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A bioactive hydrogel harnessing the regenerative potential of notochordal cells serves as instructive cell carrier for nucleus pulposus repair [scRNA-seq dog]

ABSTRACT: Natural biomatrices are popular owing to their ability to mimic tissue-specific biological properties. These properties are crucial for the intervertebral disc, a particularly demanding tissue whose degeneration is a major cause of chronic lower back pain. Degeneration starting within the core of the disc, the nucleus pulposus (NP), is marked by the loss of notochordal cells (NCs) and associated healthy extracellular matrix. The regulative potential of the NC-secretome was exploited through a PEG-based hydrogel formulated with decellularized porcine notochordal cell matrix (dNCM). Even under conditions which mimic the degenerate niche, the hydrogel supported maintenance of the phenotype of native porcine NCs, well-known for the difficulty in preserving their vacuolated morphology. dNCM-PEG hydrogel carrying human mesendodermal progenitors (hMEPCs; iPSC-derived and capable of differentiating into NCs), was injected into NP explants with enzymatically induced matrix degradation and subjected to dynamical loading. hMEPCs engrafted successfully and a healthy disc cell phenotype was observed. Injection of dNCM+hMEPCs into degenerated discs in a pilot experimental dog study indicated that ~7 % of the 0.5 million hMEPCs engrafted. Single cell RNAseq analysis showed over 30 % of the engrafted and recovered hMEPCs co-expressed Collagen Type II and Aggrecan consistent with a functional phenotype. No tumorigenic or systemic immunogenic side effects were observed. At the tissue level, TBXT expression, as well as matrix quality, were enhanced in the treated degenerate disc tissues. Together, this study highlights the translational potential of combining cell-based therapies with a bioactive material containing part of the NC secretome, warranting further development and validation.

ORGANISM(S): Canis lupus familiaris

PROVIDER: GSE313238 | GEO | 2025/12/30

REPOSITORIES: GEO

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Project description:Objective: Aging and early degeneration of the intervertebral disc (IVD) involves the substition of notochordal cells (NCs) in the nucleus pulposus (NP) by chondrocyte-like cells (CLCs). This study investigated the gene expression profiles involved in this process using NP tissue from both non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occuring IVD degeneration. Methods: Dual channel DNA microarrays were used to compare 1) healthy NP tissue, 2) NP tissue with a mixed population of NCs and CLCs, and 3) NP tissue containing solely CLCs. Canonical Wnt-signaling was validated using qPCR of relevant Wnt target genes. Caveolin-1, a known regulator of canonical Wnt signaling, was investigated further in tissue sections using qPCR and immunohistochemistry, and in cultured NCs by qPCR and immunofluorescence. Also, the NP of 3-month-old caveolin-1 knock-out mice was histopathologically evaluated and compared with wild type mice of the same age. Results: Early IVD degeneration involved significant regulations in numerous pathways, such as extracellular matrix remodeling, Bone Morphogenetic Protein- , and Wnt/beta-catenin-signaling. With regard to Wnt/beta-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant downregulation of axin2 gene expression and caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of 3-month-old WT mice were rich in viable NCs, whereas the NPs of 3-month-old caveolin-1 knock-out mice contained chondroid-like matrix with small, rounded cells, the majority of which showed morphological signs of apoptosis. Conclusions: Aging and the onset of degeneration of the IVD involve significant downregulation of canonical Wnt signaling and caveolin-1, which appears to be essential in the physiology and preservation of NCs. DNA microarrays were used to compare nucleus palposus (NP) tissue of healthy and chondrodystrophic individuals. Furthermore, the situation of the tissue was divided into three stages: NCR: notochordal cell(NC) rich; CR: tissue containing solely chondrocyte-like-cells (CLC) and T: tissue with a mixed population of NCs and CLCs. Comparisons were analysed on a 2-color platform against a common reference sample, consisting of a multitude of canine organs, including liver, spleen, kidney, lung, hart, intestine and bone.

Project description:Purpose: Degeneration and aging of the nucleus pulposus (NP) of the intervertebral disc (IVD) is accompanied by alterations in NP cell phenotype marked by reduced cellularity and a shift towards a fibroblast-like state. We have recently demonstrated an ability to manipulate the phenotypic expression of adult degenerative NP cells by culture upon poly(ethylene glycol) (PEG) based hydrogels dually functionalized with integrin- and syndecan-binding laminin-mimetic peptides. In the present study, we seek to understand the transcriptome changes elicited by NP cell interactions with the presented hydrogel system. Methods: mRNA profiles of NP from 3 human tissue samples were cultured for 4 days on either tissue culture polysyrene (TCPS) or the functionalized gel before RNA was isolated and sequenced, using Illumina NovaSeq. Unaligned reads were trimmed based on quality score (minimum quality level (Phred) = 20, minimum read length = 25) and aligned to the whole human genome (STAR 2.6.1d; hg19) using Partek Flow software. This software suite was also used to calculate the counts/normalized counts of genes and to perform gene specific analysis (GSA), principle component analysis (PCA), hierarchical clustering, and gene set enrichment. Differentially regulated genes were considered to be those with a fold change value (gel/TCPS) that was greater than or equal to 2 or less than or equal to negative 2 and a p-value of less than or equal to 0.05. Results: The data corroborate and expand on the previous findings by demonstrating that degenerative adult human NP cells cultured upon these gels have upregulations in some markers of NP and notochordal cells but also downregulations of some NP-specific markers and several fibroblastic markers. Furthermore, through gene set enrichment analysis we have shown that pathways related to cell differentiation and notochord morphogenesis were upregulated in the gel condition. Additionally, 13 genes associated with G protein-coupled receptors, many of which are known drug targets, were identified as up or downregulated following periods of culture upon the gel condition. Conclusions: The data from this RNA-sequencing demonstrate that culture on laminin-mimetic-peptide presenting gels can modulate cell phenotype and promotes upregulation of NP and notochordal markers.

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A bioactive hydrogel harnessing the regenerative potential of notochordal cells serves as instructive cell carrier for nucleus pulposus repair [scRNA-seq dog]

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