Verteporfin treatment controls morphology, phenotype, and global gene expression for cells of the human nucleus pulposus

Abstract

Cells of the nucleus pulposus (NP) are essential contributors to extracellular matrix synthesis and function of the intervertebral disc. With age and degeneration, the NP becomes stiffer and more dehydrated, which is associated with a loss of phenotype and biosynthetic function for its resident NP cells. Also, with aging, the NP cell undergoes substantial morphological changes from a rounded shape with pronounced vacuoles in the neonate and juvenile, to one that is more flattened and spread with a loss of vacuoles. Here, we make use of the clinically relevant pharmacological treatment verteporfin (VP), previously identified as a disruptor of yes-associated protein-TEA domain family member-binding domain (TEAD) signaling, to promote morphological changes in adult human NP cells in order to study variations in gene expression related to differences in cell shape. Treatment of adult, degenerative human NP cells with VP caused a shift in morphology from a spread, fibroblastic-like shape to a rounded, clustered morphology with decreased transcriptional activity of TEAD and serum-response factor. These changes were accompanied by an increased expression of vacuoles, NP-specific gene markers, and biosynthetic activity. The contemporaneous observation of VP-induced

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

“…It has widely been demonstrated that many cell types are able to robustly sense and respond to physical and chemical features of their environment 20,27,58 . These responses can be directed by using engineered substrates with defined features, such as the LMP gel studied here, to promote cell differentiation or phenotypes in vitro 38,39,[59][60][61] or for treatment of diseases in vivo 28,62-64 . Characteristics that have been shown to modulate cell behavior in 2D include the stiffness of the underlying substrate, ligand presentation, geometry of adhesive area (as controlled through micropatterning), and ligand density 9,30,35,39,43,59,[65][66][67][68] . We have recently demonstrated that a stiff gel (15% PEG) conjugated with equimolar amounts of the laminin-mimetic peptides AG73 and IKVAV could be used to promote degenerative cells to assume a phenotype consistent with healthy, juvenile NP cells 14 .…”

“…Primary adult human NP cells from each of three patients (24-year-old female, 53-year-old female, and 55-year-old male) were seeded in triplicate upon LMP gels or uncoated tissue culture polystyrene (TCPS) at a density of 100,000 cells per well in a 4-well chamber slide, consistent with previously published protocols 14,43 . Cells were cultured for 4 d at 37°C and 5% CO 2 .…”

“…Using Partek Flow® software (Partek Inc., St. Louis, MO), unaligned reads were trimmed prior to alignment to the human genome (STAR 2.6.1d and referencing to the human genome hg19). Gene Specific Analysis (GSA) was conducted in Partek Flow® and used to perform downstream analysis (identification of differentially expressed genes, principal component analysis, hierarchical clustering, and gene set enrichment) using methodology as previously described 43 . GSA was used to identify a list of upregulated and downregulated genes.…”

“…FASTQ files provided by GTAC were used in subsequent analysis. hierarchical clustering, and gene set enrichment) using methodology as previously described 43 .…”

Advancing basic and preclinical spine research: Highlights from the ORS PSRS 5th International Spine Research Symposium

“…Hernandez et al describe a new protocol for 3D imaging of nucleus pulposus (NP) cells in alginate constructs and in their native tissue environment using confocal microscopy, permitting the high‐resolution detection of phenotypical and cytoskeletal changes in these cells without the need for histological processing 1 . Fearing et al used a pharmacological‐based approach to investigate the relationships between NP cell morphology and gene expression 2 . They showed that treating degenerate NP cells with verteporfin promoted a rounded, clustered morphology and increased expression of vacuoles and NP‐specific gene markers, and biosynthetic activity.…”