Project description:The physiological function of the intervertebral disc (IVD) depends on the cellular and molecular composition of the nucleus pulposus (NP), which plays a key role in managing the mechanical loading of the IVD.We conducted single-cell RNA sequencing (scRNA-seq) analysis to better characterize the NP cells, particularly the Shh-tdTom+ NP cells, in the adult Shh-CreER; Rosa26tdTomato mice.
Project description:In order to study the effect of recombinant Shh (rShh) on nucleus pulposus cells, primary nucleus pulposus cells from SD rats were obtained.Then, nucleus pulposus cells were treated with rShh at 200 ng/ml(dissolved in dmso) for 48h. Similarly, we treated nucleus pulposus cells with the same concentration of DMSO as the control group, and analyzed the data of 3 biological repeats of RNA-seq in each group for gene expression profile analysis.
Project description:To evaluate the effect of Hypoxia-inducible factor 1-alpha inhibitor (HIF-1) in nucleus pulposus (NP) cells, human NP cells were lentivirally transduced with either control or FIH-1 targeted shRNA. Gene expression changes between samples from control and FIH-1 silenced cells were evaluated using a microarray. Primary NP cells were isolated from human discs and grown in culture. Cells were transduced with either control or FIH-1 targeting shRNA constructs. Silencing of FIH-1 was confirmed for each set using qRT-PCR and Western blot. Total RNA from NP cells was used for RNA extraction and hybridization on the Affymetrix microarray. Control samples: C1, C2, and C3 and FIH-1-silenced samples: E1, E2, and E3.
Project description:Glucose is critical to the development, homeostasis, and survival of nucleus pulposus cells. GLUT1 is a major glucose transporter, a NP phenotypic cell marker, and highly expressed in nucleus pulposus (NP) cells, however its level of importance in NP cell development and homeostasis is unknown. We used microarrays to explore the transcriptomics of differentially expressed genes between wildtype and Glut1 cKO NP cells in 9-month-old mice.
Project description:Objective: Induction of ER stress has been shown to promote NP cell apoptosis and disc degeneration. However, little is known about its regulation by hypoxia and its contribution to extracellular matrix homeostasis. Methods: NP cells were culture under hypoxia (1% O2) and normoxia (21% O2) to assess ER stress status. Tunicamycin and thapsigargin were used to induce canonical ER stress pathways and effects on cell secretome were assessed by proteomic analysis using mass spectrometry. The relevance of these findings to aging and degeneration was investigated by analyzing microarray data of NP tissues from aged mice (GSE134955) and degenerated human discs (GSE70362). Results: NP cells exhibited lower ER stress levels under hypoxia. ER stress inducers tunicamycin and thapsigargin promoted a canonical unfolded protein response. UPR further induced HIF-1 activity under hypoxia. NP cell secretome under ER stress showed an increased secretory pathway with a concomitant decrease in collagens, HAPLN1, and cell adhesion related proteins. Similar to our cell culture studies, NP tissues from aged mice and degenerated human discs presented higher levels of UPR markers and decreased levels of matrix components.
Project description:To evaluate the effect of Hypoxia-inducible factor 1-alpha inhibitor (HIF-1) in nucleus pulposus (NP) cells, human NP cells were lentivirally transduced with either control or FIH-1 targeted shRNA. Gene expression changes between samples from control and FIH-1 silenced cells were evaluated using a microarray.
Project description:The nucleus pulposus (NP) is composed of notochordal NP cells (NCs) and chondrocyte-like NP cells (CLCs). CLCs and chondrocytes have been reported to be similar. However, the interactions between CLCs and NCs remain unclear. In this study, to clarify how cells in NP and chondrocytes are regulated, we performed single-cell RNA sequencing (scRNA-seq) analysis of articular cartilage (AC) and NP of cynomolgus monkeys and found that part of CLCs and articular chondrocytes had similar gene expression profiles. These cells were enriched for genes related to GLI1, the nuclear mediator of the hedgehog pathway. In the NP, cell–cell interaction analysis revealed sonic hedgehog (SHH) expression in NCs, resulting in hedgehog signaling to CLCs, whereas no chondrocytes in our AC samples expressed hedgehog ligands.
Project description:Single cell RNA sequencing (scRNA-seq) analysis identified notochordal nucleus pulposus (NP) cells and chondrocyte-like NP cells in NP. Cells in human induced pluripotent stem cell-derived cartilage (hiPS-Cart) corresponded to chondrocyte-like NP cells but not to notochordal NP cells. hiPS-Cart cells changed their profile after implantation into intervertebral disks of nude rats, differentiating into two lineages that are metabolically distinct from each other. However, post-implanted hiPS-Cart cells corresponded to chondrocyte-like NP cells only and did not develop into notochordal NP cells.
Project description:Nucleus pulposus (NP) plays a vital role in intervertebral disc degeneration (IVDD). Previous studies have revealed cellular heterogeneity in the NP tissue during IVDD progression. Here, we used single cell RNA sequencing (scRNA-seq) to analyze the cellular and molecular alterations of diverse cell clusters during IVDD.
Project description:The aim of this transcription profiling study was to identify novel genes that could be used to distinguish bovine Nucleus pulposus (NP) cells from articular cartilage (AC) and annulus fibrosus (AF) cells and to further determine their expression in normal and degenerate human intervertebral disc (IVD). This study has identified a number of novel genes that characterise the bovine and human NP and IVD cell phenotypes and allows for discrimination between AC, AF and NP cells.<br><br>