Cell-to-Cell Culture Inhibits Dedifferentiation of Chondrocytes and Induces Differentiation of Human Umbilical Cord-Derived Mesenchymal Stem Cells.
ABSTRACT: Background:Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) possess great promise as a therapeutic to repair damaged cartilage. Direct intra-articular injection of mesenchymal stem cells has been shown to reduce cartilage damage and is advantageous as surgical implantation and associated side effects can be avoided using this approach. However, the efficacy of stem cell-based therapy for cartilage repair depends highly on the direct interactions of these stem cells with chondrocytes in the joint. In this study, we have carried out an in vitro cell-to-cell contact coculture study with human articular chondrocytes (hACs) and hUC-MSCs, with the goal of this study being to evaluate interactions between hACs and hUC-MSCs. Methods:Low-density monolayer cultures of hUC-MSCs and hACs were mixed at a ratio of 1?:?1 in direct cell-to-cell contact groups. Results were analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blot, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence. Results:A mixed coculture of hUC-MSCs and hACs was found to exhibit synergistic interactions with enhanced differentiation of hUC-MSCs and reduced dedifferentiation of chondrocytes. Mixed cultures after 21 days were found to exhibit sufficient chondrogenic induction. Conclusions:The results from this study suggest the presence of mutual effects between hUC-MSCs and hACs even culture at low density and provide further support for the use of intra-articular injection strategies for cartilage defect treatment.
Project description:Articular cartilage has limited self-regenerative capacity and the therapeutic methods for cartilage defects are still dissatisfactory in clinic. Recent studies showed that exosomes derived from mesenchymal stem cells promoted chondrogenesis by delivering bioactive substances to the recipient cells, indicating exosomes might be a novel method for repairing cartilage defect. Herein, we investigated the role and mechanism of human umbilical cord mesenchymal stem cells derived small extracellular vesicles (hUC-MSCs-sEVs) on cartilage regeneration. In vitro results showed that hUC-MSCs-sEVs promoted the migration, proliferation and differentiation of chondrocytes and human bone marrow mesenchymal stem cells (hBMSCs). MiRNA microarray showed that miR-23a-3p was the most highly expressed among the various miRNAs contained in hUC-MSCs-sEVs. Our data revealed that hUC-MSCs-sEVs promoted cartilage regeneration by transferring miR-23a-3p to suppress the level of PTEN and elevate expression of AKT. Moreover, we fabricated Gelatin methacrylate (Gelma)/nanoclay hydrogel (Gel-nano) for sustained release of sEVs, which was biocompatible and exhibited excellent mechanical property. In vivo results showed that hUC-MSCs-sEVs containing Gelma/nanoclay hydrogel (Gel-nano-sEVs) effectively promoted cartilage regeneration. These results indicated that Gel-nano-sEVs have a promising capacity to stimulate chondrogenesis and heal cartilage defects, and also provided valuable data for understanding the role and mechanism of hUC-MSCs-sEVs in cartilage regeneration.
Project description:Coculture between mesenchymal stem cells (MSCs) and chondrocytes has significant implications in cartilage regeneration. However, a conclusive understanding remains elusive. Previously, we reported that rabbit bone marrow-derived MSCs (rbBMSCs) could downregulate the differentiated phenotype of rabbit articular chondrocytes (rbACs) in a non-contact coculture system for the first time. In the present study, a systemic investigation was performed to understand the biological characteristics of chondrocytes in coculture with MSCs. Firstly, cells (MSCs and chondrocytes) from different origins were cocultured in transwell system. Different chondrocytes, when cocultured with different MSCs respectively, consistently demonstrated stimulated proliferation, transformed morphology and declined glycosaminoglycan secretion of chondrocytes. Next, cell surface molecules and the global gene expression of rbACs were characterized. It was found that cocultured rbACs showed a distinct surface molecule profile and global gene expression compared to both dedifferentiated rbACs and rbBMSCs. In the end, cocultured rbACs were passaged and induced to undergo the chondrogenic redifferentiation. Better growth and chondrogenesis ability were confirmed compared with control cells without coculture. Together, chondrocytes display comprehensive changes in coculture with MSCs and the cocultured rbACs are beneficial for cartilage repair.
