Next Generation Sequencing (NGC) Analysis of Gene Expression Proliles in Human Gingiva-derived Mesenchymal Stem Cells (GMSCs) Cultured in Methacrylated 3D-Collagen Hydrogel Compared to 2D-cultured Counterparts

ABSTRACT: Purpose: The goals of this study are to perform NGC analysis in order to compare the gene expression profiles of GMSCs cultured in 3D-collagen hydrogel and their 2D-cultured counterparts. Methods: mRNA profiles of 3D- and 2D-cultured GMSCs were generated by deep sequencing, in triplicate, using Illumina Xten. The read alignment is done using HISAT2 v.2.0.5 software with Ensembl GRCh38 genome as reference genome. Transcripts Assembly is done using StringTie v.1.3.3b software. Significant DE genes or transcripts (q-value < 0.05) were extracted by edgeR (R package) for each comparison groups. DEGs were analyzed with gene ontology enrichment analysis and KEGG by R software with clusterProfiler package. Significant GO or KEGG terms (FDR-value < 0.05) were extracted using hypergeometric distribution. qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: Using an optimized data analysis workflow, significant differentially expressed genes (DEGs) were defined as those with a log2 fold change (FC) ≥1 (GMSC-3D v.s.GMSC-2D) and a false discovery rate (FDR) of ≤ 1%. A total of 5588 DEGs, including 3476 upregulated and 2112 downregulated genes were identified between 3D-cultured GMSCs and 2D-cultured counterparts with a fold change ≥1.0 and p value <0.05. Altered expression of 17 genes reltaed to neural crest stem cells, mesenchymal cells, and NOTCh signaling pathways was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical cluster analysis revealed a different hierarchical clustering algorithm between 3D- and 2D-cultured GMSCs as illustrated in the Heatmap, which uncovered several as yet uncharacterized clusters of genes that may contribute to the conversion of GMSCs into neural crest stem-like (NCSC) or Schwann cell precursor (SCP)-like cells under a defined 3D-collagen hydrogel condition. Conclusions: Our study represents the first detailed comparative analysis of gene expression profiles between 2D- and 3D-cultured GMSCs, with biologic replicates, generated by RNA-seq technology. Our RNA-seq results show that GMSCs cultured in defined 3D-collagen hydrogel underwent conversion into neural crest stem-like (NCSC)/Schwann cell precursor-like (SCP) cells charcaterized by upregulation of a panel of neural crest-related genes, NOTCH signaling components, and downregulation of a panel of mesenchymal cell-related genes. We conclude that RNA-seq based transcriptome characterization would permit further dissection of complex molecular mechanisms underlying 3D collagen hydrogel-driven conversion of GMSCs into NCSC/SCP-like cells..

ORGANISM(S): Homo sapiens

PROVIDER: GSE180519 | GEO | 2021/10/19

REPOSITORIES: GEO