Notochordal Cells Research Articles

Induction of notochordal differentiation of bone marrow mesenchymal‑derived stem cells via the stimulation of notochordal cell‑rich nucleus pulposus tissue.

The degeneration of intervertebral disc (IVD) tissue, initiated following the disappearance of notochordal cells (NCs), is characterized by the decreased number of nucleus pulposus (NP) cells (NPCs) and extracellular matrix. Transplanting proper cells into the IVD may sustain cell numbers, resulting in the synthesis of new matrix; this represents a minimally invasive regenerative therapy. However, the lack of cells with a correct phenotype severely hampers the development of regenerative therapy. The present study aimed to investigate whether porcine NC-rich NP tissue stimulates bone marrow-derived mesenchymal stem cell (BM-MSC) differentiation toward NC-like cells, which possess promising regenerative ability, for the treatment of disc degeneration diseases. BM-MSCs were successfully isolated from porcine femurs and tibiae, which expressed CD90 and CD105 markers and did not express CD45. Differentiation induction experiments revealed that the isolated cells had osteogenic and adipogenic differentiation potential. When co-cultured with NC-rich NP tissue, the BM-MSCs successfully differentiated into NC-like cells. Cell morphological analysis revealed that the cells exhibited an altered morphology, from a shuttle-like to a circular one, and the expression of NC marker genes, including brachyury, keratin-8, and keratin-18, was enhanced, and the cells exhibited the ability to generate aggrecan and collagen II. Taken together, the findings of the present study demonstrated that the primarily isolated and cultured BM-MSCs may be stimulated to differentiate into NC-like cells by porcine NC-rich NP explants, potentially providing an ideal cell source for regenerative therapies for disc degeneration diseases.

A novel in vivo mouse intervertebral disc degeneration model induced by compressive suture

Intervertebral disc degeneration (IDD) is the root cause of many musculoskeletal disorders of the spine. However, the etiology of IDD is complex and still not well understood. Animal models of IDD would be useful in deciphering the underlying mechanisms. But the existing animal models have their limitations. Therefore, to establish a novel mouse model that can simulate the human IDD process in vivo, we proposed to carefully circumcise the 2 mm-wide tail skin and then compressively sutured the defect with a simple end-to-end suture to exert excessive pressure on the disc. After 1-week, 2-week, and 4-week compression, the mice were sacrificed and the intervertebral discs were harvested for tissue analysis. The radiological, morphological, and molecular modifications of intervertebral discs were measured to characterize this model. Radiologically, the water content of the intervertebral disc decreased significantly after 2-week compression. Morphologically, the nucleus pulposus showed a decrease in volume and the number of notochordal cells. The compressive suture also broke the balance between anabolic and catabolic enzymes in nucleus pulposus, which led to the remodeling of the extracellular matrix in nucleus pulposus as the content of aggrecan and collagen II decreased. The compressive suture could induce intervertebral discs degeneration in a more reasonable way, which was solely influenced by mechanical loading, as the mice caudal vertebrae still moved freely after the operation. This kind of animal model could be adapted as a reliable in vivo mouse IDD model for the research regarding the etiology and treatments of IDD.

Delayed notochordal cell disappearance through integrin α5β1 mechanotransduction during ex-vivo dynamic loading-induced intervertebral disc degeneration.

The loss of nucleus pulposus (NP) notochordal cells is one of the key initial hallmarks of age-related intervertebral disc degeneration. Although the transmembrane mechanoreceptor integrin α5β1 is important in the process of disc degeneration, the relationship between integrin α5β1 and notochordal cell disappearance remains unclear. The purpose of this study was to elucidate the roleof integrin α5β1 in the homeostasis of notochordal cells using an ex-vivo dynamic loading culture system that we developed. Rat tail functional spinal units (n = 80 from 40 rats) were cultured under unloading or 1.3-MPa, 1.0-Hz dynamic compressive loading for 48 or 144 h with or without an integrin α5β1 inhibitor. Disc histomorphology, cell viability, apoptosis, senescence, and phenotypic expression were investigated. Consequently, histological degenerative disc changes with decreased cell viability and increased cell apoptosis and senescence were observed with an extended loading duration. Immunofluorescence revealed that the expression of notochordal cell markers, CD24 and brachyury, and chondrocyte markers, collagen type II and SRY-box 9, declined with loading. In particular, reduction in notochordal cell marker expression was more dramatic than that in chondrocyte marker expression. Apoptotic terminal deoxynucleotidyl transferase dUTP nick-end labelingpositivity was also higher in brachyury-positive notochordal cells. Furthermore, all these changes were delayed by inhibiting integrin α5β1. Findings of our dynamic loading regimen with a relatively high pressure suggest reproducibility of the cellularity and phenotypic disappearance of NP notochordal cells during adolescence, the susceptibility of notochordal cells tomechanical stimuli partially through the integrin α5β1 pathway, and future potential treatment of integrin regulation for intervertebral disc disease.

