Engineered Peptide-Functionalized Hydrogels Modulate RNA Transcriptome of Human Nucleus Pulposus Cells In Vitro
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PROVIDER: PRJNA644918 | ENA |
REPOSITORIES: ENA
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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.
2021-02-01 | GSE154044 | GEO
Project description:Biologically ligands (e.g., RGDS from fibronectin: Fn-RGD) play a critical role in the development of chemically defined biomaterials. However, there has been limited progress in recent decades towards discovering novel extracellular-matrix-protein-derived ligands for translational applications. Here, by combining motif analysis of evolutionarily conserved RGD-containing regions in laminin (Ln) with functional microarray screening, we identified a Ln-derived angiogenic peptide (LDAP) that showed enhanced proangiogenic activities over commonly used Fn-RGD. Mechanistic studies using RNA-sequencing of LDAP functionalized hydrogels showed an improved angiogenic transcriptome over Fn-RGD functionalized hydrogels and high similarity to Matrigel, attributed to the ability of LDAP to engage both Ln- and Fn-binding integrins. Injectable hydrogels functionalized with LDAP, along with MMP-QK (a VEGF-mimetic peptide), exhibited increased functional recovery over decellularized extracellular matrix (dECM) and alginates functionalized with Fn-RGD and MMP-QK in a mouse ischemic hindlimb model, illustrating the power of the strategy to rapidly develop potent chemically-defined biomaterials for therapeutic applications.
2020-09-21 | GSE126717 | GEO
Project description:Abstract: Stem cell therapies are severely limited by poor cell survival and engraftment rates. A major hurdle to the use of materials for cell delivery is the lack of understanding of material properties that govern transplanted stem cell functionality. Here, we show that synthetic hydrogels presenting integrin-specific peptides enhance the survival, persistence, and osteo-reparative functions of human mesenchymal stem cells (hMSC) transplanted in murine bone defects. Integrin-specific hydrogels regulated hMSC adhesion, paracrine signaling, and osteoblastic differentiation in vitro. Hydrogels presenting GFOGER, a triple helical peptide targeting α2β1 integrin, prolonged hMSC survival and engraftment in a segmental bone defect and resulted in improved bone repair compared to other adhesive and control peptides. Moreover, integrin-specific hydrogels had diverse pleiotropic effects on hMSC reparative activities, modulating in vitro cytokine secretion and in vivo gene expression for effectors associated with inflammation, vascularization, and bone formation. These results demonstrate that integrin specificity can be engineered into synthetic hydrogel systems resulting in improved tissue healing by directing mesenchymal stem cell survival, engraftment, paracrine immunomodulatory potential, and reparative activities. Radial segmental defects were treated with hydrogels functionalized with adhesive peptide and encapsulated hMSC. The tissue within the defect space was explanted at 1 week post-transplantation and stored in RNAlater solution (Qiagen) until further processing. Samples were placed in Qiazol solution (Qiagen), lysed by probe sonication, and homogenized in QIAshredder columns (Qiagen). Total RNA was isolated using an RNAeasy Plus Micro kit (Qiagen), and RNA content and purity were measured by spectrophotometry (NanoDrop 1000). cDNA synthesis was performed on total RNA (100 ng) using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher). Quantitative PCR was performed using Fluidigm 96×96 nanofluidic arrays targeting a set of 96 transcripts (human or murine) to observe changes in bone, survival, inflammation, vascularization, and matrix markers. Primers used are listed in Supplementary Table 4. The genes were pre-amplified in a single 13-cycle PCR reaction for each sample with EvaGreen Mastermix (Fluidigm BioMark) following the manufacturer’s protocol. Sixty-three gene targets resulted in detectable qPCR results. All subsequent statistical analyses were carried out using JMP-Genomics (SAS Institute) using the basic gene expression workflow 62. Raw Ct values were imported into JMP-Genomics and normalized to mean Ct values across all genes for each sample for principal components analysis (PCA), assessment of the biological principal variance component contributions (PVCA), and hierarchical clustering using Ward’s method to identify sub-types of expression profile. Finally, one-way ANOVA was used to detect statistical differences followed by False Discovery Rate analysis using two-state linear step-up procedure of Benjamini, Krieger and Yekutieli. Results are presented as raw Ct values normalized to mean Ct values across all genes for a sample.
