Role of collagen XXII in the development, maintenance, and repair of articular cartilage

Abstract

Articular cartilage (AC) enables synovial joint movement, but its inefficient repair can lead to osteoarthritis (OA). The mechanisms behind cartilage repair and its inefficiency are not well-understood. Progenitor cells with mesenchymal stromal cell (MSC) properties can be isolated from healthy and OA cartilage and have shown some success in enhancing cartilage repair. However, questions remain about the ideal cell type and the niche environment necessary for proper repair. This thesis investigates potential progenitor cells for AC in developing synovial joints, expressing key markers Lgr5 and Col22a1. LGR5 facilitates Wnt signalling and is a known marker for proliferative adult stem cells. Col22a1 codes for collagen XXII, an extracellular matrix (ECM) protein with an unknown function. Collagen XXII is localized to the superficial layer of AC during joint development, suggesting its role in the ECM niche supporting cells at this layer and their progressive differentiation to articular chondrocytes. To understand the role of collagen XXII in AC formation and maintenance, I studied homozygous mice with a null allele for Col22a1 (Col22a1PLAP/PLAP). Inactivation of Col22a1 did not impair cavitation progress in development or joint formation. However, distinct changes were observed in cell attachment at the superficial layer and cellular content, shape, and organization within the underlying superficial zone. Immunohistological analyses revealed impaired ECM composition of the superficial layer and decreased joint progenitor cell markers (Lgr5, Creb5, Cdh13) and focal adhesion (Intgβ-1). Increased cell proliferation (Ki67) suggests collagen XXII's role in the ECM organization of the superficial layer and cell-matrix interactions that influence progressive differentiation to articular chondrocytes. Nevertheless, AC markers (Prg4 and Cilp) remained unchanged, with the most dramatic change being abnormal meniscus formation. The cause is unclear but may relate to abnormal gait observed in adult Col22a1-null mice. Further studies are needed as gait parameters are complex and Col22a1 is expressed in tendon-muscular junctions. Having established Col22a1-expressing cells' relevance in development, we hypothesized that such cells would have an advantage in joint repair. Previous studies showed positive effects of supplementing interzone cells in AC repair. In the present study, modified H1 ESCs equipped with FailSafeTM and Immunocloaking system (H1-CFS) have been used to eliminate the risk of immune rejection and tumorigenicity. I utilized CRISPR/Cas9 technology to engineer H1-CFS with reporter fluorescent genes (GFP and EBFP) for Lgr5 and Co22a1 expression. Using these cells, I developed a two-phase in vitro differentiation protocol to generate joint progenitor cells (JPCs) expressing these genes. Phase I involved H1 cell differentiation toward a mesoderm lineage, as indicated by reduced pluripotency marker expression (Oct4, Nanog) and increased mesoderm markers (Tbx6, Meox1). Phase II used a combination of GDF5, R-Spondin 2 and 3, and CHIR99021 to promote JPC differentiation. Promising data showed enhanced levels of Lgr5, Col22a1, Creb5, Cdh13, but overall efficiency remains low and requires further optimization. In summary, this research provides novel insights into collagen XXII's function in AC maintenance and its potential role as an ECM niche for JPCs. Optimizing in vitro JPC differentiation could present opportunities for developing therapeutic products for cartilage repair, ensuring safety and universal usage.