Notochordal-Cell-Derived Exosomes Induced by Compressive Load Inhibit Angiogenesis via the miR-140-5p/Wnt/β-Catenin Axis

Angiogenesis is a pathological signature of intervertebral disc degeneration (IDD). Accumulating evidence has shown that notochordal cells (NCs) play an essential role in maintaining intervertebral disc development and homeostasis with inhibitive effect on blood vessel in-growth. However, the anti-angiogenesis mechanism of NCs is still unclear. In the current study, we, for the first time, isolated NC-derived exosomes (NC-exos) and showed their increased concentration following compressive load cultures. We further found that NC-exos from 0.5 MPa compressive load cultures (0.5 MPa/NC-exos) inhibit angiogenesis via transferring high expressed microRNA (miR)-140-5p to endothelial cells and regulating the downstream Wnt/β-catenin pathway. Clinical evidence showed that exosomal miR-140-5p expression of the nucleus pulposus is negatively correlated with angiogenesis in IDD. Finally, 0.5 MPa/NC-exos were demonstrated to have a therapeutical impact on the degenerated disc with an anti-angiogenesis effect in an IDD model. Consequently, our present findings provide insights into the anti-angiogenesis mechanism of NC-exos, indicating their therapeutic potential for IDD.

Clinical, magnetic resonance imaging and histologic findings of canine thoracolumbar discal pseudocyst: 8 cases (2014‐2019)

To report the clinical and MRI features, and histologic findings of thoracolumbar discal pseudocyst in dogs. The records of eight dogs with thoracolumbar discal cyst-like structures were retrospectively collected to record their clinical signs, MRI features and surgical and histologic findings. Eight dogs with surgically and histologically confirmed thoracolumbar discal pseudocysts were included in the case series. Six dogs presented with acute onset and two dogs presented with subacute onset of thoracolumbar myelopathy. MRI showed compressive thoracolumbar myelopathy due to a round to oval-shaped epidural mass lesion communicating with the intervertebral disc, iso/hypointense on T1WI and mostly hyperintense on T2WI, associated with a variable contrast-enhancing wall, compatible with a cyst-like structure. These structures were surgically visualised and removed through a mini-hemilaminectomy or hemilaminectomy and submitted for histologic investigation. One dog also underwent cytologic examination of the cystic content. Similar to that in humans, histology revealed a cyst-like nature with a wall consisting of dense fibrous connective tissue containing clusters of chondroid cells accompanied by groups of notochordal cells and occasional erythrocytes; however, a real epithelial lining was missing and the term pseudocyst seemed more appropriate. This report describes clinical signs, and MRI and histologic findings of discal pseudocysts in dogs with thoracolumbar myelopathy. Despite being rare, discal pseudocysts should be considered as a differential diagnosis in dogs with acute onset thoracolumbar myelopathy.

Granulocyte-colony stimulating factor improves intervertebral disc degeneration in experimental adult male rats: A microscopic and radiological study.

