Kinetics of Gene Expression Changes in Equine Fetal Interzone and Anlagen Cells Over 14 Days of Induced Chondrogenesis.

Chan Hee Mok,James N Macleod

doi:10.3389/fvets.2021.722324

Abstract

Within developing synovial joints, interzone and anlagen cells progress through divergent chondrogenic pathways to generate stable articular cartilage and transient hypertrophic anlagen cartilage, respectively. Understanding the comparative cell biology between interzone and anlagen cells may provide novel insights into emergent cell-based therapies to support articular cartilage regeneration. The aim of this study was to assess the kinetics of gene expression profiles in these skeletal cell lines after inducing chondrogenesis in culture. Interzone and anlagen cells from seven equine fetuses were isolated and grown in a TGF-β1 chondrogenic inductive medium. Total RNA was isolated at ten time points (0, 1.5, 3, 6, 12, 24, 48, 96, 168, and 336 h), and gene expression for 93 targeted gene loci was measured in a microfluidic RT-qPCR system. Differential transcriptional responses were observed as early as 1.5 h after the initiation of chondrogenesis. Genes with functional annotations that include transcription regulation responded to the chondrogenic stimulation earlier (1.5–96 h) than genes involved in signal transduction (1.5–336 h) and the extracellular matrix biology (3–336 h). Between interzone and anlagen cell cultures, expression levels of 73 out of the 93 targeted genes were not initially different at 0 h, but 47 out of the 73 genes became differentially expressed under the chondrogenic stimulation. While interzone and anlagen cells are both chondrogenic, they display clear differences in response to the same TGF-β1 chondrogenic stimulation. This study provides new molecular insight into a timed sequence of the divergent developmental fates of interzone and anlagen cells in culture over 14 days.

Highlights

IntroductionArticular cartilage develops from interzone located at the presumptive sites of synovial joints within the cartilaginous anlagen of bones
During early fetal skeletogenesis, articular cartilage develops from interzone located at the presumptive sites of synovial joints within the cartilaginous anlagen of bones
By comparing a timed sequence of the cellular response to TGF-β1 induced chondrogenesis between interzone and anlagen cell cultures, the present study investigated how gene expression patterns change over time and molecular details of chondrogenic divergence in these two cell types

Summary

Introduction

Articular cartilage develops from interzone located at the presumptive sites of synovial joints within the cartilaginous anlagen of bones. While anlagen cartilage is transient, progressing through endochondral ossification, articular cartilage remains stable and functions throughout life to facilitate biomechanical load distribution and low friction movement between adjoining bones [1, 2]. Despite the important functional properties of articular cartilage, its intrinsic ability to restore structural defects is limited in mature mammals [3] in stark contrast. Fractured bones repair quite well by recapitulating endochondral ossification; provided the fracture ends are brought together, stabilized, and not compromised by infection or loss of blood supply. Research on the comparative cell biology between interzone and anlagen cells, as well as their developmental chondrogenic pathways may provide novel information relevant to improving mammalian articular cartilage regenerative treatments

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