Distinct Lineage Derived Chondrocytes From Isogenic Human Peripheral Blood Induced Pluripotent Stem Cells For Articular Cartilage Regeneration
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE152080 | GEO | 2021/05/26
REPOSITORIES: GEO
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Project description:Objectives Traditional approaches to study Progressive Pseudorheumatoid Arthropathy of Childhood (PPAC) failed to uncover how loss-of-function mutations in Wnt inducible secreted protein 3 (WISP3) cause premature cartilage failure in children. We developed a human induced pluripotent stem cell (hiPSC)-based model of cartilage development to elucidate pathological changes in WISP3-deficient articular cartilage tissues compared to WISP3-sufficient isogenic cartilage tissues. Methods We generated iPSCs from 2 patients with PPAC, and we corrected the disease-causing mutation in one of these lines using CRISPR/Cas9. We also created a WISP3-knockout human embryonic stem cell line. We generated articular cartilage tissues from these hPSCs by established directed differentiation methods. Bioinformatic analyses were used to identify differentially expressed genes (DEGs) and cell type abundances, which were validated by quantitative RT-qPCR and in situ hybridization. Results WISP3-deficient articular cartilage tissues exhibited significantly different transcriptomic profiles and cellular composition compared with their isogenic controls. WISP3-deficient cartilage transcriptomes exhibited enriched biological processes such as TGFb signaling and epithelial to mesenchymal transition. We validated increased expression of TGFb-related genes in WISP3-deficient cartilage, and determined that the abundance of chondrocyte subtypes was altered compared with isogenic controls. Conclusions We developed a robust and reproducible model of iPSC-derived cartilage development to better understand the role of WISP3 in cartilage biology, and the pathology of cartilage failure associated with loss of WISP3 in patients with PPAC. We identified several altered biological processes and signaling pathways in WISP3-deficient cartilage that can be validated in the future with additional patient-derived cell lines, or large animal models.
2023-08-24 | GSE230195 | GEO
Project description:ObjectiveOsteoarthritis is a degenerative joint disease whose molecular mechanism is currently unknown. Wnt/beta-catenin signaling has been demonstrated to play a critical role in the development and function of articular chondrocytes. To determine the role of beta-catenin signaling in articular chondrocyte function, we generated Col2a1-ICAT-transgenic mice to inhibit beta-catenin signaling in chondrocytes.MethodsThe expression of the ICAT transgene was determined by immunostaining and Western blot analysis. Histologic analyses were performed to determine changes in articular cartilage structure and morphology. Cell apoptosis was determined by TUNEL staining and the immunostaining of cleaved caspase 3 and poly(ADP-ribose) polymerase (PARP) proteins. Expression of Bcl-2, Bcl-x(L), and Bax proteins and caspase 9 and caspase 3/7 activities were examined in primary sternal chondrocytes isolated from 3-day-old neonatal Col2a1-ICAT-transgenic mice and their wild-type littermates and in primary chicken and porcine articular chondrocytes.ResultsExpression of the ICAT transgene was detected in articular chondrocytes of the transgenic mice. Associated with this, age-dependent articular cartilage destruction was observed in Col2a1-ICAT-transgenic mice. A significant increase in cell apoptosis in articular chondrocytes was identified by TUNEL staining and the immunostaining of cleaved caspase 3 and PARP proteins in these transgenic mice. Consistent with this, Bcl-2 and Bcl-x(L) expression were decreased and caspase 9 and caspase 3/7 activity were increased, suggesting that increased cell apoptosis may contribute significantly to the articular cartilage destruction observed in Col2a1-ICAT-transgenic mice.ConclusionInhibition of beta-catenin signaling in articular chondrocytes causes increased cell apoptosis and articular cartilage destruction in Col2a1-ICAT- transgenic mice.
| S-EPMC2667964 | biostudies-literature
Project description:Gene expression profiling of primary mouse articular chondrocyte treated with interleukin-1β. In this study, we have attempted to explore the effects of interleukin-1β on mouse transcriptome and have identified numerous genes which are involved in osteoarthritis pathogenesis.