Intervertebral disc degeneration (IVDD) is a major contributor to low back pain (LBP). Granulocyte-colony stimulating factor (GCSF) is known to mobilize hematopoietic stem cells (HSCs) that may be implicated in intervertebral disc (IVD) regeneration. Rats were divided into the following three groups: (i) control group; (ii) IVDD group-the rats underwent Co5/Co6 and Co7/Co8 IVDD operation; and (iii) GCSF-treated group-the rats received daily GCSF subcutaneous injections starting 6 weeks after the IVDD operation and continued for 5 days. All of the rats were euthanized after 8 weeks, and IVDs were assessed by tail X-ray and histopathological, immunohistochemical, and transmission electron microscopy (TEM) analyses. The X-rays showed disc narrowing in the IVDD group that was significantly widened in the GCSF-treated rats. Histologically, the IVDD group showed disarrangement of the annulus fibrosis lamellae, complete degeneration of the nucleus pulposus, and loss of proteoglycan content. These changes were improved after GCSF treatment. Vertebral endplate thickness and cellularity were significantly decreased with IVDD and significantly increased after GCSF treatment. Stromal cell-derived factor-1α (SDF-1α) immune expression was significantly increased in the IVDD group but decreased in the GCSF-treated group. However, the caspase-3 expression percentage showed no significant difference among the studied groups. TEM showed excessive collagen deposits around the notochordal cells in the IVDD group, which were attenuated in the GCSF-treated group. These results indicate that GCSF improves IVDD and promotes its recovery based on radiological, histological and TEM findings.

213. Study comparing NTG-101, a growth factor-based therapy with mesenchymal stem cell (MSC) based treatment of degenerative disc disease (DDD) in pre-clinical rodent model

BACKGROUND CONTEXT Currently there is significant interest in the use of cellular therapies (such as stem cells) to treat degenerative disc disease (DDD). We recently explored the potential use of bone marrow derived stem cells (BMSCs) and cartilage-derived stem cells (CDSCs) in a small animal model of DDD as compared to our recently developed molecular therapy ‘NTG-101’. PURPOSE To compare the therapeutic efficacy of NTG-101 with BMSCs or CDSCs to treat DDD in rodent model. STUDY DESIGN/SETTING In vivo laboratory experimentation involving female Wistar rats aged 12-weeks. PATIENT SAMPLE No human subjects. Animal Care approved study involving female Wistar rats using validated rat-tail injury and injection model. OUTCOME MEASURES Histology, immunohistochemistry, Western blotting, gene expression. METHODS CDSCs were derived from the tibial and femoral plateaus of Wistar rats and sphere forming cells were passaged further in serum free, stem cell specific media. BMSCs were obtained from the femurs of these animals and cultured as adherent cells in media containing 8% fetal bovine serum (FBS) following standard protocols. Stemness was confirmed by differentiation into multiple lineages. In parallel, we induced DDD in caudal intervertebral discs (IVDs) of 12 weeks old Wistar rats (N=30) using image-guided needle puncture injury. Ten weeks later, animals were randomized in six treatment groups (n=5 animals/group) and received an intradiscal injection (8µl) containing:G1-phosphate buffered saline (PBS, 1X, pH=7.2); G2-NTG-101(1X);G3-CDSCs (150,000 cells/disc) suspended in PBS; G4-BMSCs (150,000 cells/disc) suspended in PBS;G5-CDSCs (150,000 cells/disc) suspended in NTG-101(1X); G6-BMSCs (150,000 cells/disc) suspended in NTG-101(1X). At the end of experiment (ie, 10 weeks post-injection, 20 weeks post-injury), animals were humanely euthanized and IVDs were harvested for histology, immunohistochemistry and Western blotting. RESULTS Gene expression analysis using quantitative real time PCR confirmed expression of pluripotency (Oct4, Nanog, Sox2) and mesenchymal stem cell surface markers (CD29, CD44, CD90, CD105, CD133, CD166) in all stem cells. CDSC and BMSCs differentiation into chondrogenic, osteogenic and adipogenic lineages was confirmed, thereby validating their stemness potential. Discs injected with PBS developed a degenerative phenotype with the loss of NP morphology, cellularity and the development of a degenerative fibrocartilaginous phenotype. On the other hand, IVDs injected with NTG-101 or CDSCs alone showed a significant recovery. The morphology and cellularity of discs injected with BMSCs alone or in combination with NTG-101 was not significantly different from those discs injected with PBS. NTG-101 injected discs showed a healthy NP rich in notochordal and resident stem cells as revealed by nuclear Brachyury and Oct4 respectively, whereas discs injected with stem cells alone or stem cells plus NTG-101 did not. CONCLUSIONS Our preclinical data demonstrated that single intradiscal injection of NTG-101 is a superior and effective therapy for regeneration of IVD-NP in comparison to mesenchymal stem cell-based therapies for treatment of DDD. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.

Adolescent idiopathic scoliosis: The mechanobiology of differential growth.

