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Project description:Objectives: Articular cartilage damage has become a universal health burden. Despite the recent progresses in cell-based cartilage regeneration, chondrocyte dedifferentiation has compromised the clinical outcomes, which is frequently seen in chondrocyte expansion and osteoarthritis, involving functional phenotype loss. However, its concept remains elusive with unknown intermediate process and mechanisms. Here we demonstrated a time-lapse atlas of chondrocyte dedifferentiation, to provide molecular details and informative biomarkers for clinical chondrocyte evaluation. Methods: Single-cell RNA sequencing (scRNA-seq) was employed to dissect the chondrocyte dedifferentiation and identify distinct cellular subpopulations. To validate the findings, live-cell metabolic assay, high-resolution imaging and ¡°assay for transposase-accessible chromatin with high throughput sequencing¡± (ATAC-seq) were conducted. Using a chemical inhibitor BTB06584 affecting on mitochondrial F1F0ATPase, we revealed the recovery potential of early and late dedifferentiated chondrocytes. Results:Our findings shape a biphasic model consist of early and late dedifferentiation stages. Early dedifferentiated chondrocytes uniquely obtained a transient glycolytic phenotype with an activation of metabolic and anti-oxidant genes; late dedifferentiated chondrocytes exhibited ultrastructural changes including mitochondrion damages and stress-associated chromatin remodeling. Using a chemical inhibitor BTB06584, we revealed that early and late dedifferentiated chondrocytes possessed distinguished recovery potential from functional phenotype loss. BTB06584 treatment ameliorated early phenotype loss through inhibiting MAPK pathway. Notably, this two-stage transition was validated in human chondrocyte culture. An image-based approach was established for clinical utilization, to efficiently predict chondrocytes plasticity with stage-specific biomarkers. Conclusion: Thus this study lays a foundation for future clinical chondrocyte quality regulation and provides insights for deeper understanding of chondrocyte dedifferentiation.
2022-01-20 | GSE193742 | GEO
Project description:Objectives: Articular cartilage damage has become a universal health burden. Despite the recent progresses in cell-based cartilage regeneration, chondrocyte dedifferentiation has compromised the clinical outcomes, which is frequently seen in chondrocyte expansion and osteoarthritis, involving functional phenotype loss. However, its concept remains elusive with unknown intermediate process and mechanisms. Here we demonstrated a time-lapse atlas of chondrocyte dedifferentiation, to provide molecular details and informative biomarkers for clinical chondrocyte evaluation. Methods: Single-cell RNA sequencing (scRNA-seq) was employed to dissect the chondrocyte dedifferentiation and identify distinct cellular subpopulations. To validate the findings, live-cell metabolic assay, high-resolution imaging and ¡°assay for transposase-accessible chromatin with high throughput sequencing¡± (ATAC-seq) were conducted. Using a chemical inhibitor BTB06584 affecting on mitochondrial F1F0ATPase, we revealed the recovery potential of early and late dedifferentiated chondrocytes. Results:Our findings shape a biphasic model consist of early and late dedifferentiation stages. Early dedifferentiated chondrocytes uniquely obtained a transient glycolytic phenotype with an activation of metabolic and anti-oxidant genes; late dedifferentiated chondrocytes exhibited ultrastructural changes including mitochondrion damages and stress-associated chromatin remodeling. Using a chemical inhibitor BTB06584, we revealed that early and late dedifferentiated chondrocytes possessed distinguished recovery potential from functional phenotype loss. BTB06584 treatment ameliorated early phenotype loss through inhibiting MAPK pathway. Notably, this two-stage transition was validated in human chondrocyte culture. An image-based approach was established for clinical utilization, to efficiently predict chondrocytes plasticity with stage-specific biomarkers. Conclusion: Thus this study lays a foundation for future clinical chondrocyte quality regulation and provides insights for deeper understanding of chondrocyte dedifferentiation.
2022-01-20 | GSE193743 | GEO
Project description:A High-Resolution Route Map Reveals Distinct Stages of Chondrocyte Dedifferentiation
| PRJNA797446 | ENA
Project description:A High-Resolution Route Map Reveals Distinct Stages of Chondrocyte Dedifferentiation (RNA-Seq)
| PRJNA797442 | ENA
Project description:A High-Resolution Route Map Reveals Distinct Stages of Chondrocyte Dedifferentiation (ATAC-Seq)
| PRJNA797450 | ENA
Project description:Due to the limited self-repair capacity of articular cartilage, the surgical restoration of defective cartilage remains a major clinical challenge. The cell-based approach, which is known as autologous chondrocyte transplantation (ACT), has limited success, presumably because the chondrocytes acquire a fibroblast-like phenotype in monolayer culture. This unwanted dedifferentiation process is typically addressed by using three-dimensional scaffolds, pellet culture, and/or the application of exogenous factors. Alternative mechanical unloading approaches are suggested to be beneficial in preserving the chondrocyte phenotype. In this study, we examined if the random positioning machine (RPM) could be used to expand chondrocytes in vitro such that they maintain their phenotype. Bovine chondrocytes were exposed to (a) eight days in static monolayer culture; (b) two days in static monolayer culture, followed by six days of RPM exposure; and, (c) eight days of RPM exposure. Furthermore, the experiment was also conducted with the application of 20 mM gadolinium, which is a nonspecific ion-channel blocker. The results revealed that the chondrocyte phenotype is preserved when chondrocytes go into suspension and aggregate to cell clusters. Exposure to RPM rotation alone does not preserve the chondrocyte phenotype. Interestingly, the gene expression (mRNA) of the mechanosensitive ion channel TRPV4 decreased with progressing dedifferentiation. In contrast, the gene expression (mRNA) of the mechanosensitive ion channel TRPC1 was reduced around fivefold to 10-fold in all of the conditions. The application of gadolinium had only a minor influence on the results. This and previous studies suggest that the chondrocyte phenotype is preserved if cells maintain a round morphology and that the ion channel TRPV4 could play a key role in the dedifferentiation process.
