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Co-culture Aids Robust Cartilage Differentiation



"Coculture-Driven Mesenchymal Stem Cell-Differentiated Articular Chondrocyte-Like Cells Support Neocartilage Development"

Mesenchymal stem cells (MSCs) are an attractive source for repair and regeneration of tissue or organ defects. Transplantation of MSCs into defective knee joints to restore cartilage function has yielded some success, although they have yet to pass the pre-clinical and phase I stages towards proper therapeutic use (Koga et al.). A greater understanding of in vivo MSC differentiation mechanisms could aid the widespread use of MSCs through the precise control of cell lineage during in vitro differentiation. Several bioactive agents, such as transforming growth factor-b (TGF-b), insulin-like growth factor-1 (IGF-1), bone morphogenetic protein-2 (BMP-2), and basic fibroblast growth factor (FGF-2), are essential for the chondrogenic differentiation of MSCs (Heng et al.). The mode of delivery of these factors to MSCs has been scrutinised using gradual delivery through means such as gene therapy (Pagnotto et al.) and polymeric vehicles for molecule release (Macdonald et al.and Shah et al.) which have been posited to more closely replicate in vivo conditions (Macdonald et al.). However, an easier method may be the co-culture of MSCs with primary chondrocytes which would secrete proteins such as TGF-b, IGF-1, BMP-2 and FGF-2 slowly over time at physiological concentrations. This has now been studied in detail by researchers from the laboratory of Gilda A. Barabino at the Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA and published in Stem Cells Translational Medicine. The researchers found that co-culture mediated differentiation of MSCs with chondrocytes led to the development of robust neocartilage in a three-dimensional agarose system that resisted hypertrophic maturation and calcification (Yang, Lee and Barabino).

Non-contact co-culture systems were set up using bone marrow derived-primary MSC monolayers with chondrocytes grown in transwell inserts which allowed for factor diffusion but not cell migration. Initial experiments used differing concentrations of chondrocytes with MSCs grown in serum-free, growth factor free inductive medium. Low concentrations of chondrocytes relative to MSCs (7:1 ratio) led to MSCs with a flat irregular morphology with cells spread over the growth substrate, whereas MSCs grown in medium (31:1 and 15:1) and high (100:1 and 63:1) chondrocyte to MSC ratios formed aggregates of different sizes. MSCs harvested from these differing systems were analysed for gene expression of chondrogenic markers (type II collagen, aggrecan, and SOX9). Both collagen II and aggrecan levels in the high and medium chondrocyte systems were significantly higher than MSCs grown with no chondrocytes as control, with gene expression levels generally showing a positive correlation with chondrocyte number. SOX9 expression was increased in all but the 7:1 system when compared to both the MSC only and the chondrocyte only control systems. Analysis of osteogenic markers (type I collagen and RUNX2) found no increase in any of the co-culture systems while hypertrophic markers (type X collagen and MMP-13) were elevated only in the high chondrocyte system when compared to MSC control.

MSC surface markers (CD166 and CD44) showed an inverse correlation with increased chondrocyte number, moving from 99.3% to 9.2% for CD166 and 98.6% to 3.54% respectively when the chondrocyte to MSC ratio was changed from low (7:1) to high (100:1). This correlated with a change in cell size and granularity; moving away from the large granular MSC phenotype of the cells which had fibroblast-like spindle shape that is commonly observed during the MSC expansion processes (Soleimani and Nadri), to a phenotype of smaller cell size and lower granularity, which formed chondrocyte-like clusters.

Of note were the cells grown in the 63:1 ratio system which yielded an MSC-differentiated cell population that resembled articular chondrocytes in morphology, phenotype, and behavior and were used for neocartilage formation experiments in comparison with primary chondrocytes. For this analysis, cells were encapsulated in agarose hydrogels and cell laden disks of these were cultivated in a regular chondrocyte medium. The relative stiffness/compressive strength of both chondrocyte and co-cultured MSC constructs were close to the level of cell-free agarose discs and both were increased to a higher level of stiffness/compressive strength at 2 weeks, and at 4 weeks the co-cultured MSCs demonstrated a stronger compressive strength compared with chondrocyte cultures. This was associated with an increase in increased cell number and proliferation rate of cells in the co-cultured MSC constructs. Sulfated glycosaminoglycan (GAG) deposition, typically observed in cartilage, although lower in co-cultured MSC constructs at early time points as compared to chondrocytes, rose throughout the 28 day period to a higher level than that observed for the chondrocytes. Collagen accumulation was higher at both day 14 and 28 for the co-cultured MSC construct (mostly collagen II) while collagen synthetic activity was higher throughout in the co-cultured MSC construct.

Alkaline phosphatase (ALP) activity, which facilitates endochondral bone formation (Steck et al.) and is correlated to the hypertrophic potential of cells within engineered cartilage was subsequently analysed. While ALP levels rose over time in both constructs, the co-cultured MSC construct did so at a lower rate than the chondrocyte construct, although neither construct showed evidence of mineral deposition as measured by von Kossa staining. CD44, which is reduced in differentiating MSCs and is a receptor for hyaluronan that highly relates to cartilage tissue properties (Culty et al.and Knudson and Knudson), had a significantly reduced expression level in the co-cultured MSC construct compared to the chondrocyte construct at day 0 but increased in both constructs over time. At 28 days, the co-cultured MSC construct contained higher levels of CD44 than the chondrocyte construct although both stained intensely for CD44.

Similar studies have been published on co-cultivation mediated chondrogenesis (See paper for extended references), but this study is the first to be able to analyse signals only from a primary MSC population as direct contact between the cells was avoided. Further, the elimination of serum and exogenous stimulators allowed that the differentiation process was driven mainly by paracrine signaling. Together the researchers have demonstrated that this process allowed for bone marrow derived MSCs to efficiently differentiate into a cell population that highly mimics articular chondrocytes therefore providing a basis to improve protocols for MSC differentiation toward the development of cartilage tissue replacements suitable for implantation.



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Study originally appeared in Stem Cells Translational Medicine.

Correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.