Adolescent idiopathic scoliosis (AIS) has been linked to neurological, genetic, hormonal, microbial, and environmental cues. Physically, however, AIS is a structural deformation, hence an adequate theory of etiology must provide an explanation for the forces involved. Earlier, we proposed differential growth as a possible mechanism for the slow, three‐dimensional deformations observed in AIS. In the current perspective paper, the underlying mechanobiology of cells and tissues is explored. The musculoskeletal system is presented as a tensegrity‐like structure, in which the skeletal compressive elements are stabilized by tensile muscles, ligaments, and fasciae. The upright posture of the human spine requires minimal muscular energy, resulting in less compression, and stability than in quadrupeds. Following Hueter‐Volkmann Law, less compression allows for faster growth of vertebrae and intervertebral discs. The substantially larger intervertebral disc height observed in AIS patients suggests high intradiscal pressure, a condition favorable for notochordal cells; this promotes the production of proteoglycans and thereby osmotic pressure. Intradiscal pressure overstrains annulus fibrosus and longitudinal ligaments, which are then no longer able to remodel and grow, and consequently induce differential growth. Intradiscal pressure thus is proposed as the driver of AIS and may therefore be a promising target for prevention and treatment.

Effects of lentivirus-mediated insulin-like growth factor 1 and platelet derived growth factor genes on nucleus pulposus tissue of human degenerated intervertebral disc

To observe and compare the cytological and biological differences between human normal and degenerated nucleus pulposus (NP), and to investigate the repair effect of insulin-like growth factor 1 (IFG-1) and platelet derived growth factor (PDGF) on human degenerated NP. Human degenerative and normal NP tissues were obtained from operative patients, a portion of which were processed into tissue sections and HE staining was performed to observe the morphological changes of nucleus pulposus cells (NPCs) before and after degeneration of NP. Immunohistochemistry staining was used to determine the expression levels of collagen type Ⅰ, collagen type Ⅱ, B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X (Bax) proteins. Another portion of tissues were isolated and cultured and NPCs morphology was observed under inverted microscope. Western blot analysis was used to detect collagen type Ⅱ protein expression. Then, the gene transfection experiments were launched, including 4 groups, with group A designed as degenerated NPCs only, and groups B, C, and D of degenerated NPCs transfected with IGF-1 gene lentiviral particles, PDGF gene lentiviral particles, and lentiviral particles carrying IGF-1 and PDGF double genes, respectively. At 21 days after transfection, the cell morphology of each group was observed under inverted microscope, the positive rates of IGF-1 and PDGF of each group were measured by flow cytometry, and the expression of collagen type Ⅱ protein was detected by using immunohistochemistry staining and Western blot. HE staining showed that there were a large number of notochordal cells and a small number of chondrocytes in the central NP tissue of normal group, while the NPCs in degeneration group were significantly reduced, and a large proportion of fibrocartilage tissues were found in NP tissue. Immunohistochemistry staining showed that the percentages of collagen type Ⅰ and Bax protein-positive cells in degeneration group were significantly higher than those of normal group, while the percentages of collagen type Ⅱ and Bcl-2 protein-positive cells were significantly lower than those of normal group ( P<0.05). Western blot showed that the relative expression level of collagen type Ⅱ protein in degeneration group was significantly lower than that in normal group ( t=65.493, P=0.000). At 21 days after gene transfection, compared with group A, the cell viability of groups B, C, and D increased and the morphology became more regular. Flow cytometry showed that the percentages of IGF-1-positive cells in groups B and D were significantly higher than that in group A, and the percentages of PDGF-positive cells in groups C and D were significantly higher than that in group A ( P<0.05). Immunohistochemistry staining showed that the positive stainings of collagen type Ⅱ in groups A, B, C, and D was (±), (+), (+), and (++), respectively. Western blot showed that the relative expression of collagen type Ⅱ protein in groups A, B, C, and D increased by degrees, and the differences between groups were significant ( P<0.05). Both IGF-1 and PDGF can reverse the degeneration of intervertebral discs NPCs and they have synergistic effects, providing experimental basis for its application in clinical treatment approaches for degenerative disc disease.