| S-EPMC5983850 | biostudies-other
Project description:The extent to which alternative splicing and long intergenic noncoding RNAs (lincRNAs) contribute to the specialized functions of cells within an organ is poorly understood. We generated a comprehensive dataset of gene expression from individual cell types of the Arabidopsis root. Comparisons across cell types revealed that alternative splicing tends to remove parts of coding regions from a longer, major isoform, providing evidence for a progressive mechanism of splicing. Cell-type-specific intron retention suggested a possible origin for this common form of alternative splicing. Coordinated alternative splicing across developmental stages pointed to a role in regulating differentiation. Consistent with this hypothesis, distinct isoforms of a transcription factor were shown to control developmental transitions. lincRNAs were generally lowly expressed at the level of individual cell types, but co-expression clusters provided clues as to their function. Our results highlight insights gained from analysis of expression at the level of individual cell types.
| S-EPMC5125536 | biostudies-literature
Project description:Over the course of development, the vertebrate heart undergoes a series of complex morphogenetic processes that transforms it from a simple myocardial epithelium to the complex 3D structure required for its function. One of these processes leads to the formation of trabeculae to optimize the internal structure of the ventricle for efficient conduction and contraction. Despite the important role of trabeculae in the development and physiology of the heart, little is known about their mechanism of formation. Using 3D time-lapse imaging of beating zebrafish hearts, we observed that the initiation of cardiac trabeculation can be divided into two processes. Before any myocardial cell bodies have entered the trabecular layer, cardiomyocytes extend protrusions that invade luminally along neighboring cell-cell junctions. These protrusions can interact within the trabecular layer to form new cell-cell contacts. Subsequently, cardiomyocytes constrict their abluminal surface, moving their cell bodies into the trabecular layer while elaborating more protrusions. We also examined the formation of these protrusions in trabeculation-deficient animals, including erbb2 mutants, tnnt2a morphants, which lack cardiac contractions and flow, and myh6 morphants, which lack atrial contraction and exhibit reduced flow. We found that, compared with cardiomyocytes in wild-type hearts, those in erbb2 mutants were less likely to form protrusions, those in tnnt2a morphants formed less stable protrusions, and those in myh6 morphants extended fewer protrusions per cell. Thus, through detailed 4D imaging of beating hearts, we have identified novel cellular behaviors underlying cardiac trabeculation.
| S-EPMC3899815 | biostudies-other
Project description:Turning genes on and off is essential for development and homeostasis, yet little is known about the sequence and causal role of chromatin state changes during the repression of active genes. This is surprising, as defective gene silencing underlies developmental abnormalities and disease. Here we delineate the sequence and functional contribution of transcriptional repression mechanisms at high temporal resolution. Inducible entry of the NuRD-interacting transcriptional regulator Ikaros into mouse pre-B cell nuclei triggered immediate binding to target gene promoters. Rapid RNAP2 eviction, transcriptional shutdown, nucleosome invasion, and reduced transcriptional activator binding required chromatin remodeling by NuRD-associated Mi2beta/CHD4, but were independent of HDAC activity. Histone deacetylation occurred after transcriptional repression. Nevertheless, HDAC activity contributed to stable gene silencing. Hence, high resolution mapping of transcriptional repression reveals complex and interdependent mechanisms that underpin rapid transitions between transcriptional states, and elucidates the temporal order, functional role and mechanistic separation of NuRD-associated enzymatic activities.
| S-EPMC5373822 | biostudies-literature
Project description:Approximately 3,000 cattle bacterial artificial chromosome (BAC)-end sequences were added to the Illinois-Texas 5,000-rad RH (RH, radiation hybrid) map. The BAC-end sequences selected for mapping are approximately 1 Mbp apart on the human chromosomes as determined by blastn analysis. The map has 3,484 ordered markers, of which 3,204 are anchored in the human genome. Two hundred-and-one homologous synteny blocks (HSBs) were identified, of which 27 are previously undiscovered, 79 are extended, 26 were formed by previously unrecognized breakpoints in 18 previously defined HSBs, and 23 are the result of fusions. The comparative coverage relative to the human genome is approximately 91%, or 97% of the theoretical maximum. The positions of 64% of all cattle centromeres and telomeres were reassigned relative to their positions on the previous map, thus facilitating a more detailed comparative analysis of centromere and telomere evolution. As an example of the utility of the high-resolution map, 22 cattle BAC fingerprint contigs were directly anchored to cattle chromosome 19 [Bos taurus, (BTA) 19]. The order of markers on the cattle RH and fingerprint maps of BTA19 and the sequence-based map of human chromosome 17 [Homo sapiens, (HSA) 17] were found to be highly consistent, with only two minor ordering discrepancies between the RH map and fingerprint contigs. The high-resolution Illinois-Texas 5,000-rad RH and comparative maps will facilitate identification of candidate genes for economically important traits, the phylogenomic analysis of mammalian chromosomes, proofing of the BAC fingerprint map and, ultimately, aid the assembly of cattle whole-genome sequence.
| S-EPMC1317968 | biostudies-literature