2017-10-10 | GSE104793 | GEO
Project description:Osteoarthritis (OA) impacts hundreds of millions of people worldwide, with those affected incurring significant physical and financial burdens. Injury to the articular surface is a major contributing risk factor for the development of OA. Current cartilage repair strategies are moderately effective at reducing pain but often replace damaged tissue with biomechanically inferior fibrocartilage. Here we describe the development, transcriptomic ontogenetic characterization and quality assessment at the single cell level, as well as the scaled manufacturing of an allogeneic pluripotent stem cell-derived articular chondrocyte formulation that exhibits long-term functional repair of porcine articular cartilage. These results define a new potential clinical paradigm for articular cartilage repair and mitigation of the associated risk of OA.
2021-12-10 | GSE142045 | GEO
Project description:Sonic hedgehog (Shh) is involved in the induction of early cartilaginous differentiation of mesenchymal cells in the limb. We investigated whether Shh could promote redifferentiation of dedifferentiated chondrocytes and have a favorable effect on the regeneration of cartilage. Articular chondrocytes of rats were separated and cultured. The redifferentiation of dedifferentiated chondrocytes transfected with Shh was evaluated using monolayer and pellet culture system. The signaling molecules (Ptc 1, Gli 1 and Sox9) of the hedgehog pathway were investigated. A rat model of articular cartilage defect was used to evaluate cartilage repair after transplantation with dedifferentiated chondrocytes. After Shh gene transfer, the hedgehog pathway was upregulated in dedifferentiated chondrocytes. Real time-PCR and western blot analysis verified the stronger expression of Ptc1, Gli1 and Sox9 in Shh transfected cells. Shh upregulates the Shh signaling pathway and multiple cytokines (bone morphogenetic protein 2 and insulin-like growth factor 1) in dedifferentiated chondrocytes. After transplantation in the joint, histologic analysis of the regenerative tissues revealed that significantly better cartilage repair in rats transplanted with Shh transfected cells. These data suggest that Shh could induce redifferentiation of dedifferentiated chondrocytes through up-regulating Shh signaling pathway, and have considerable therapeutic potential in cartilage repair.
| S-EPMC3922882 | biostudies-literature
Project description:Gene expression profiling of primary mouse articular chondrocyte infected with recombinant adenovirus expressing mouse ZFP36 Ring Finger Protein Like 1(Zfp36l1) or recombinant adenovirus expressing mouse small hairpin RNA of Zfp36l1. In this study, we have attempted to explore the effects of Zfp36l1 gene overexpression or knockdown on mouse transcriptome and have identified numerous genes which are involved in osteoarthritis pathogenesis.
2018-02-14 | GSE110581 | GEO
Project description:Based on our findings that PHD2 is a negative regulator of chondrocyte differentiation and that hypoxia signaling is implicated in the pathogenesis of osteoarthritis, we investigated the consequence of disruption of the Phd2 gene in chondrocytes on the articular cartilage phenotype in mice. Immunohistochemistry detected high expression of PHD2 in the superficial zone (SZ), while PHD3 and HIF-1α (target of PHD2) are mainly expressed in the middle-deep zone (MDZ). Conditional deletion of the Phd2 gene (cKO) in chondrocytes accelerated the transition of progenitors to hypertrophic (differentiating) chondrocytes as revealed by reduced SZ thickness, and increased MDZ thickness, as well as increased chondrocyte hypertrophy. Immunohistochemistry further revealed decreased levels of progenitor markers but increased levels of hypertrophy markers in the articular cartilage of the cKO mice. Treatment of primary articular chondrocytes, in vitro, with IOX2, a specific inhibitor of PHD2, promoted articular chondrocyte differentiation. Knockdown of Hif-1α expression in primary articular chondrocytes using lentiviral vectors containing Hif-1α shRNA resulted in reduced expression levels of Vegf, Glut1, Pgk1, and Col10 compared to control shRNA. We conclude that Phd2 is a key regulator of articular cartilage development that acts by inhibiting the differentiation of articular cartilage progenitors via modulating HIF-1α signaling.
