The temporal transcriptomic signature of cartilage formation

Abstract

Chondrogenesis is a multistep process whereby cartilage progenitor cells generate a tissue with distinct structural and functional properties. Although several approaches to cartilage regeneration rely on the differentiation of implanted progenitor cells, the temporal transcriptomic landscape of in vitro chondrogenesis in different models has not been reported. Using RNA sequencing, we examined the differences between gene expression patterns during early cartilage formation in micromass cultures of embryonic limb bud-derived progenitors. Principal component analysis indicated a progressively different and distinct transcriptome during chondrogenesis. Differentially expressed genes (DEGs) based on pairwise comparisons of samples from consecutive days were classified into clusters and analysed. We confirmed the involvement of the top DEGs in chondrogenic differentiation with pathway analysis. We discovered several chondrogenesis-associated transcription factors and new collagen subtypes not previously linked to cartilage formation. These results provide, for the first time, a detailed insight into the molecular mechanism of in vitro chondrogenesis and provide a new gold standard for the temporal transcriptomic landscape of chondrogenic differentiation that may serve as a platform for new approaches in cartilage tissue engineering.

Isolation and Micromass Culturing of Primary Chicken Chondroprogenitor Cells for Cartilage Regeneration

“…It allows for monitoring the temporal pattern of chondrogenesis in vitro (Takacs et al., 2023). It is also a highly adaptable and well‐characterized model (Takacs et al., 2023) that has been used for assessing a variety of conditions including the application of soluble factors or mechanical stimuli (Rolfe et al., 2022). However, there are some major disadvantages of this method that are worth noting.…”

“…Another unfavorable feature of this method is that the hyaline cartilage which forms in the cell cultures is primarily transient cartilage, which undergoes chondrocyte hypertrophy; essentially recapitulating the main steps of endochondral ossification in the developing limbs. Gene expression profiling supports rapid chondrogenesis and transition to hypertrophy (Rolfe et al., 2022; Takacs et al., 2023). Cartilage formed in micromass cultures does not exhibit the unique organization of articular hyaline cartilage matrix.…”

“…The high seeding density creates a microenvironment which supports chondrogenesis in the micromass cultures and prevents differentiation of the other progenitor cells, such as epithelial or muscle progenitors. In chicken embryo‐derived micromass cultures, overt chondrogenesis is completed in 6 days in culture (Takacs et al., 2023).…”

“…A well‐accepted in vitro model to recapitulate embryonic chondrogenesis is the high density micromass cell culture established from embryonic limb buds (Ahrens et al., 1977; Takacs et al., 2023). Limb bud‐derived chondrogenic progenitor cells aggregate and then spontaneously differentiate into chondroblasts and chondrocytes, which produce hyaline cartilage ECM in micromass cell cultures in vitro (Fig.…”