Collagen X Is Dispensable for Hypertrophic Differentiation and Endochondral Ossification of Human iPSC-Derived Chondrocytes

Collagen X is a non-fibril collagen produced by hypertrophic chondrocytes and was believed to associate with the calcification process of growth plate cartilage. The homozygous loss of Col10a1 gene in mice, however, demonstrated no remarkable effects on growth plate formation or skeletal development. To investigate the role of collagen X in human chondrocytes, we established human induced pluripotent stem cells (hiPSCs) with heterozygous (COL10A1⁺/⁻) or homozygous (COL10A1⁻/⁻) deletions of COL10A1 gene using the dual sgRNA CRISPR/Cas9 system. Several mutant clones were established and differentiated into hypertrophic chondrocytes by a previously reported 3D induction method. No remarkable differences were observed during the differentiation process between parental and mutant cell lines, which differentiated into cells with features of hypertrophic chondrocytes, indicating that collagen X is dispensable for the hypertrophic differentiation of human chondrocytes in vitro. To investigate the effects of collagen X deficiency in vivo, chondrocyte pellets at the proliferating or prehypertrophic stage were transplanted into immunodeficient mice. Proliferating pellet-derived tissues demonstrated the zonal distribution of chondrocytes with the transition to bone tissues mimicking growth plates, and the proportion of bone tended to be larger in COL10A1⁻/⁻ tissues. Prehypertrophic pellet-derived tissues produced trabecular bone structures with features of endochondral ossification, and there was no clear difference between parental- and mutant-derived tissues. A transcriptome analysis of chondrocyte pellets at the hypertrophic phase showed a lower expression of proliferating-phase genes and a higher expression of calcification-phase genes in COL10A1⁻/⁻ pellets compared with parental cell pellets. These in vitro and in vivo data suggested that collagen X is dispensable for the hypertrophic differentiation and endochondral ossification of human iPSC-derived chondrocytes, though it may facilitate the differentiation process. Thus, COL10A1⁻/⁻ iPSC lines are useful for investigating the physiological role of collagen X in chondrocyte differentiation. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research

Oxidative phosphorylation is a pivotal therapeutic target of fibrodysplasia ossificans progressiva

酸化的リン酸化の抑制がFOPの新たな治療法に繋がる可能性. 京都大学プレスリリース. 2024-03-11.Heterotopic ossification (HO) is a non-physiological bone formation where soft tissue progenitor cells differentiate into chondrogenic cells. In fibrodysplasia ossificans progressiva (FOP), a rare genetic disease characterized by progressive and systemic HO, the Activin A/mutated ACVR1/mTORC1 cascade induces HO in progenitors in muscle tissues. The relevant biological processes aberrantly regulated by activated mTORC1 remain unclear, however. RNA-sequencing analyses revealed the enrichment of genes involved in oxidative phosphorylation (OXPHOS) during Activin A–induced chondrogenesis of mesenchymal stem cells derived from FOP patient–specific induced pluripotent stem cells. Functional analyses showed a metabolic transition from glycolysis to OXPHOS during chondrogenesis, along with increased mitochondrial biogenesis. mTORC1 inhibition by rapamycin suppressed OXPHOS, whereas OXPHOS inhibitor IACS-010759 inhibited cartilage matrix formation in vitro, indicating that OXPHOS is principally involved in mTORC1-induced chondrogenesis. Furthermore, IACS-010759 inhibited the muscle injury–induced enrichment of fibro/adipogenic progenitor genes and HO in transgenic mice carrying the mutated human ACVR1. These data indicated that OXPHOS is a critical downstream mediator of mTORC1 signaling in chondrogenesis and therefore is a potential FOP therapeutic target

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Taha Toros Arşivi, Dosya Adı: Galatasarayİstanbul Kalkınma Ajansı (TR10/14/YEN/0033) İstanbul Development Agency (TR10/14/YEN/0033