Effects of Glucose Deprivation on ATP and Proteoglycan Production of Intervertebral Disc Cells under Hypoxia

As the most common cause of low back pain, the cascade of intervertebral disc (IVD) degeneration is initiated by the disappearance of notochordal cells and progressive loss of proteoglycan (PG). Limited nutrient supply in the avascular disc environment restricts the production of ATP which is an essential energy source for cell survival and function such as PG biosynthesis. The objective of this study was to examine ATP level and PG production of porcine IVD cells under prolonged exposure to hypoxia with physiological glucose concentrations. The results showed notochordal NP and AF cells responded differently to changes of oxygen and glucose. Metabolic activities (including PG production) of IVD cells are restricted under the in-vivo nutrient conditions while NP notochordal cells are likely to be more vulnerable to reduced nutrition supply. Moreover, provision of energy, together or not with genetic regulation, may govern PG production in the IVD under restricted nutrient supply. Therefore, maintaining essential levels of nutrients may reduce the loss of notochordal cells and PG in the IVD. This study provides a new insight into the metabolism of IVD cells under nutrient deprivation and the information for developing treatment strategies for disc degeneration.

Pcdh18a regulates endocytosis of E-cadherin during axial mesoderm development in zebrafish

The notochord defines the axial structure of all vertebrates during development. Notogenesis is a result of major cell reorganization in the mesoderm, the convergence and the extension of the axial cells. However, it is currently not fully understood how these processes act together in a coordinated way during notochord formation. The prechordal plate is an actively migrating cell population in the central mesoderm anterior to the trailing notochordal plate cells. We show that prechordal plate cells express Protocadherin 18a (Pcdh18a), a member of the cadherin superfamily. We find that Pcdh18a-mediated recycling of E-cadherin adhesion complexes transforms prechordal plate cells into a cohesive and fast migrating cell group. In turn, the prechordal plate cells subsequently instruct the trailing mesoderm. We simulated cell migration during early mesoderm formation using a lattice-based mathematical framework and predicted that the requirement for an anterior, local motile cell cluster could guide the intercalation and extension of the posterior, axial cells. Indeed, a grafting experiment validated the prediction and local Pcdh18a expression induced an ectopic prechordal plate-like cell group migrating towards the animal pole. Our findings indicate that the Pcdh18a is important for prechordal plate formation, which influences the trailing mesodermal cell sheet by orchestrating the morphogenesis of the notochord.

Therapeutic Potential of Extracellular Vesicles in Degenerative Diseases of the Intervertebral Disc.

Extracellular vesicles (EVs) are lipid membrane particles carrying proteins, lipids, DNA, and various types of RNA that are involved in intercellular communication. EVs derived from mesenchymal stem cells (MSCs) have been investigated extensively in many different fields due to their crucial role as regeneration drivers, but research for their use in degenerative diseases of the intervertebral disc (IVD) has only started recently. MSC-derived EVs not only promote extracellular matrix synthesis and proliferation in IVD cells, but also reduce apoptosis and inflammation, hence having multifunctional beneficial effects that seem to be mediated by specific miRNAs (such as miR-233 and miR-21) within the EVs. Aside from MSC-derived EVs, IVD-derived EVs (e.g., stemming from notochordal cells) also have important functions in IVD health and disease. This article will summarize the current knowledge on MSC-derived and IVD-derived EVs and will highlight areas of future research, including the isolation and analysis of EV subpopulations or exposure of MSCs to cues that may enhance the therapeutic potential of released EVs.

Influence of hypoxia on human induced pluripotent stem cells differentiation into notochordal-like cells for intervertebral disc regeneration

Purpose: The intervertebral disc (IVD) degeneration is one of the main causes of chronic low back pain. The central part of the IVD, the nucleus pulposus (NP) contains notochordal cells (NC), a progenitor cell population together with the mature NP cells responsible for the synthesis of the extracellular matrix. There is now considerable evidence that NC has a significant influence on IVD homeostasis and their loss has been correlated with IVD degeneration. Due to the lack of spontaneous repair, the supplementation of NP by functional regenerative cells is considered as promising strategy to limit the progression of IVD degeneration.