Project description:Coculture of mesenchymal stem cells with chondrocytes increases production of cartilaginous matrix. Chondrocytes isolated from late stage osteoarthritic patients usually lost their phenotype of producing cartilaginous matrix. Fibroblast growth factor 18 is believed to redifferentiate OA chondrocyte into functionally active chondrocytes. The aim of this study is to investigate the supportive effects of MSCs on OA chondrocytes and test if FGF18 could enhance the responsiveness of OA chondrocytes to the support of MSCs in a coculture system. Both pellet and transwell co-cultures were used. GAG quantification, hydroxyproline assay, and qPCR were performed. An ectopic models of cartilage formation was also applied. Our data indicated that, in pellets coculture of MSCs and OA chondrocytes, matrix production was increased in the presence of FGF18, comparing to the monoculture of chondrocytes. Results from transwell coculture study showed that expression of matrix producing genes in OA chondrocytes increased when cocultured with MSCs with FGF18 in culture medium, while hypertrophic genes were not changed by coculture. Finally, coimplantation of MSCs with OA chondrocytes produces more matrix than chondrocytes only. In conclusion, FGF18 can restore the responsiveness of OA chondrocytes to the trophic effects of MSCs. Coimplantation of MSCs and OA chondrocytes treated with FGF18 may be a good alternative cell source for regenerating cartilage tissue that is degraded during OA pathological changes.
Project description:Coculture between mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) represents a promising strategy for cartilage regeneration. This study aimed at elaborating how ACs were regulated by MSCs. Rabbit ACs (rACs) and rabbit MSCs (rMSCs) were seeded separately in a Transwell system to initiate non-contact coculture in growth medium without chondrogenic factors. Cell morphology, cell proliferation, production of extracellular matrix (ECM), and gene expression of rACs were characterized. Upon coculture, rACs underwent a morphological transition from a rounded or polygonal shape into a fibroblast-like one and proliferation was provoked simultaneously. Such effects were dependent on the amount of rMSCs. Along with these changes, ECM production and gene expression of rACs were also perturbed. Importantly, when a ROCK inhibitor (Y27632) was supplemented to coculture, the effects except that on cell proliferation were inhibited, suggesting the involvement of RhoA/ROCK signaling. By applying an inhibitor (BIBF1120) of VEGFR1/2/3, FGFR1/2/3 and PDGFR?/? in coculture, or supplementing FGF-1, VEGF-A and PDGFbb in monoculture, it was confirmed that the paracrine factors by rMSCs mediated the compounding effects on rACs. These findings shed light on MSCs-ACs interactions and might confer an insight view on cell-based cartilage regeneration.
Project description:Bone marrow mesenchymal stem cells (BM-MSCs) are considered a good source for cellular therapy in cartilage repair. But, their potential to repair the extracellular matrix, in an osteoarthritic environment, is still controversial. In osteoarthritis (OA), anti-inflammatory action and extracellular matrix production are important steps for cartilage healing. This study examined the interaction of BM-MSC and OA-chondrocyte on the production of hyaluronan and inflammatory cytokines in a Transwell system. We compared cocultured BM-MSCs and OA-chondrocytes with the individually cultured controls (monocultures). There was a decrease in BM-MSCs cell count in coculture with OA-chondrocytes when compared to BM-MSCs alone. In monoculture, BM-MSCs produced higher amounts of hyaluronan than OA-chondrocytes and coculture of BM-MSCs with OA-chondrocytes increased hyaluronan production per cell. Hyaluronan synthase-1 mRNA expression was upregulated in BM-MSCs after coculture with OA-chondrocytes, whereas hyaluronidase-1 was downregulated. After coculture, lower IL-6 levels were detected in BM-MSCs compared with OA-chondrocytes. These results indicate that, in response to coculture with OA-chondrocytes, BM-MSCs change their behavior by increasing production of hyaluronan and decreasing inflammatory cytokines. Our results indicate that BM-MSCs per se could be a potential tool for OA regenerative therapy, exerting short-term effects on the local microenvironment even when cell:cell contact is not occurring.