| S-EPMC5368651 | biostudies-literature
Project description:Chondrocyte gene expression was analyzed to study mechanisms involved in the structural and functional adaptation of articular cartilage during postnatal maturation. Transcriptional profiling was used to compare articular chondrocytes between four neonatal and four adult horses. Expressional differences featured matrix proteins and matrix-modifying enzymes reflecting the transition from cartilage growth to cartilage homeostasis. Keywords: articular cartilage, maturation, horse, cDNA microarray
2008-11-10 | GSE10303 | GEO
Project description:Objective: To examine the role of carbonic anhydrase II (CA2 gene), the most efficient isoform in the CA family of zinc-containing metalloenzymes, in chondrocyte metabolism. Methods: CA2 expression was measured in human and mouse osteoarthritic cartilage. RNA-seq was performed in C28/I2 cells in which CA2 was silenced. Normal human chondrocytes were cultured under normoxia (21% oxygen) or hypoxia (2% oxygen) following CA2 knockdown. Cell metabolism was studied by measuring extracellular lactate production, glucose consumption, intracellular ADP/ATP, intracellular pH, and ROS production. Glycolysis was measured using Seahorse XF96 analyzer. The effect of CA2 knockdown on anabolic and catabolic markers was measured following treatment with IL-1. Effects of CA2 was examined on cell proliferation, cell-cycle distribution, colony-formation, and migration. Results: CA2 was highly elevated in human and murine osteoarthritic cartilage. RNA-seq analysis revealed that processes related to glycolysis, apoptosis and TNF signaling were perturbed in cells lacking CA2. CA2 expression was 10-fold higher under hypoxia and its knockdown caused decreased extracellular lactate production, increased ADP/ATP ratio, impaired glycolysis, decreased glycolytic capacity and lowered the expression of glycolysis rate-limiting enzymes, but did not affect intracellular pH and ROS production. CA2 deficiency disturbed chondrocyte anabolic and catabolic equilibrium. CA2 knockdown suppressed chondrocyte migration and proliferation and induced cell-cycle arrest. Conclusion: This study unravels a novel role of CA2 in chondrocyte metabolism and inflammation. These findings indicate that CA2 is required for the maintenance of chondrocyte metabolic homeostasis. Future work is needed to further illuminate the mechanistic and functional role of CA2 in osteoarthritis development.
2022-03-17 | GSE184062 | GEO
Project description:Donor-site morbidity, limited numbers of cells, loss of phenotype during ex vivo expansion, and age-related decline in chondrogenic activity present critical obstacles to the use of autologous chondrocyte implantation for cartilage repair. Chondrocytes from juvenile cadaveric donors may represent an alternative to autologous cells. Hypothesis/The authors hypothesized that juvenile chondrocyte would show stronger and more stable chondrogenic activity than adult cells in vitro and that juvenile cells pose little risk of immunologic incompatibility in adult hosts.Controlled laboratory study.Cartilage samples were from juvenile (<13 years old) and adult (>13 years old) donors. The chondrogenic activity of freshly isolated human articular chondrocytes and of expanded cells after monolayer culture was measured by proteoglycan assay, gene expression analysis, and histology. Lymphocyte proliferation assays were used to assess immunogenic activity.Proteoglycan content in neocartilage produced by juvenile chondrocytes was 100-fold higher than in neocartilage produced by adult cells. Collagen type II and type IX mRNA in fresh juvenile chondrocytes were 100- and 700-fold higher, respectively, than in adult chondrocytes. The distributions of collagens II and IX were similar in native juvenile cartilage and in neocartilage made by juvenile cells. Juvenile cells grew significantly faster in monolayer cultures than adult cells (P = .002) and proteoglycan levels produced in agarose culture was significantly higher in juvenile cells than in adult cells after multiple passages (P < .001). Juvenile chondrocytes did not stimulate lymphocyte proliferation.These results document a dramatic age-related decline in human chondrocyte chondrogenic potential and show that allogeneic juvenile chondrocytes do not stimulate an immunologic response in vivo.Juvenile human chondrocytes have greater potential to restore articular cartilage than adult cells, and may be transplanted without the fear of rejection, suggesting a new allogeneic approach to restoring articular cartilage in older individuals.
| S-EPMC3774103 | biostudies-literature