Chondrogenesis

Transforming growth factor-βs (TGF-βs) and bone morphometric proteins (BMPs) belong to the TGF-β superfamily and perform essential functions during osteoblast and chondrocyte lineage commitment and differentiation, skeletal development, and homeostasis. TGF-βs and BMPs transduce signals through SMAD-dependent and -independent pathways; specifically, they recruit different receptor heterotetramers and R-Smad complexes, resulting in unique biological readouts. BMPs promote osteogenesis, osteoclastogenesis, and chondrogenesis at all differentiation stages, while TGF-βs play different roles in a stage-dependent manner. BMPs and TGF-β have opposite functions in articular cartilage homeostasis. Moreover, TGF-β has a specific role in maintaining the osteocyte network. The precise activation of BMP and TGF-β signaling requires regulatory machinery at multiple levels, including latency control in the matrix, extracellular antagonists, ubiquitination and phosphorylation in the cytoplasm, nucleus-cytoplasm transportation, and transcriptional co-regulation in the nuclei. This review weaves the background information with the latest advances in the signaling facilitated by TGF-βs and BMPs, and the advanced understanding of their diverse physiological functions and regulations. This review also summarizes the human diseases and mouse models associated with disordered TGF-β and BMP signaling. A more precise understanding of the BMP and TGF-β signaling could facilitate the development of bona fide clinical applications in treating bone and cartilage disorders.

The nuclear factor-κB (NF-κB) is a transcription factor that regulates the expression of genes that control cell proliferation and apoptosis, as well as genes that respond to inflammation and immune responses. There are two means of NF-κB activation: the classical pathway, which involves the degradation of the inhibitor of κBα (IκBα), and the alternative pathway, which involves the NF-κB-inducing kinase (NIK, also known as MAP3K14). The mouse growth plate consists of the resting zone, proliferative zone, prehypertrophic zone, and hypertrophic zone. The p65 (RelA), which plays a central role in the classical pathway, is expressed throughout the cartilage layer, from the resting zone to the hypertrophic zone. Inhibiting the classical NF-κB signaling pathway blocks growth hormone (GH) or insulin-like growth factor (IGF-1) signaling, suppresses cell proliferation, and suppresses bone morphogenetic protein 2 (BMP2) expression, thereby promoting apoptosis. Since the production of autoantibodies and inflammatory cytokines, such as tumor necrosis factor-α (TNFα), interleukin (IL)-1β, IL-6, and IL-17, are regulated by the classical pathways and are increased in rheumatoid arthritis (RA), NF-κB inhibitors are used to suppress inflammation and joint destruction in RA models. In osteoarthritis (OA) models, the strength of NF-κB-activation is found to regulate the facilitation or suppression of OA. On the other hand, RelB is involved in the alternative pathway, and is expressed in the periarticular zone during the embryonic period of development. The alternative pathway is involved in the generation of chondrocytes in the proliferative zone during physiological conditions, and in the development of RA and OA during pathological conditions. Thus, NF-κB is an important molecule that controls normal development and the pathological destruction of cartilage.

The therapeutic application of human induced pluripotent stem cells (hiPSCs) for cartilage regeneration is largely hindered by the low yield of chondrocytes accompanied by unpredictable and heterogeneous off-target differentiation of cells during chondrogenesis. Here, we combine bulk RNA sequencing, single cell RNA sequencing, and bioinformatic analyses, including weighted gene co-expression analysis (WGCNA), to investigate the gene regulatory networks regulating hiPSC differentiation under chondrogenic conditions. We identify specific WNTs and MITF as hub genes governing the generation of off-target differentiation into neural cells and melanocytes during hiPSC chondrogenesis. With heterocellular signaling models, we further show that WNT signaling produced by off-target cells is responsible for inducing chondrocyte hypertrophy. By targeting WNTs and MITF, we eliminate these cell lineages, significantly enhancing the yield and homogeneity of hiPSC-derived chondrocytes. Collectively, our findings identify the trajectories and molecular mechanisms governing cell fate decision in hiPSC chondrogenesis, as well as dynamic transcriptome profiles orchestrating chondrocyte proliferation and differentiation.

Campomelic Dysplasia (CD) is a rare congenital disease caused by haploinsufficiency (HI) in SOX9. Patients with CD typically present with skeletal abnormalities and 75% of them have sex reversal. In this study, we use CRISPR/Cas9 to generate a human induced pluripotent stem cell (hiPSC) model from a heathy male donor, based on a previously reported SOX9 splice site mutation in a CD patients. This hiPSCs-derived chondrocytes from heterozygotes (HT) and homozygotes (HM) SOX9 mutation carriers showed significant defects in chondrogenesis. Bulk RNA profiling revealed that the BMP-SMAD signaling pathway, ribosome-related, and chromosome segregation-related gene sets were altered in the HT chondrocytes. The profile also showed significant noggin upregulation in CD chondrocytes, with ChIP-qPCR confirming that SOX9 binds to the distal regulatory element of noggin. This suggests SOX9 plays a feedback role in the BMP signaling pathway by modulating noggin expression rather than acting solely as a downstream regulator. This provides further insights into its dosage sensitivity in chondrogenesis. Overexpression of SOX9 showed promising results with improved sulfated glycosaminoglycans (GAGs) aggregation and COL2A1 expression following differentiation. We hope this finding could provide a better understanding of the dosage-dependent role of SOX9 in chondrogenesis and contribute to the development of improved therapeutic targets for CD patients.