Molecular characterization of physis tissue by RNA sequencing

Paradise, Christopher R.; Galeano-Garces, Catalina; Galeano-Garces, Daniela; Dudakovic, Amel; Milbrandt, Todd A.; Saris, Daniël B.F.; Krych, Aaron J.; Karperien, Marcel; Ferguson, Gabriel B.; Evseenko, Denis; Riester, Scott M.; Wijnen, André J. van; Larson, A. Noelle

doi:10.1016/j.gene.2018.05.034

Cited by 18 publications

(9 citation statements)

References 39 publications

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“…This protocol mimics the process of chondrocyte differentiation in the growth plate, with cells characteristic of hypertrophic chondrocytes in their morphology as well as their gene expression being obtained after 56 days in 3D culture. Genes that are highly upregulated in the physis but not articular cartilage, such as MATN3 and COL9A2 (Paradise et al, 2018), were expressed in the course of induction, supporting the validity of our model to research disorders of the growth plate.…”

Section: Discussionsupporting

confidence: 61%

Differentiation of Hypertrophic Chondrocytes from Human iPSCs for the In Vitro Modeling of Chondrodysplasias

et al. 2021

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Summary 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.

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Section: Discussionsupporting

confidence: 61%

Differentiation of Hypertrophic Chondrocytes from Human iPSCs for the In Vitro Modeling of Chondrodysplasias

et al. 2021

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“…Differential gene expression analysis was conducted using RPKM values as previously described. 53 Genes with an average RPKM <0.1 in both treatment groups were excluded from the analysis. GO term overlap was conducted using the Compute Overlap tool in the Molecular Signature Database (MSigDB) v6.2 suite on the Gene Set Enrichment Analysis (GSEA) website.…”

Section: Rna Seqmentioning

confidence: 99%

Knockdown of formin mDia2 alters lamin B1 levels and increases osteogenesis in stem cells

et al. 2019

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Nuclear actin plays a critical role in mediating mesenchymal stem cell (MSC) fate commitment. In marrow-derived MSCs, the principal diaphanous-related formin Diaph3 (mDia2) is present in the nucleus and regulates intranuclear actin polymerization, whereas Diaph1 (mDia1) is localized to the cytoplasm and controls cytoplasmic actin polymerization. We here show that mDia2 can be used as a tool to query actinlamin nucleoskeletal structure. Silencing mDia2 affected the nucleoskeletal lamin scaffold, altering nuclear morphology without affecting cytoplasmic actin cytoskeleton, and promoted MSC differentiation. Attempting to target intranuclear actin polymerization by silencing mDia2 led to a profound loss in lamin B1 nuclear envelope structure and integrity, increased nuclear height, and reduced nuclear stiffness without compensatory changes in other actin nucleation factors. Loss of mDia2 with the associated loss in lamin B1 promoted Runx2 transcription and robust osteogenic differentiation and suppressed adipogenic differentiation. Hence, mDia2 is a potent tool to query intranuclear actin-lamin nucleoskeletal structure, and its presence serves to retain multipotent stromal cells in an undifferentiated state.

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“…Moreover, when comparing DE genes in GP from different ages, we observed that through aging, more shared DE genes were found between 21 and 35 days and between 35 and 42 days of age, than when 21 and 42 days were compared ( Figure 4 ). When looking at the 12 exclusively DE genes between normal and FHS-affected broilers at 21 days of age ( SHISA3, FNDC1, ANGPTL7, LEPR, OXTR, COL16A1, BOC, E NSGALG00000049529, RASD2, GDF10 ENSGALG00000045154, and THSD7B) , they were mainly related to WNT and FGF signaling ( Amanatullah et al, 2014 ), bone metabolism ( Nilsson et al, 2007 ; Di Benedetto et al, 2014 ; Lui et al, 2018 ), angiogenesis ( Shang et al, 2015 ; Huang et al, 2021 ), chondrogenesis ( Sekiya et al, 2002 ; Paradise et al, 2018 ) and Hedgehog signaling pathway ( Kavran et al, 2010 ; Xavier et al, 2016 ). In this way, it is possible to highlight that DE genes between normal and affected broilers at 21 days of age were mainly related to endochondral ossification, while when GP and AC at 35 and 42 days of age were evaluated ( Peixoto et al, 2019 ; Oliveira et al, 2020 ; Hul et al, 2021 ; Goldoni et al, 2022 ), there were several DE genes related to other biological processes, such as inflammation, defense response, chemotaxis, within others.…”

Section: Discussionmentioning

confidence: 99%

Downregulation of growth plate genes involved with the onset of femoral head separation in young broilers

et al. 2022

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Femoral head separation (FHS) is characterized by the detachment of growth plate (GP) and articular cartilage, occurring in tibia and femur. However, the molecular mechanisms involved with this condition are not completely understood. Therefore, genes and biological processes (BP) involved with FHS were identified in 21-day-old broilers through RNA sequencing of the femoral GP. 13,487 genes were expressed in the chicken femoral head transcriptome of normal and FHS-affected broilers. From those, 34 were differentially expressed (DE; FDR ≤0.05) between groups, where all of them were downregulated in FHS-affected broilers. The main BP were enriched in receptor signaling pathways, ossification, bone mineralization and formation, skeletal morphogenesis, and vascularization. RNA-Seq datasets comparison of normal and FHS-affected broilers with 21, 35 and 42 days of age has shown three shared DE genes (FBN2, C1QTNF8, and XYLT1) in GP among ages. Twelve genes were exclusively DE at 21 days, where 10 have already been characterized (SHISA3, FNDC1, ANGPTL7, LEPR, ENSGALG00000049529, OXTR, ENSGALG00000045154, COL16A1, RASD2, BOC, GDF10, and THSD7B). Twelve SNPs were associated with FHS (p < 0.0001). Out of those, 5 were novel and 7 were existing variants located in 7 genes (RARS, TFPI2, TTI1, MAP4K3, LINK54, and AREL1). We have shown that genes related to chondrogenesis and bone differentiation were downregulated in the GP of FHS-affected young broilers. Therefore, these findings evince that candidate genes pointed out in our study are probably related to the onset of FHS in broilers.

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Molecular characterization of physis tissue by RNA sequencing

Cited by 18 publications

References 39 publications

Differentiation of Hypertrophic Chondrocytes from Human iPSCs for the In Vitro Modeling of Chondrodysplasias

Differentiation of Hypertrophic Chondrocytes from Human iPSCs for the In Vitro Modeling of Chondrodysplasias

Knockdown of formin mDia2 alters lamin B1 levels and increases osteogenesis in stem cells

Downregulation of growth plate genes involved with the onset of femoral head separation in young broilers

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