Development and characterization of a novel non-surgical mouse model of intervertebral disc degeneration

Purpose: In the spinal column, the purpose of the intervertebral discs (IVD) is to provide the spine with flexibility and load-bearing capacity. Each IVD is comprised of a highly hydrated nucleus pulposus (NP) core that is enclosed by the concentric collagenous fibers of the annulus fibrosus (AF). Maintenance of this composite structure and its biomechanical function requires proper activity of disc-resident cells, with balanced synthesis and degradation of macromolecular components. Despite that, our understanding of the role of senescence and cell death on the progression of disc degeneration is limited. IVD degeneration is one of the main contributors to low back pain and is known to precede several prevalent and debilitating spinal diseases, such as disc herniation and spondylosis. Although the causes of IVD degeneration are poorly understood, dysfunction and progressive decline in the number of resident cells have been found with aging and disease. Most animal models currently used to investigate the pathophysiology of IVD degeneration rely on surgical intervention, where invasiveness coupled with an inherent inflammatory response, limits our ability to mimic the pathogenesis of non-traumatic human disc degeneration. Hence, we sought to develop a non-surgical mouse model of IVD degeneration using genetically-induced targeted ablation of disc-resident cells that express aggrecan (Acan), the main proteoglycan component found in the extracellular matrix of the IVDs. Methods: We crossed heterozygous mice that express Cre recombinase under the control of the aggrecan gene promoter (AcanCreERT2) with mice that express diphtheria toxin (DTA) following Cre recombination (Gt(ROSA)DTA). The Acan+/Cre ;DTA+/- offspring received tamoxifen (4-OHT) interperitoneally for 5 (five) consecutive days to promote the targeted ablation of Acan-expressing cells. Animals were euthanized, and spines were harvested at 1-, 5-, 9- and 12-weeks post-DTA induction for histology. Sagittal sections were stained with Safranin-O/Fast-green and compared to littermate controls lacking the Cre allele (Acan+/+;DTA+/-) who received the same treatment, to allow a detailed spatiotemporal analysis of changes in the IVDs structure and composition. Long-term histological assessment was also performed at 7-months post-DTA induction, to investigate IVD degenerative changes with aging. Results: Histological analysis revealed mild degenerative changes of the IVDs by 5-weeks post-DTA induction in the Acan+/Cre:DTA mice, with the cells in the NP demonstrating a hypertrophic phenotype by 12-weeks, with few to no notochordal cells identified. Lumbar IVDs exhibited profound histological changes by 7-months post-DTA induction when compared to controls (Figure 1). These changes included decreased cellularity, loss of NP/AF borders, altered AF lamellar fiber structure, and disruption of NP matrix with loss of proteoglycan staining, and are typical characteristics of severe disc degeneration. Conclusions: Targeted cell depletion within the IVD resulted in progressive histological changes in the structure and composition of the NP, with clear degeneration of the AF compared to controls, reaching severe IVD degeneration by 7-months post-DTA induction. This novel mouse model of IVD degeneration does not require direct injury to the IVD; thus, it may contribute to further understanding the mechanisms underlying spontaneous and/or age-related degeneration of IVD, which more closely mimic the progression of human disease. Considering the structure-function relationship of this tissue, future studies should explore the mechanical behavior of IVDs to assess how the overall structural degeneration seen histologically translates into the biomechanical properties of the tissue.

NOTO Transcription Factor Directs Human Induced Pluripotent Stem Cell-Derived Mesendoderm Progenitors to a Notochordal Fate.

The founder cells of the Nucleus pulposus, the centre of the intervertebral disc, originate in the embryonic notochord. After birth, mature notochordal cells (NC) are identified as key regulators of disc homeostasis. Better understanding of their biology has great potential in delaying the onset of disc degeneration or as a regenerative-cell source for disc repair. Using human pluripotent stem cells, we developed a two-step method to generate a stable NC-like population with a distinct molecular signature. Time-course analysis of lineage-specific markers shows that WNT pathway activation and transfection of the notochord-related transcription factor NOTO are sufficient to induce high levels of mesendoderm progenitors and favour their commitment toward the notochordal lineage instead of paraxial and lateral mesodermal or endodermal lineages. This study results in the identification of NOTO-regulated genes including some that are found expressed in human healthy disc tissue and highlights NOTO function in coordinating the gene network to human notochord differentiation.

