Evaluation of engraftable human pluripotent stem cell-derived chondrocytes in a long-term preclinical pig model of focal cartilage injury

Purpose: The pathogenesis of OA often begins from an injury to articular cartilage, which establishes chronic, low-grade inflammation that eventually promotes matrix degradation leading to the destruction of cartilage. Currently, there are no agents that efficiently slow or inhibit this process. Pluripotent stem cell-derived chondrocytes (PDC) represent a promising new tool for cartilage repair, but in vivo, speci- fication of these cells remains unclear. Methods: Detailed molecular analysis of human primary articular chondrocytes was carried out at embryonic, fetal, adolescent and adult stages. Human pluripotent feeder-free stem cell line (hES17) was propagated and differentiated using protocols previously established in our lab. Generated PDCs were evaluated using single-cell RNA- sequencing to assess cellular heterogeneity and purity. Full thickness, 6- mm in diameter, critical size cartilage lesions were generated in articular cartilage of male Yucatan minipigs. In pig model, PDC were transplanted using 2 formulations: cell aggregates (chondrospheres) at a high dose (group 1), and a low dose (1 group 2), and cells on collagen 1/ 3 membrane at a high dose (group 3), and a low dose (group 4). Control groups included vehicle only (fibrin glue) and clinical grade collagen 1/3 membrane transplanted with no cells on it. Analysis was performed 1 and 6 months after transplantation using Mach 1 bio-indenter (Biomomentum, Canada), immunohistochemistry (IHC), flow cytometry, PCR and routine histology. Detection of human cells was carried out using human specific TERT PCR assay, CD29 antibody ( flow cytometry) or Ku80 (IHC). Statistical methods: Student t-test, one-way ANOVA followed by the Newman-Keuls test. Results: Our studies have shown that transcriptional, epigenetic and functional signatures of PDC closely mimic their primary counterparts, but PDC remain immature in vitro. Single cell sequencing showed that more than 90% of PDCs expressed high levels of cartilage specific markers. Our data also demonstrated that implanted human PDC engrafted and generated human articular cartilage tissue in pig joints. After 1 month (N =4 per group), analysis of engrafted tissue clearly showed the presence of human cells post transplantation as detected by both FACS and IHC staining using human specific antibodies. High dose membrane formulation demonstrated the best repair potential as shown by both biomechanical and histological assessment. Defects repaired with low dose formulations and high dose chondrospheres showed some islands of cartilage tissue formation in the defect area mixed with fibrotic tissue and also highly vascularized loose connective tissue. At 6 months after transplantation (n =5 per group) lesions treated with high dose membrane formulation showed significantly better repair compared to control groups. IHC analysis using antibodies against PRG4/lubricin and other markers showed the presence of all 4 populations of zonally organized human chondrocytes in pig cartilage lesions. Safranin O staining and IHC for cartilage markers collagen type 2 and SOX9 showed the presence of human cartilage tissue in pig defects minimally expressing collagens type 1 and 10. In contrast, control animals healed cartilage defects with a highly disorganized fibrotic tissue marked by collagen type 1. No signs of neoplastic growth were detected in any of the transplanted pig joints as well as the liver, kidney, or lung tissues. Conclusions: Transplanted human PDC enhance repair in Yucatan minipig model of cartilage injury and demonstrate clear ability to form human cartilage cells in vivo. The study showed that although some human cells were present in the site of injury 6 months post-transplantation the vast majority of repaired tissue was composed of pig cells. These findings indicate a prominent paracrine effect of implanted PDC constructs in addition to direct integration of implanted human chondrocytes into pig tissue. Overall, current study is the first to demonstrate long-term safety and ef ficacy of engraftable allogenic mass-produced pluripotent stem cell-derived chondrocytes in preclinical large animal model of articular cartilage repair.