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Engineered Peptide-Functionalized Hydrogels Modulate RNA Transcriptome of Human Nucleus Pulposus Cells In Vitro

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE154044 | GEO | 2021/02/01

REPOSITORIES: GEO

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Project description:Purpose: It has been observed that the drug verteporfin (VP), a known disruptor of YAP-TEAD signaling, causes cell rounding and changes in phenotype and biosynthesis in human nucleus pulposus cells. In this study the time-dependent effects of VP treatment on the transcriptome of human NP cells was explored. Methods: mRNA profiles of NP cells treated for 24 hours, 48 hours, or 4 days with VP or vehicle (DMSO) control were generated for cells from 3 adult human tissue samples, 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 (VP/DMSO) for a given time point that was greater than or equal to 2 or less than or equal to negative 2 and a FDR-adjusted p-value of less than or equal to 0.05. Results: Using a data analysis workflow buit in Partek Flow, we identified 1810 differentially regulated genes at 24 hours, 4287 differentially regulated genes at 48 hours, and 9272 differentially regulated genes at 4 days. Hierarchical clustering and PCA analysis demonstrated clustering of the data by patient and time point, but separation by treatment group. We further observed that while generally the expression of fibroblastic markers decreased with VP treatment, a number of markers of NP phenotype were increased. We also identified enriched gene sets (ex. 2026 at the 4 day time point) which represented a variety of cellular components, processes, and functions potentially implicated by VP treatment. Conclusions: The data from this RNA-sequencing demonstrate that VP treatment which induces cell rounding in NP cells was also accompanied by pronounced and time-dependent changes in the transcriptome of adult NP cells in culture. The enriched gene sets and differentially regulated genes suggest a major role for cell adhesion, cytoskeletal remodeling, vacuolar lumen, and M APK activity in the NP phenotypic and functional response to changes in cell shape.

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.

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Engineered Peptide-Functionalized Hydrogels Modulate RNA Transcriptome of Human Nucleus Pulposus Cells In Vitro

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