Leptin receptor–expressing cells represent a distinct subpopulation of notochord-derived cells and are essential for disc homoeostasis

Background/objectiveIntervertebral disc degeneration (IDD) remains to be an intractable clinical challenge. Although IDD is characterised by loss of notochordal cells (NCs) and dysfunction of nucleus pulposus (NP) cells, little is known about the origin, heterogeneity, fate and maintenance of NCs and NP cells, which further stunts the therapeutic development. Thus, effective tools to spatially and temporally trace specific cell lineage and clarify cell functions in intervertebral disc (IVD) development and homoeostasis are urgently required. MethodsIn this study, NP specimens were obtained from 20 patients with degenerative disc disease or scoliosis. LepR-Cre mice was crossed with R26R-Tdtomato mice to generate LepR-Cre; R26R-Tdtomato mice, which enabled fate-mapping of NPs from embryo stage to late adult. LMNA G609G/G609G mice was used to determine the effect of premature-aging induced IDD on LepR NPs. X-ray imaging was used to measure lumber disc height of mice. ResultsHere, we provide the first evidence that the leptin receptor (LepR) is preferentially expressed in NCs at embryonic stages and notochord-derived cells in the postnatal IVD. By using R26R-Tdtomato fluorescent reporter mice, we systematically analysed the specificity of activity and targeting efficiency of leptin receptor-Cre (LepR-Cre) in IVD tissues from the embryonic stage E15.5 to 6-month-old LepR-Cre; Rosa26-Tdtomato (R26R-Tdtomato) mice. Specifically, LepR-Cre targets a distinct subpopulation of notochord-derived cells closely associated with disc homoeostasis. The percentage of LepR-expressing NP cells markedly decreases in the postnatal mouse IVD and, more importantly, in the human IVD with the progression of IDD. Moreover, both spine instability–induced and premature ageing–induced IDD mouse models display the phenotype of IDD with decreased percentage of LepR-expressing NP cells. These findings uncover a potential role of LepR-expressing notochord-derived cells in disc homoeostasis and open the gate for therapeutically targeting the NP cell subpopulation. ConclusionIn conclusion, our data prove LepR-Cre mice useful for mapping the fate of specific subpopulations of IVD cells and uncovering the underlying mechanisms of IDD. The translational potential of this articleThe translation potential of article is that we first identified LepR as a candidate marker of subpopulation of nucleus pulposus (NP) cells and provided LepR as a potential target for the treatment of intervertebral disc degeneration (IDD), which have certain profound significance.

Derivation of notochordal cells from human embryonic stem cells reveals unique regulatory networks by single cell-transcriptomics.

Intervertebral disc degeneration (IDD) is a public health dilemma as it is associated with low back and neck pain, a frequent reason for patients to visit the physician. During IDD, nucleus pulposus (NP), the central compartment of intervertebral disc (IVD) undergo degeneration. Stem cells have been adopted as a promising biological source to regenerate the IVD and restore its function. Here, we describe a simple, two-step differentiation strategy using a cocktail of four factors (LDN, AGN, FGF, and CHIR) for efficient derivation of notochordal cells from human embryonic stem cells (hESCs). We employed a CRISPR/Cas9 based genome-editing approach to knock-in the mCherry reporter vector upstream of the 3' untranslated region of the Noto gene in H9-hESCs and monitored notochordal cell differentiation. Our data show that treatment of H9-hESCs with the above-mentioned four factors for 6 days successfully resulted in notochordal cells. These cells were characterized by morphology, immunostaining, and gene and protein expression analyses for established notochordal cell markers including FoxA2, SHH, and Brachyury. Additionally, pan-genomic high-throughput single cell RNA-sequencing revealed an efficient and robust notochordal differentiation. We further identified a key regulatory network consisting of eight candidate genes encoding transcription factors including PAX6, GDF3, FOXD3, TDGF1, and SOX5, which are considered as potential drivers of notochordal differentiation. This is the first single cell transcriptomic analysis of notochordal cells derived from hESCs. The ability to efficiently obtain notochordal cells from pluripotent stem cells provides an additional tool to develop new cell-based therapies for the treatment of IDD.