2019-12-09 | GSE141517 | GEO
Project description:Human salivary gland organoids have opened tremendous possibilities for regenerative medicine in patients undergoing radiotherapy for the treatment of head and neck cancer. However, their clinical translation is greatly limited by the current use of Matrigel for organoid derivation and expansion. Here, we envisage that the use of a fully, synthetic hydrogel based on the oligo(-ethylene glycol) functionalized polymer polyisocyanopeptides (PICs) can provide an environment suitable for the generation and expansion of salivary gland organoids (SGOs) after optimization of PIC polymer properties. We demonstrate that PIC hydrogels decorated with the cell-binding peptide RGD allow SGO formation from salivary gland (SG)-derived stem cells. This self-renewal potential is preserved for only 4 passages. It was found that SGOs differentiated prematurely in PIC hydrogels affecting their self-renewal capacity. Similarly, SGOs show decreased expression of immediate early genes (IEGs) after culture in PIC hydrogels. Activation of multiple signalling pathways involved in IEG expression by β-adrenergic agonist isoproterenol, led to increased stem cell self-renewal capacity as measured by organoid forming efficiency (OFE). These results indicate that PIC hydrogels are promising 3D matrices for SGOs, with the option to be used clinically, after further optimization of the hydrogel and culture conditions.
2022-12-10 | GSE220084 | GEO
Project description:scRNA-seq was used to characterise hiPSC-derived kidney organoids differentiated within fully synthetic self-assembling peptide hydrogels of variable mechanical strengths and compare these to organoids differentiated within the animal-derived matrix, Matrigel. Organoids were matured in the respective matrices until day 24 of differentiation and 6 organoids per support matrix were then pooled and dissociated using the cold-active protease from Bacillus licheniformis. Cells were processed on the 10x Genomics Chromium platform using the Single-Cell 3’ v3.1 protocol. The NextSeq500 (Illumina) was used to sequence the libraries generated and initial processing of the data was carried out using the 10X Genomic Cell Ranger v3.1.0 pipeline.
2022-10-31 | E-MTAB-11149 | biostudies-arrayexpress
Project description:Human neural organoid models have become an important tool for studying neurobiology. In this work, we compared Matrigel to an N-cadherin peptide-functionalized gelatin methacryloyl hydrogel (termed GelMA-Cad) for culturing cortical neural organoids. Specifically, we compare five materials: (1) Matrigel, (2) GelMA-Cad with high crosslinker (HC), (3) GelMA-Cad with low crosslinker (LC), (4) GelMA HC and (5) GelMA LC. We determined that both mechanical properties and peptide presentation can tune cell fate and diversity in gelatin-based matrices during differentiation. Of particular note, cortical organoids cultured in GelMA-Cad produce higher numbers of neurogenic and ciliated radial glia and upper-layer excitatory neurons—an important population for modeling neurodegenerative disease—compared to GelMA and Matrigel controls.
2024-02-28 | E-MTAB-13828 | biostudies-arrayexpress
Project description:Synovial and bone marrow mesenchymal stem cells after intradiscally injection show regenerative effects of nucleus pulposus. Microarray analyses of rats were performed to investigate the closeness of the gene profiles between the nucleus pulposus cells and the synovial or bone marrow mesenchymal stem cells. To investigate interaction between synovial mesenchymal stem cells and nucleus pulposus cells, human synovial mesenchymal stem cells and rat nucleus pulposus cells were co-cultured, and species specific microarray were performed. Synovium of knee joints, bone marrow and nucleus pulposus were harvested from rat or human, and cells were isolated for RNA extraction and hybridization on Affymetrix microarrays. To compare the gene profiles each other, isolated cells were mono-cultured respectively, and human synovial mesenchymal stem cells and rat nucleus pulposus cells were co-cultured.
2010-10-11 | E-GEOD-24612 | biostudies-arrayexpress
Project description:MicroRNAs expression profiling of human nucleus pulposus cells derived from patients with disc degeneration in comparison with those derived from patients with scoliosis as control. Two-condition experiment: control nucleus pulposus cells vs. degenerative nucleus pulposus cells. Biological replicates: 3 control, 3 degenerated, independently harvested. Four replicates per array. The supplementary file 'GSE19943_fold_pvalue.txt' contains fold-changes and p-values.
2012-01-01 | E-GEOD-19943 | biostudies-arrayexpress
Project description:Integrin-specific hydrogels modulate transplanted human mesenchymal stem cell survival, engraftment, and reparative activities
| PRJNA593767 | ENA
Project description:Natural materials can reversibly self-assemble into hierarchical networks with diverse structural and functional properties. Recreating these dynamic architectures using peptide-based synthetic materials has been an elusive goal, hindered by challenges in relating sequence to structure to function. Here we report on the de novo discovery of short peptides based on the “tryptophan zipper” (Trpzip) motif that self-assemble into hierarchically structured hydrogels. Trpzip hydrogels have a tunable modulus and show self-healing, stress relaxation, antimicrobial properties, and biocompatibility. The low yield point allows syringe extrusion with cytoprotection, and cell harvest with a flick of the wrist. Integrating a pendant cell adhesion motif promotes human intestinal organoid growth and differentiation, with polarity control and improved shape fidelity. Considering these unique characteristics, we anticipate Trpzip hydrogels will prove a versatile reagent for biotechnology and medicine.
2023-10-25 | PXD038870 | Pride