Project description:We generated iPSCs from human intervertebral disc cells which were obtained during spine fusion surgery of patients with spinal cord injury. The disc cell-derived iPSCs (diPSCs) showed similar characteristics to human embryonic stem cells (hESCs) and were efficiently differentiated into neural progenitor cells (NPCs) with the capability of differentiation into mature neurons in vitro. To examine whether the transplantation of NPCs derived from the diPSCs showed therapeutic effects, the NPCs were transplanted into mice at 9 days post-spinal cord injury. We detected a significant amelioration of hind limb dysfunction during the follow up recovery periods. Histological analysis at 5 weeks post-transplantation, we could identify undifferentiated human NPCs (Nestin+) as well as early (TUJ1+) and mature neurons (MAP2+) derived from the NPCs. Furthermore, the NPC transplantation demonstrated a preventive effect on the spinal cord degeneration resulting from the secondary injury. This study revealed that the intervertebral disc, a “to-be-waste” tissue, removed from the surgical procedure, could provide a unique opportunity to study iPSCs derived from hardly accessible somatic cells in normal situation and also be a useful therapeutic resource to generate autologous neural cells to treat patients suffering from spinal cord injury.
Project description:We generated iPSCs from human intervertebral disc cells which were obtained during spine fusion surgery of patients with spinal cord injury. The disc cell-derived iPSCs (diPSCs) showed similar characteristics to human embryonic stem cells (hESCs) and were efficiently differentiated into neural progenitor cells (NPCs) with the capability of differentiation into mature neurons in vitro. To examine whether the transplantation of NPCs derived from the diPSCs showed therapeutic effects, the NPCs were transplanted into mice at 9 days post-spinal cord injury. We detected a significant amelioration of hind limb dysfunction during the follow up recovery periods. Histological analysis at 5 weeks post-transplantation, we could identify undifferentiated human NPCs (Nestin+) as well as early (TUJ1+) and mature neurons (MAP2+) derived from the NPCs. Furthermore, the NPC transplantation demonstrated a preventive effect on the spinal cord degeneration resulting from the secondary injury. This study revealed that the intervertebral disc, a M-bM-^@M-^\to-be-wasteM-bM-^@M-^] tissue, removed from the surgical procedure, could provide a unique opportunity to study iPSCs derived from hardly accessible somatic cells in normal situation and also be a useful therapeutic resource to generate autologous neural cells to treat patients suffering from spinal cord injury. Total RNA was isolated using the NucleoSpin RNA II Kit (Macherey-Nagel, Duren, Germany, www.mn-net.com) according to the manufacturerM-bM-^@M-^Ys suggestions and was utilized for a genome-wide gene expression profiling experiment using the Illumina array (Illumina, San Diego, CA, USA, www.illumina.com) at Macrogen (Macrogen, Seoul, Korea, www.macrogen.com).
Project description:Low back pain is a major cause of disability especially for people between 20 and 50 years of age. As a costly healthcare problem, it imposes a serious socio-economic burden. Current surgical therapies have considerable drawbacks and fail to replace the normal disc in facilitating spinal movements and absorbing load. Therefore, the focus of regenerative medicine is on identifying biomarkers and signalling pathways to improve our understanding about the cascades of disc degeneration and allow for the design of specific therapies. We hypothesized that comparing microarray profiles from degenerative and non-degenerative discs will lead to the identification of dysregulated signalling and pathophysiological targets. Microarray data sets were generated from human annulus fibrosus cells and analysed using IPA ingenuity pathway analysis system. Gene expression values were validated by qRT-PCR, and respective proteins were identified by immunohistochemistry. Microarray analysis revealed 17 dysregulated molecular markers and various dysregulated cellular functions, including cell proliferation and inflammatory response, in the human degenerative annulus fibrosus. The most significant canonical pathway induced in degenerative annulus fibrosus was found to be the interferon signalling pathway. In conclusion, this study indicates interferon-alpha signalling pathway activation with IFIT3 and IGFBP3 up-regulation which may affect cellular function in human degenerative disc. 48 samples of intervertebral disc tissue - annulus fibrosus and nucleus pulposus - displaying varying degrees (grades) of degeneration
Project description:In our study, MS-based high-resolution proteomic analysis of CSF have been employed in order to gain deeper insights into changes occurring during IVDH in dogs, monitoring the pathophysiological processes at the protein and metabolite levels. The proteomics study involved canine patients classified within two groups. Healthy dogs (control group) (N=6) were mix breeds (20 kg average body weight), age 2-6 years, while the intervertebral disc herniation group (N=9) were mixed breeds (body weight 6-15 kg), age 7-12 years. In IVDH dogs, disc herniation was detected in T13/L1/L2 discs by CT. The dogs in IVDH group were paraplegic with preserved deep pain reflex and underwent a surgery procedure for decompression of the spinal cord.