Knockdown of cytokeratin 8 overcomes chemoresistance of chordoma cells by aggravating endoplasmic reticulum stress through PERK/eIF2\u03b1 arm of unfolded protein response and blocking autophagy

Chordoma is a malignant primary osseous spinal tumor with pronounced chemoresistance. However, the mechanisms of how chordoma cells develop chemoresistance are still not fully understood. Cytokeratin 8 (KRT8) is a molecular marker of notochordal cells, from which chordoma cells were believed to be originated. In this study, we showed that either doxorubicin or irinotecan promoted KRT8 expression in both CM319 and UCH1 cell lines, accompanied by an increased unfolded protein response and autophagy activity. Then, siRNA-mediated knockdown of KRT8 chemosensitized chordoma cells in vitro. Mechanistic studies showed that knockdown of KRT8 followed by chemotherapy aggravated endoplasmic reticulum stress through PERK/eIF2α arm of unfolded protein response and blocked late-stage autophagy. Moreover, suppression of the PERK/eIF2α arm of unfolded protein response using PERK inhibitor GSK2606414 partially rescued the apoptotic chordoma cells but did not reverse the blockage of the autophagy flux. Finally, tumor xenograft model further confirmed the chemosensitizing effects of siKRT8. This study represents the first systematic investigation into the role of KRT8 in chemoresistance of chordoma and our results highlight a possible strategy of targeting KRT8 to overcome chordoma chemoresistance.

Pathologic Features of the Intervertebral Disc in Young Nova Scotia Duck Tolling Retrievers Confirms Chondrodystrophy Degenerative Phenotype Associated With Genotype.

Chondrodystrophy results in predictable and progressive biochemical and structural changes to the intervertebral disc, resulting in early onset degeneration and dystrophic mineralization of the disc. Accelerated degeneration and mineralization of the intervertebral disc are common in multiple dog breeds and can result in compromised function, herniation, pain, and a variety of neurological sequelae. A mutation responsible for chondrodystrophy in dogs has been identified as an aberrant fibroblast growth factor 4 (FGF4) retrogene insertion on chromosome 12 (CFA12) and is associated with short stature of the Nova Scotia Duck Tolling Retriever. Segregation of the CFA12 FGF4 retrogene in this dog breed provides an opportunity to examine the effect of retrogene presence on radiographic and histologic appearance of chondrodystrophic disc degeneration within a single breed. Here we found that in the intervertebral discs isolated from 2 dogs with the CFA12 FGF4 genotype, the nucleus pulposus was largely replaced by cartilaginous tissue, and physaliferous notochordal cells were rarely if ever identified. These findings are in contrast to the normal histologic findings in 2 breed-matched dogs lacking the mutation. The findings are consistent with premature chondroid degeneration of the intervertebral disc and suggest that the presence of the CFA12 FGF4 retrogene is sufficient to cause the chondrodystrophic phenotype.

Effects of Oxygen Concentration and Culture Time on Porcine Nucleus Pulposus Cell Metabolism: An in vitro Study.

Low back pain is a common ailment that affects millions of individuals each year and is linked to degeneration of the intervertebral discs in the spine. Intervertebral disc degeneration is known to result from an imbalance in anabolic and catabolic activity by disc cells. Due to the avascular nature of the intervertebral disc, oxygen deficiency may occur in the central nucleus pulposus (NP). The resulting hypoxia affects matrix regulation and energy metabolism of disc cells, although the mechanisms are not fully understood. This study investigates in vitro glucose consumption and gene expression by NP cells over time under varying oxygen tensions. Notochordal porcine NP cells were cultured in agarose discs at 21, 5, or 1% oxygen tension for 1, 5, or 10 days. The expression of 10 key matrix genes, as well as Brachyury (T), by NP cells was analyzed using RT-PCR. Glucose consumption was measured using a two-point method. Results show that culture time and oxygen tension significantly affect glucose consumption rates by porcine NP cells. There were also significant changes in T expression based on oxygen level and culture time. The 1% oxygen tension had a significantly higher T expression on day 10 than the other two groups, which may indicate a better maintenance of the notochordal phenotype. MMP 1 and 13 expression increased over time for all groups, while only the 5% group showed an increase over time for MMP 3. TIMP expression followed the direction of MMPs but to a lesser magnitude. Five percent and twenty-one percent oxygen tensions led to decreases in anabolic gene expression while 1% led to increases. Oxygen concentration and culture time significantly impacted glucose consumption rate and the gene expression of matrix regulatory genes with hypoxic conditions most accurately maintaining the proper NP phenotype. This information is valuable not only for understanding disc pathophysiology, but also for harnessing the potential of notochordal NP cells in therapeutic applications.