Biology subjects

Kaneda, G.

Publications and source records attributed to Kaneda, G..

5 recordsLinked to original sources

bioRxiv2025

A Porcine Model of Intervertebral Disc Injury Recapitulates Human Discogenic Pain via Notochordal Cell Loss and Pain-inducing Nucleus Pulposus Cell Emergence

Lower back pain (LBP) is one of the most common causes of disability, with up to 40% of LBP cases attributed to intervertebral disc (IVD) degeneration. While small animal models are widely used to study IVD and LBP, the small size of their IVDs limits translational and biological relevance. Large animal models more accurately emulate human disease; however, methods of measuring LBP are not well established. Pigs were also considered unfit for LBP research, due to notochordal cells (NCs) persistence through life, unlike humans. We developed a comprehensive porcine model with quantitative measure of discogenic pain via biobehavioral testing (BBT), MRI, and multi-omics tissue analysis of IVD and DRGs. MRI demonstrated the progression of IVD degeneration beginning at 4 weeks post-injury. BBTs showed the development of significant pain responses by week 4 post-injury, supported by transcriptomics of injury matched DRGs. Single cell transcriptomics, trajectory and cell-cell communication analyses suggest that, with injury, NCs are differentiating to nucleus pulposus cells (NPCs). Furthermore, NPCs showed upregulation of cellular stress, neural outgrowth, and inflammation pathway, consistent with pain-inducing distress signals found in human samples. This study establishes novel MRI and BBT-based methods for quantifying LBP in pigs and supports its translational relevance to human discogenic LBP. The identification of LBP-associated clusters mirrors our previous finding in humans. Moreover, the shift of NC to NPC phenotype further supports that the porcine model is relevant to human pathology, as the injury induced accelerated aging and loss of NCs with IVD degeneration and discogenic pain.

developmental biology↗

bioRxiv2025

Single Cell Proteomics Reveals Novel Cell Phenotypes in Marfan Mouse Aneurysm

BackgroundSingle-cell omics technology is a powerful tool in biomedical research. However, single cell proteomics has lagged due to an inability to amplify peptides in a similar fashion to nucleotide strings. Single cell proteomics is important because proteins are the main functional unit in cells, and they often poorly correlate with mRNA quantities. In this paper we describe the first single cell proteomic analysis of complex tissue, comparing aneurysmal and normal mouse aorta from males and females. We also compare and integrate our single cell proteomic profiles with a matching single cell transcriptomics dataset. MethodsWe compared single cell proteomes between male and female, wild-type and Fbn1C1041G/+ Marfan mice (N=3 per group). Individual cells from mouse aortic root single cell suspensions were deposited in 384 well plates and subjected to ultra-sensitive nanoflow liquid chromatography-ion mobility-time of flight-mass spectrometry. The data were analyzed with leiden clustering to identify cell types. Statistical analyses were performed to detect differential proteins within cell types and multi-omics analysis integrated single cell proteomics with published single cell RNA-seq. ResultsWe identified all major aortic cell types including 7 distinct smooth muscle cell subtypes. The proportion of these cells varied based on sex and the Fbn1C1041G/+ genotype. Differentially expressed proteins between male and female in addition to wild-type and Marfan samples uncovered enhanced endothelial to mesenchymal transition patterns in endothelial cells from male Marfan mice. Comparisons between single cell RNA and single cell proteomic profiles showed similarities in major subtypes but not smooth muscle cell subtypes. Multi-omics analysis of these two single cell platforms demonstrated a potential novel role for smooth muscle cell derived angiotensin signaling in the Marfan phenotype. ConclusionsSingle cell proteomics identified new subpopulations of vascular smooth muscles cells and novel cell type specific protein signatures related to sex differences and aneurysm formation. AbbreviationsNext generation sequencing (NGS), Mass spectrometer (MS), Single cell proteomics by Mass Spectrometry (ScOPE-MS), Marfans syndrome (MFS), Fibrillin 1 (FBN1), Transforming growth factor {beta} (TGF{beta}), Smooth muscle cell (SMC), Single cell proteomic (scProteomic), Differentially expressed proteins (DEPs), Wild-type (WT), Hanks balanced salt solution (HBSS), Fetal bovine serum (FBS), Dulbeccos Modified Eagle Medium (DMEM), Data-independent acquisition parallel accumulation-serial fragmentation (DIA-PASEF), Magnetic assisted cell sorted (MACS), Single Cell Analysis in Python (Scanpy), Kyoto Encyclopedia of Genes and Genomes (KEGG), Principal component analysis (PCA), Uniform manifold projection (UMAP), Single cell transcriptomic (scTranscriptomic), Smoothelin (Smtn), Transgelin (Tagln), Myosin heavy chain 11 (Myh11), Platelet endothelial cell adhesion molecule 1 (Pecam1), Dipeptidase 1 (Dpep1), Uncoupling protein 1 (Ucp1), Low-density lipoprotein receptor-related protein (Lrp1), DNA ligase 3 (Lig3), Capsaicin channel transient receptor potential vanilloid 1 (Trpv1), Endothelial to mesenchymal transition (endMT), Intercellular adhesion molecule 1 (Icam1), Intercellular adhesion molecule 2 (Icam2), Endothelial cell-selective adhesion molecule (Esam), Calponin 1 (Cnn1), Vimentin (Vim), Zinc finger E-box-binding homeobox 1 (Zeb1), Snail family transcriptional repressor 1 (Snai1), Tropomyosin alpha-4 chain (Tpm4), Angiotensin converting enzyme (Ace)

