Time- and cell-specific activation of BMP signaling restrains chondrocyte hypertrophy
Differentiated chondrocytes from populations of human bone marrow stromal cells including skeletal stem cells (hBMSCs/SSCs) and induced pluripotent stem cells (hiPSCs) have the potential to restore damaged cartilage, yet chondrocyte hypertrophy is a barrier for translational therapy. However, hBMSCs/SSCs attached to a hyaluronic acid-coated fibrin microbead scaffold (HyA-FMBs) produce cartilage that resists hypertrophy. We show that HyA-FMBs initially suppress Bone Morphogenic Protein (BMP) signaling early in chondrogenic differentiation of hBMSCs/SSCs, then restore BMP signaling in a stable chondrogenic population enriched for Insulin-like Growth Factor Binding Protein 5 and Matrix Gla Protein. We subsequently developed a serumfree hiPSC differentiation strategy that inhibited, then activated BMP signaling in a purified subpopulation derived from ?chondrospheroids? that exhibited stable expression of Type II Collagen, Aggrecan (ACAN), and Lubricin (PRG4) and minimal e  xpression of Type X Collagen. Overall, timed, and selective BMP signaling restrains chondrocyte hypertrophy, supporting the potential of chondrospheroid-derived cells for clinical utility in osteoarthritis. Overall design: Gene expression profiling of human bone marrow stromal cells/skeletal stem cells (hBMSCs/SSCs) attached to fibrin microbead scaffolds coated with hyaluronic acid (HyA-FMBs). Differentiated chondrocytes from human bone marrow stromal cells including skeletal stem cells (hBMSCs/SSCs) have the potential to permanently restore damaged cartilage in arthritic joints, yet chondrocyte hypertrophy is a major barrier for translational therapy.  With chondrogenic differentiation, hBMSCs/SSCs undergo hypertrophy in vitro and mineralization in vivo, leading to inferior fibrocartilage and bone formation.  However, hBMSCs/SSCs attached to a fibrin microbead scaffold coated with hyaluronic acid (HyA-FMBs) produce hyaline-like cartilage for up to 28 weeks in vivo.  Therefore, the goal of the present study was to examine the signaling pathways that govern the development of hypertrophic-resistant chondrocytes using the HyA-FMB model system.  hBMSCs/SSCs attached to HyA-FMBs formed organoids during chondrogenic differentiation in vitro, and these organoids were digested at days 1, 3, 5, and 10 for single-cell RNA sequencing.  hBMSCs/SSCs attached to HyA-FMBs were also transplanted, and tissues were harvested after 8 weeks for single-cell RNA sequencing. Passage 2 hBMSC/SSCs from three donors were used in these studies.  Organoids generated from hBMSCs/SSCs during days 1, 3, 5, and 10 of chondrogenic differentiation were dissociated and analyzed by scRNAseq using 10x Genomics platform.  Naïve hBMSCs/SSCs (no chondrogenic differentiation) were included as controls. Transplanted hBMSCs/SSCs attached to HyA-FMBs were also dissociated after 8 weeks for comparison. Gene expression profiling of human induced pluripotent stem cell (hiPSC)-derived chondrocytes supplemented with TGFb and BMP ligands. Human induced pluripotent stem cell (hiPSC)-derived sclerotome differentiated in chondrogenic medium with TGFb forms organoids that retain strong Collagen I expression, scattered Aggrecan expression, and absence of PRG4 expression.  However, hiPSC-derived sclerotome differentiated with BMP2 and GDF5 (i.e., in the formation of chondrospheroids) forms cartilage tissues with homogenous Aggrecan and Collagen 2 expression with uniform PRG4 expression, which remains expressed upon transplantation at the joint site in NSG mice and SRG rats.  To confirm the role of BMP activation in strengthening this chondrogenic phenotype, we performed single cell RNA sequencing of tissues exposed to TGFb alone or supplemented with BMP ligands. contributor: NIDCD/NIDCR Genomics and Computational Biology Core