Project description:Seed cells of articular cartilage tissue engineering face many obstacles in their application because of the dedifferentiation of chondrocytes or unstable chondrogenic differentiation status of pluripotent stem cells. To overcome mentioned dilemmas, a simulation of the articular cartilage microenvironment was constructed by primary articular cartilage cells (pACs) and acellular cartilage extracellular matrix- (ACECM-) oriented scaffold cocultured with human umbilical cord Wharton's jelly-derived mesenchymal stem cells (hWJMSCs) in vitro. The coculture groups showed more affluent cartilage special matrix ingredients including collagen II and aggrecan based on the results of histological staining and western blotting and cut down as many pACs as possible. The RT-PCR and cell viability experiments also demonstrated that hWJMSCs were successfully induced to differentiate into chondrocytes when cultured in the simulated cartilage microenvironment, as confirmed by the significant upregulation of collagen II and aggrecan, while the cell proliferation activity of pACs was significantly improved by cell-cell interactions. Therefore, compared with monoculture and chondrogenic induction of inducers, coculture providing a simulated native articular microenvironment was a potential and temperate way to regulate the biological behaviors of pACs and hWJMSCs to regenerate the hyaline articular cartilage.
Project description:Chondrogenic mesenchymal stem cells (MSCs) have not yet been used to address the clinical demands of large osteochondral joint surface defects. In this study, self-assembling tissue intermediates (TIs) derived from human periosteum-derived stem/progenitor cells (hPDCs) were generated and validated for stable cartilage formation in vivo using two different animal models.hPDCs were aggregated and cultured in the presence of a novel growth factor (GF) cocktail comprising of transforming growth factor (TGF)-?1, bone morphogenetic protein (BMP)2, growth differentiation factor (GDF)5, BMP6, and fibroblast growth factor (FGF)2. Quantitative polymerase chain reaction (PCR) and immunohistochemistry were used to study in vitro differentiation. Aggregates were then implanted ectopically in nude mice and orthotopically in critical-size osteochondral defects in nude rats and evaluated by microcomputed tomography (µCT) and immunohistochemistry.Gene expression analysis after 28 days of in vitro culture revealed the expression of early and late chondrogenic markers and a significant upregulation of NOGGIN as compared to human articular chondrocytes (hACs). Histological examination revealed a bilayered structure comprising of chondrocytes at different stages of maturity. Ectopically, TIs generated both bone and mineralized cartilage at 8 weeks after implantation. Osteochondral defects treated with TIs displayed glycosaminoglycan (GAG) production, type-II collagen, and lubricin expression. Immunostaining for human nuclei protein suggested that hPDCs contributed to both subchondral bone and articular cartilage repair.Our data indicate that in vitro derived osteochondral-like tissues can be generated from hPDCs, which are capable of producing bone and cartilage ectopically and behave orthotopically as osteochondral units.
