High-depth transcriptomic profiling reveals the temporal gene signature of human mesenchymal stem cells during chondrogenesis

Abstract

Mesenchymal stem/stromal cells (MSCs) provide an attractive cell source for cartilage repair and cell therapy; however, the underlying molecular pathways that drive chondrogenesis of these populations of adult stem cells remain poorly understood. We generated a rich data set of high-throughput RNA sequencing of human MSCs throughout chondrogenesis at 6 different time points. Our data consisted of 18 libraries with 3 individual donors as biologic replicates, with each library possessing a sequencing depth of 100 million reads. Computational analyses with differential gene expression, gene ontology, and weighted gene correlation network analysis identified dynamic changes in multiple biologic pathways and, most importantly, a chondrogenic gene subset, whose functional characterization promises to further harness the potential of MSCs for cartilage tissue engineering. Furthermore, we created a graphic user interface encyclopedia built with the goal of producing an open resource of transcriptomic regulation for additional data mining and pathway analysis of the process of MSC chondrogenesis.-Huynh, N. P. T., Zhang, B., Guilak, F. High-depth transcriptomic profiling reveals the temporal gene signature of human mesenchymal stem cells during chondrogenesis.

Keywords: RNA-Seq; chondrocyte; lncRNA; miRNA; pericyte.

Figure 1

Distribution of MSC surface markers. A) Experimental design. MSCs were formed into pellets after passage 4 and cultured under chondrogenic conditions for up to 21 d. Pellets were collected at d 0, 1, 3, 7, 14, and 21. B) Representative histograms from 3 donors. Control samples stained with isotypes are depicted in red; samples stained with surface markers are depicted in blue.

Figure 2

MSC chondrogenesis in pellet culture. A) Biochemical assays measuring GAG and DNA over time course of analysis. There was increase in GAG accumulation from d 3 to later time points (left and right), while DNA levels remained similar across days (middle). Mean ± sd. n = 3 independent specimens per donor (3 donors as biologic replicates). Two-way ANOVA followed by Tukey post hoc test (α = 0.05). Groups of different letters are statistically different from one another. B) Safranin O/Fast Green staining on pellets from 3 donors at different time points. C–E) Immunohistochemistry staining on pellets from 3 donors at different time points: collagen type II (C); collagen type I (D); collagen type X (E). Scale bars, 500 µm.

Figure 3

Differential expression (DE) analysis. A) PCA of 18 MSC-derived pellet samples. Samples were closely related by days, and there was minor donor-to-donor variation. B) PCA of MSC-derived pellet samples vs. human cartilage at different developmental stages. C) Expression patterns of MSC markers and markers of potential lineages. Gene expression was represented as log₂ fold change compared to d 0 samples. D) K-means clustering grouped DE genes into 4 patterns. Gene expression was represented as log₂ fold change compared to d 0 samples. E) Bar graphs of enriched pathways from gene ontology analysis. Datasets for human cartilage were retrieved using GEO accession number GSE106292 (79).

Figure 4

Modules and their relationships to biochemical changes. A) Dendrogram of RNA-Seq samples and corresponding changes in traits. Lowest values are depicted in white; highest values are depicted in red; missing values are depicted in gray. B) Eigengene adjacency heat map with blocks of metamodules. C) Modules whose eigengenes are highly correlated with changes in GAG per DNA and their corresponding Pearson correlation values.

Figure 5

Characteristics of module steel blue. A) Coexpression network of 20 top hubs. B) Coexpression network of involved TFs (red) and their top 3 connected genes (steel blue). C) Heat map of steel blue modular gene expression. D) Eigengene vector of module steel blue. E) Enriched Gene Ontology terms and pathways from modular genes with gene ontology analysis.

Figure 6

Characteristics of module magenta. A) Coexpression network of 20 top hubs. B) Coexpression network of involved TFs (red) and their top 3 connected genes (magenta). C) Heat map of magenta modular gene expression. D) Eigengene vector of module magenta. E) Enriched Gene Ontology terms and pathways from modular genes with gene ontology analysis.