC infusion, the authors administered, at arthritis onset, a short course with a proteasome inhibitor, bortezomib, whereas MSC were infused when established disease was expected. The bortezomib plus MSC group demonstrated a signifi cantly decreased arthritis score over arthritic, MSC-only, bortezomib-only groups, also confi rmed by histology and immunohistochemistry. The bortezomib plus MSC combination restored TLR expression and Treg frequency in blood and normalised FLS and splenocyte proliferation, apoptosis and cytokine secretion. Conclusion MSC lose their immunomodulatory properties when infused in the infl ammatory micromilieu of autoimmune arthritis. Conditioning of the recipient with bortezomib alters the disease microenvironment enabling MSC to modulate arthritis. Should milieu limitations also operate in human disease, this approach could serve as a strategy to treat RA by MSC.