Project description:Articular cartilage has poor healing ability and cartilage injuries often evolve to osteoarthritis. Cell-based strategies aiming to engineer cartilaginous tissue through the combination of biocompatible scaffolds and articular chondrocytes represent an alternative to standard surgical techniques. In this context, perfusion bioreactors have been introduced to enhance cellular access to oxygen and nutrients, hence overcoming the limitations of static culture and improving matrix deposition. Here, we combined an optimized cocktail of soluble factors, the BIT (BMP-2, Insulin, Thyroxin), and clinical-grade collagen sponges with a bidirectional perfusion bioreactor, namely the oscillating perfusion bioreactor (OPB), to engineer in vitro articular cartilage by human articular chondrocytes (HACs) obtained from osteoarthritic patients. After amplification, HACs were seeded and cultivated in collagen sponges either in static or dynamic conditions. Chondrocyte phenotype and the nature of the matrix synthesized by HACs were assessed using western blotting and immunohistochemistry analyses. Finally, the stability of the cartilaginous tissue produced by HACs was evaluated in vivo by subcutaneous implantation in nude mice. Our results showed that perfusion improved the distribution and quality of cartilaginous matrix deposited within the sponges, compared to static conditions. Specifically, dynamic culture in the OPB, in combination with the BIT cocktail, resulted in the homogeneous production of extracellular matrix rich in type II collagen. Remarkably, the production of type I collagen, a marker of fibrous tissues, was also inhibited, indicating that the association of the OPB with the BIT cocktail limits fibrocartilage formation, favoring the reconstruction of hyaline cartilage.
Project description:The role of rabbit synovial fluid-derived mesenchymal stem cells (rbSF-MSCs) in cartilage defect repair remains undefined. This work evaluates the in vivo effects of rbSF-MSCs to repair knee articular cartilage defects in a rabbit model.Cartilage defects were made in the patellar grooves of New Zealand white rabbits. The rbSF-MSCs were generated from the knee cavity by arthrocentesis. Passage 5 rbSF-MSCs were assayed by flow cytometry. The multipotency of rbSF-MSCs was confirmed after 3 weeks induction in vitro and the autologous rbSF-MSCs and predifferentiated rbSF-MSCs were injected into the synovial cavity. The intra-articular injection was performed once a week for 4 weeks. The animals were euthanized and the articular surfaces were subjected to macroscopic and histological evaluations at 8 and 12 weeks after the first intra-articular injection.Hyaline-like cartilage was detected in the defects treated with rbSF-MSCs, while fibrocartilage tissue formed in the defects treated with chondrocytes induced from rbSF-MSCs.Our results suggest that autologous undifferentiated rbSF-MSCs are favorable to articular cartilage regeneration in treating cartilage defects.
Project description:A major problem with chondrocytes derived in vitro from stem cells is undesired hypertrophic degeneration, to which articular chondrocytes (ACs) are resistant. As progenitors of all adult tissues, induced pluripotent stem cells (iPSCs) are in theory able to form stable articular cartilage. In vitro differentiation of iPSCs into chondrocytes with an AC-phenotype and resistance to hypertrophy has not been demonstrated so far. Here, we present a novel protocol that succeeded in deriving chondrocytes from human iPSCs without using pro-hypertrophic bone-morphogenetic-proteins. IPSC-chondrocytes had a high cartilage formation capacity and deposited two-fold more proteoglycans per cell than adult ACs. Importantly, cartilage engineered from iPSC-chondrocytes had similar marginal expression of hypertrophic markers (COL10A1, PTH1R, IBSP, ALPL mRNAs) like cartilage from ACs. Collagen X was barely detectable in iPSC-cartilage and 30-fold lower than in hypertrophic cartilage derived from mesenchymal stromal cells (MSCs). Moreover, alkaline phosphatase (ALP) activity remained at basal AC-like levels throughout iPSC chondrogenesis, in contrast to a well-known significant upregulation in hypertrophic MSCs. In line, iPSC-cartilage subjected to mineralizing conditions in vitro showed barely any mineralization, while MSC-derived hypertrophic cartilage mineralized strongly. Low expression of Indian hedgehog (IHH) like in ACs but rising BMP7 expression like in MSCs suggested that phenotype stability was linked to the hedgehog rather than the bone morphogenetic protein (BMP) pathway. Taken together, unlimited amounts of AC-like chondrocytes with a high proteoglycan production reminiscent of juvenile chondrocytes and resistance to hypertrophy and mineralization can now be produced from human iPSCs in vitro. This opens new strategies for cartilage regeneration, disease modeling and pharmacological studies.