The meniscus is a fibrocartilaginous tissue comprised of an inner avascular and an outer vascular region. To distinguish it from other mesenchymal tissues, studies have uncovered a set of genes to define the meniscus phenotype. Using the following genes - IGF-BP3, R-Spondin-2, CILP2, CHAD, CSRP2, ID2. Dermatopontin, Collagen XV - we investigated their specificity on meniscus tissue, and on avascular and vascular meniscus cells in monolayer and pellet culture.
Human meniscus tissue (n = 3) had a portion snap frozen, whilst remaining tissue was split into avascular and vascular regions for cell isolation. Avascular and vascular meniscus cells were cultured in either a 20% oxygen or 2% oxygen incubator. At passage 1, QIAzol was applied to monolayer cells at each oxygen tension and then frozen. At passage 2, avascular and vascular meniscus pellets were created and cultured for 21 days at each oxygen tension using previously described protocols. RNA was isolated from tissue, cells and pellets using QIAzol method and subsequent qPCR analysis for stated genes was performed using a Biorad CFX96 system.
In meniscus tissue, there was elevated expression levels of all selected genes, with a significant upregulation in CLIP2, CSRP2, Dermatopontin and collagen XV (*p < 0.05). In monolayer, all genes were downregulated, whereas upon pellet redifferentiation, only IGF-BP3, CHAD (*p > 0.05) and Dermatopontin were upregulated in avascular and vascular meniscus cells. There was no significant difference in gene expression with respect to oxygen tension in monolayer and pellets for both meniscus cell types.
The selected genes were all upregulated in meniscus tissue. Monolayer expansion led to downregulation of all genes, whilst subsequent pellet redifferentiation resulted in upregulation of only IGF-BP3, CHAD and Dermatopontin. Future studies should identify optimal in vitro conditions to maintain expression of meniscus-specific genes, thus preventing phenotype drift during cell expansion.