Differentiation of hypertrophic chondrocytes from human iPSCs for the in vitro modeling of chondrodysplasias

iPS細胞から肥大軟骨細胞への誘導法を確立し、成長板疾患の病態再現に成功. 京都大学プレスリリース. 2021-02-26.Reprogramming children's cells to study cartilage diseases. 京都大学プレスリリース. 2021-02-26.Chondrodysplasias are hereditary diseases caused by mutations in the components of growth cartilage. Although the unfolded protein response (UPR) has been identified as a key disease mechanism in mouse models, no suitable in vitro system has been reported to analyze the pathology in humans. Here, we developed a three-dimensional culture protocol to differentiate hypertrophic chondrocytes from induced pluripotent stem cells (iPSCs) and examine the phenotype caused by MATN3 and COL10A1 mutations. Intracellular MATN3 or COL10 retention resulted in increased ER stress markers and ER size in most mutants, but activation of the UPR was dependent on the mutation. Transcriptome analysis confirmed a UPR with wide-ranging changes in bone homeostasis, extracellular matrix composition, and lipid metabolism in the MATN3 T120M mutant, which further showed altered cellular morphology in iPSC-derived growth-plate-like structures in vivo. We then applied our in vitro model to drug testing, whereby trimethylamine N-oxide led to a reduction of ER stress and intracellular MATN3

A de novo dominant-negative variant is associated with OTULIN-related autoinflammatory syndrome

稀少遺伝性自己炎症性疾患: OTULIN関連自己炎症症候群の新たな病態を解明～既報の疾患に新たな視点を追加し、未診断患者の診断や炎症・細胞死研究の進展に期待～. 京都大学プレスリリース. 2024-04-25.OTULIN-related autoinflammatory syndrome (ORAS), a severe autoinflammatory disease, is caused by biallelic pathogenic variants of OTULIN, a linear ubiquitin-specific deubiquitinating enzyme. Loss of OTULIN attenuates linear ubiquitination by inhibiting the linear ubiquitin chain assembly complex (LUBAC). Here, we report a patient who harbors two rare heterozygous variants of OTULIN (p.P152L and p.R306Q). We demonstrated accumulation of linear ubiquitin chains upon TNF stimulation and augmented TNF-induced cell death in mesenchymal stem cells differentiated from patient-derived iPS cells, which confirms that the patient has ORAS. However, although the de novo p.R306Q variant exhibits attenuated deubiquitination activity without reducing the amount of OTULIN, the deubiquitination activity of the p.P152L variant inherited from the mother was equivalent to that of the wild-type. Patient-derived MSCs in which the p.P152L variant was replaced with wild-type also exhibited augmented TNF-induced cell death and accumulation of linear chains. The finding that ORAS can be caused by a dominant-negative p.R306Q variant of OTULIN furthers our understanding of disease pathogenesis

A Japanese predicate argument structure analysis using decision lists

Maintaining high annotation consistency in large corpora is crucial for statistical learning; however, such work is hard, especially for tasks containing semantic elements. This paper describes predi-cate argument structure analysis using transformation-based learning. An advan-tage of transformation-based learning is the readability of learned rules. A dis-advantage is that the rule extraction pro-cedure is time-consuming. We present incremental-based, transformation-based learning for semantic processing tasks. As an example, we deal with Japanese pred-icate argument analysis and show some tendencies of annotators for constructing a corpus with our method.

Density-dependent induction of TNF-α release from human monocytes by immobilized P-selectin

AbstractP-selectin purified from human platelets, when immobilized on a solid surface, induced monocytes to release tumor necrosis factor-α (TNF-α). The induction of TNF-α release was dependent on the concentration of P-selectin used for the immobilization, and the maximal stimulation was observed when the plate was coated with 0.3 μg/ml of P-selectin. Use of either a higher or a lower concentration of P-selectin for the plate-coating was found to elicit less TNF-α release, although the higher concentration of P-selectin caused a stronger adhesion of HL-60 leukemic cells. The expression of mRNA for TNF-α roughly paralleled the TNF-α secretion, as assessed by RT-PCR. These results indicate that monocytes are activated by immobilized P-selectin in a density-dependent manner