The trans-well coculture of human synovial mesenchymal stem cells with chondrocytes leads to self-organization, chondrogenic differentiation, and secretion of TGFβ

Abstract

BackgroundSynovial mesenchymal stem cells (SMSC) possess a high chondrogenic differentiation potential, which possibly supports natural and surgically induced healing of cartilage lesions. We hypothesized enhanced chondrogenesis of SMSC caused by the vicinity of chondrocytes (CHDR).MethodsHuman SMSC and CHDR interactions were investigated in an in-vitro trans-well monolayer coculture over a time period of up to 21 days. Protein expression was analyzed using histology, immunostaining, or enzyme-linked immunosorbent assay. Additionally, mRNA expression was assessed by quantitative PCR.ResultsAfter 7 days, phase-contrast microscopy revealed cell aggregation of SMSC in coculture with CHDR. Afterwards, cells formed spheres and lost adherence. However, this phenomenon was not observed when culturing SMSC alone. Fluorescence labeling showed concurrent collagen type II expression. Addition of transforming growth factor beta (TGFβ) to the cocultures induced SMSC aggregation in less time and with higher intensity. Additionally, alcian blue staining demonstrated enhanced glycosaminoglycan expression around SMSC aggregates after 1 and 2 weeks. Although TGFβ mRNA was expressed in all SMSC, the protein was measured with constantly increasing levels over 21 days only in supernatants of the cocultures. Considering the enhanced mRNA levels following supplementation with TGFβ, a positive feedback mechanism can be supposed. In line with the development of a chondrogenic phenotype, aggrecan mRNA expression increased after 7 and 14 days in the cocultures with and without TGFβ. Coculture conditions also amplified collagen type II mRNA expression after 2 weeks without and already after 1 week with TGFβ. There was no difference in collagen type I and type X expression between SMSC alone and the coculture with CHDR. Expression of both collagens increased following addition of TGFβ. mRNA data correlated with the intensity of immunofluorescence staining.ConclusionsParacrine effects of CHDR induce a chondrogenic phenotype in SMSC possibly mimicking joint homeostasis. Coculture approaches may lead to a better understanding of cellular interactions with potential implications for cartilage repair procedures.

Infrapatellar Fat Pad Stem Cells: From Developmental Biology to Cell Therapy

“…A possible strategy in the field of cartilage repair is to use cocultures of different cell types, usually with articular chondrocytes, instead of choosing only one [ 92 ]. In this regard, several promising attempts have been performed with bone-marrow [ 93 – 96 ], synovium [ 97 ], and adipose-derived [ 96 , 98 ] stem cells. IFP stem cells also present an enhanced chondrogenic potential when cocultured with articular chondrocytes [ 99 ] and most especially when cocultured in a structured manner (i.e., on top of the articular chondrocytes), instead of homogeneously mixing them, in an attempt to recapitulate the zonal characteristics of a progenitor population on top of the native articular cartilage [ 100 ].…”

Fargesin ameliorates osteoarthritis via macrophage reprogramming by downregulating MAPK and NF-κB pathways

“…Activated synovial macrophages showed that enhanced in ammation mediated the chondrocyte microenvironment and promoted MMP accumulation, which resulted in degradation of extracellular matrix (ECM). [28][29][30] Consistently, ECM degradation acted as a DAMP, stimulating macrophage activation and increasing synovial in ammation, which resulted in positive feedback regulation of in ammation and cartilage degradation. Samavedi [31] et al demonstrated that chondrocytes co-cultured with macrophage activation expressed signi cantly enhanced in ammatory factors, resulting in ECM degradation.…”

Concise Review: Mesenchymal Stem Cells for Functional Cartilage Tissue Engineering: Taking Cues from Chondrocyte-Based Constructs

“…Chondrocytes have been reported to secrete a range of soluble factors that promote chondrogenesis of MSCs during in vitro culture, regulating matrix remodeling, cell proliferation, and synthesis of extracellular matrix components by stem cells. For example, when combined in close proximity, mixed cell culture systems produced engineered cartilage with increased mechanical properties (Young's modulus and dynamic modulus), GAG levels, and collagen content . Interestingly, these chondrocytes were also able to decrease the deposition of collagen X , a marker of MSC hypertrophy, perhaps by secreting PTHrP .…”

“…It is important to note that a more thorough characterization of the influence of these two cell systems on one another is necessary to fully realize the potential of coculture systems. While a number of studies have found positive effects from chondrocytes on MSC chondrogenic differentiation , other studies have found that the beneficial effects are due to the trophic role of MSCs in stimulating chondrocyte proliferation and matrix deposition . Notably, using a xenogenic system and species‐specific gene expression analysis to determine the contribution of various cell populations to cartilage formation, Wu et al showed that following coculture, micromass pellets contained predominantly DNA from the species of origin of the primary chondrocytes, indicating an overgrowth of chondrocytes or loss of MSCs during the culture period.…”