Project description:The intervertebral disc is a specialized fibrocartilage structure of the spinal column that is pivotal for spinal mobility and function. It is composed of 3 distinct anatomical components: the annulus fibrosus, nulceus pulposus and cartilage endplates. We used 10x single cell seq to identify the various cell components of the disc as well as discover novel cell populations and signaling networks.
Project description:Herniation of the intervertebral disc (IVDH) is the most common cause of neurological and intervertebral disc degeneration-related diseases. Since the disc starts to degenerate before it can be observed by currently available diagnostic methods, there is an urgent need for novel diagnostic approaches. To identify molecular networks and pathways which may play important roles in intervertebral disc herniation, as well as to reveal the potential features which could be useful for monitoring disease progression and prognosis, multi-omics profiling including high-resolution LC-MS-based metabolomics and proteomics was performed. Furthermore, multi-omics data were integrated to decipher a complex interaction between individual omic layers leading to improved prediction model. Together with metabolic pathways related to amino acids and lipid metabolism, and coagulation cascades, our integromics prediction model identified the key features in IVDH, namely the proteins FSTL1, SCG5, NUCB1 and CRSP2 and the metabolites N-acetyl-D-glucosamine and adenine, involved in neuropathic pain, myelination, neurotransmission and inflammatory response, respectively. Their clinical application is to be further investigated. The utilization of novel integrative interdisciplinary strategy may provide opportunities to apply the innovative diagnostic and monitoring methods for degenerative spinal disorders.
Project description:Intervertebral disc degeneration is the main cause of low back pain and the mechanism of which is far from fully revealed. Although multiple factors are related to the intervertebral disc degeneration, inflammation and matrix metabolism dysregulation are the two key factors that play an important role in degeneration. Here, we found that CHSY3 is highly related to the nucleus pulposus degeneration. We generated CHSY3 knockout mice using Crisper/Cas9 system, and the NP cells are studied in this experiment.
Project description:Failure of intervertebral disc components, e.g. the nucleus pulposus causes intervertebral disc disease and associated low-back pain. Despite the high prevalence of disc disease, the changes in intervertebral disc cells and their regenerative potential with ageing and degeneration are not fully elucidated. Understanding the cell lineage, cell differentiation and maintenance of nucleus pulposus may have therapeutic application for the regeneration of degenerative disc, with significant impact for healthy ageing. Here we found that TAGLN expressing cells are present in human healthy nucleus pulposus, but diminish in degenerative disc. By lineage analyses in mice, we found cells in the nucleus pulposus are derived from a peripherally located population of notochord-derived Tagln expressing cells (PeriNP cells). The PeriNP cells are proliferative and can differentiate into the inner part of the nucleus pulposus. The Tagln+ cells and descendants diminish during aging and puncture induced disc degeneration. The maintenance and differentiation of PeriNP cells is partially regulated by Smad4 dependent signaling. Removal of Smad4 by nucleus pulposus specific Cre (Foxa2mNE-Cre), results in decreased Tagln+ cells and abnormal disc morphology, leading to disc degeneration. Our findings propose that the PeriNP Tagln expressing cells are a pool of notochord-derived progenitors that are important for maintenance of the nucleus pulposus and provide insights for regenerative therapy against intervertebral disc degeneration.
Project description:We compare transcriptomic profiles of human induced pluripotent stem cells (iPSCs), motor neurons (MNs) in vitro differentiated from iPSCs or human ESCs containing a HB9::GFP reporter for MNs, and human fetal spinal cords. The purpose of this comparison is to assess the extent of molecular similarities between in vitro differentiated MNs and in vivo fetal or adult spinal cord MNs. Data for adult spinal cord MNs are published from other studies: GSE3526, GSE19332, GSE20589, and GSE40438. Human induced pluripotent stem cells, pluripotent stem cell derived motor neurons, and fetal spinal cords for RNA extraction and hybridization on Affymetrix arrays.
Project description:Mice lacking equilibrative nucleoside transporter 1 demonstrate progressive calcification of spinal tissues including the annulus fibrosus (AF) of the intervertebral disc (IVD). To identify cellular pathways altered by loss of ENT1, we conducted microarray analysis of AF tissue from wild-type (WT) and ENT1-null mice before calcification (2 months) and associated with calcification (6 months).