systems biology↗

bioRxiv2023

Environmentally stressed human nucleus pulposus cells trigger the onset of discogenic low back pain

Low back pain (LBP) is often associated with the degeneration of human intervertebral discs (IVDs). However, the pain-inducing mechanism in degenerating discs remains to be elucidated. Here, we identified a subtype of locally residing nucleus pulposus cells (NPCs), generated by the environmental stress in degenerating discs, that triggered the onset of discogenic LBP. Single-cell transcriptomic analysis of human tissues showed a strong correlation between this specific pain-triggering subtype and the pain conditions in human degenerated discs. Next, we recreated this pain-triggering subtype by applying known exogenous stressors to healthy NPCs in vitro. The recreated pain phenotype activated functional sensory neurons response in vitro and induced local inflammatory responses, hyperalgesia, and mechanical sensitivity in a healthy rat IVD in vivo. Our findings provide strong evidence of a previously unknown pain-inducing mechanism mediated by NPCs in degenerating IVDs. This newly defined pathway will aid in the development of NPC-targeted therapeutic strategies for clinically unmet need to attenuate discogenic LBP. One Sentence SummaryDiscogenic low back pain can be initiated by a stress-induced subtype of nucleus pulposus cells present in human degenerating intervertebral discs

molecular biology↗

bioRxiv2021

Directing iPSC Differentiation into iTenocytes using Combined Scleraxis Overexpression and Cyclic Loading

Regenerative therapies for tendon are falling behind other tissues due to the lack of an appropriate and potent cell therapeutic candidate. This study aimed to induce cell tenogenesis using stable Scleraxis (Scx) overexpression in combination with uniaxial mechanical stretch of mesenchymal stromal cells (MSCs) of different origins. Scleraxis (Scx) is the single direct molecular regulator of tendon differentiation known to date. Mechanoregulation is known to be a central element guiding tendon development and healing. Cells explored were bone marrow-derived (BM-)MSCs as well as MSCs differentiated from induced pluripotent stem cells (iMSCs). Mechanical stimulation combined with Scx overexpression resulted in morphometric and cytoskeleton-related changes, upregulation of early and late tendon markers, increased ECM deposition and alignment, and tenomodulin perinuclear localization in iMSCs, which was greater compared to BM-MSCs and controls. Our findings suggest that these cells can be differentiated into tenocytes and may be a better candidate for tendon cell therapy applications than BM-MSCs.

cell biology↗

bioRxiv2021

Single-cell Atlas Unveils Cellular Heterogeneity and Novel Markers in Human Neonatal and Adult Intervertebral Discs

The origin, composition, distribution, and function of cells in the human intervertebral disc (IVD) has not been fully understood. Here, cell atlases of both human neonatal and adult IVDs have been generated and further assessed by gene ontology pathway enrichment, pseudo-time trajectory, histology, and immunofluorescence. Comparison of cell atlases revealed the presence of two sub-populations of notochordal cells (NCs) and their associated markers in both the neonatal and adult IVDs. Developmental trajectories predicted 7 different cell states that describe the developmental process from neonatal to adult cells in IVD and analyzed the NCs role in the IVD development. A high heterogeneity and gradual transition of annulus fibrosus cells (AFCs) in the neonatal IVD was detected and their potential relevance in IVD development assessed. Collectively, comparing single-cell atlases between neonatal and adult IVDs delineates the landscape of IVD cell biology and may help discovering novel therapeutic targets for IVD degeneration.

developmental biology↗