Short heat shock has a long-term effect on mesenchymal stem cells' transcriptome

Abstract

The adverse effects of heat stress (HS) on physiological systems are well documented, yet the underlying molecular mechanisms behind it remain poorly understood. To address this knowledge gap, we conducted a comprehensive investigation into the impact of HS on mesenchymal stem cells (MSCs), focusing on their morphology, phenotype, proliferative capacity, and fate determination. Our in-depth analysis of the MSCs' transcriptome revealed a significant influence of HS on the transcriptional landscape. Notably, even after a short period of stress, we observed a persistent alteration in cell identity, potentially mediated by the activation of bivalent genes. Furthermore, by comparing the differentially expressed genes following short HS with their transcriptional state after recovery, we identified the transient upregulation of MLL and other histone modifiers, providing a potential mechanistic explanation for the stable activation of bivalent genes. This could be used to predict and modify the long-term effect of HS on cell identity.

Keywords: Biological sciences; Cell biology; Stem cells research.

Graphical abstract

Figure 1

Transcriptome changes in response to heat shock Related to Figures S1–S3. (A) A schematic timeline showing the HS treatments from which RNA-Seq libraries were prepared. MSCs were plated and 24 h later exposed to heat shock (HS, 40.5°C) for: 6 h (ShortHS), 6 h + 3 days recovery at 37°C (ShortHS_Recovery), 72 h (LongHS), followed by collection for RNA extraction. Control cells were parallelly plated and maintained in normal temperature (NT, 37°C). Each treatment has color code, which is be used throughout the article: ShortNT (red), ShortHS (green), LongNT (olive), ShortHS_Recovery (blue), LongHS (purple). (B) Scatterplot for principal component analysis of global gene expression (RNA-seq) demonstrates clear separation of gene transcripts between groups. (C) Number of differentially expressed genes (DEGs) with padj ≤0.05 and log2(FC)≥1 that change between the treatment groups. (D) Heatmap of K-means clustering results in 3667 DEGs between the treatment groups. Expression in blue indicates downregulation and red indicates upregulation.

Figure 2

Downregulation of cell cycle and immune response genes while developmental pathways genes are induced following long heat shock Related to Figure S4. (A) Illustration of long heat shock treatment. (B) Cells after LongHS stained with G3BP1 (green), actin filaments stained with phalloidin (red) and nuclei stained with DAPI (blue). Imaging was done using Nikon ECLIPSE TI-DH Florescent Microscope, scale bar = 10 μm. Turquoise nuclei marked with arrows contain the stress granules. No stress granules were found in LongNT. (C and D) (C) Results of Gene set enrichment analysis (GSEA) Hallmark analysis showing significantly enriched gene sets (FDR q < 25% and p < 0.05). A positive normalized enrichment score value, in red, indicates enrichment in the LongHS phenotype (upregulated gene sets in LongHS). Negative enrichment, in blue, indicate enrichment in the LongNT phenotype (downregulated in LongHS). Upregulated heat stress genes and redox maintenance related genes are marked by red and green errors, respectively, while downregulated genes are pointed at by blue errors (D) Differentiation pathways significantly (padj < 0.05) upregulated in LongHS vs. LongNT as per g:Profiler GO analysis, and their associated genes are presented in GOChord plot. The plot’s left half shows the DEGs shared by at least 3 differentiation pathways and the right half displays the GO terms related to differentiation pathways marked in various colors. The colored bands connect a gene to a specific GO term. The scale bar presents gene’s log2FC. (E) KEGG pathways significantly enriched in upregulated DEGs of LongHS vs. LongNT presented by ClueGo pathway analysis and visualization. Colors represented p value: light red 0.05, red 0.01, brown 0.001. The node size represents the term enrichment significance. (F) Significantly enriched (FDR q < 25% and p < 0.05) upregulated GSEA GO terms related to histone modifications and PcG complex following LongHS.

Figure 3

Induced stress response and reduced cellular functions following short heat shock (A) Illustration of short heat shock treatment. (B) Heatmap of the top 30 marker genes for each phenotype in the comparison of ShortHS (left columns) vs. ShortNT (right columns). Expression values are represented as colors and range from red (high expression) to dark blue (lowest expression). (C) Results of GSEA Hallmark analysis showing enriched gene sets (FDR q < 25% and p < 0.05). A positive normalized enrichment score values, marked in red, indicate enrichment of upregulated hallmarks in the ShortHS phenotype, while negative enrichment, marked in blue, indicate enrichment in ShortNT. green, red, and blue arrows indicate. (D) Results of GSEA biological processes significantly enriched (FDR q < 0.25 and p < 0.01) in the comparison of ShortHS vs. ShortNT. Due to high number of GOs, the GOs with similar process were combined under one general phrase (Table S3). A positive normalized enrichment score is the average of combined terms. (E and F) (E) Significantly (padj < 0.05) enriched GO terms, KEGG and Reactome pathways in downregulated and (F) upregulated DEGs in ShortHS vs. ShortNT cells using g:Profiler.

Figure 4

Transient upregulation of chromatin modifiers after short heat shock and stable activation of lineage commitment genes, maintained after three day recovery Related to Figures S5 and S6. (A) Illustration of ShortHS_Recovery vs. ShortHS treatment. (B) Heatmap of K-means clustering results for differentially expressed genes (padj <0.05) between ShortHS and ShortNT. (C) Enrichment of biological processes from g:Profiler enriched in cluster 2. (D) Enrichment of biological processes from g:Profiler in cluster 3. (E) Population doubling time for prolonged treated groups. Data are the mean ± s.e.m. (n = 6). All p values were calculated using Kruskal-Wallis non parametric test, ∗∗∗p < 0.001. (F) Enrichment of biological processes from g:Profiler in cluster 4. (G) Hypergeometric enrichment analysis of MLL (GO:0044665, GO:0044666), HAT (GO:0000123) and HMT (GO:0035097) complexes showing upregulated complexes following ShortHS that return to normal or downregulated during recovery period. The node size represents the p value. (H) Hypergeometric enrichment analysis of bivalent genes, showing bivalent genes upregulation following ShortHS that remain in this state even after recovery period.

Figure 5

Cell cycle and metabolism are commonly reduced following all heat shock protocols, while other processes related to key characteristics of MSC are differently regulated Related to Figures S7–S9. (A) Venn diagram of upregulated genes (padj <0.05) between the treatment groups. (B) Venn diagram of downregulated genes (padj <0.05) between the treatment groups. (C) Heat shock common GSEA hallmark gene sets (FDR q < 25% and p < 0.05) and their enrichment score. (D) Hypergeometric enrichment analysis of cell cycle related biological processes, effected by HS, and their enrichment score. N00455: CDC25-Cell cycle G2/M (KEGG pathway), GO:0007049: Cell cycle (MGI database), R-BTA-69002 – DNA Replication Pre-Initiation (Reactome). Size node represents p value as indicated in E. (E) Hypergeometric enrichment analysis of biological processes related to development effected by HS. GO:0060070: Canonical WNT signaling, GO 0048863: Stem cell differentiation, GO:0048468: Cell development, GO:0009888: Tissue development, GO:0007492: Endoderm development. GO:0007498: Mesoderm development, GO:0048762: mesenchymal cell differentiation. The legends in this graph – enriched groups and p values - are applicable also to D and F. (F) Hypergeometric analysis of biological processes related to immune system effected by HS. GO:0002526: Acute Inflammatory response, GO:1905517: Macrophage migration, GO:0032635: IL-6 production, Cytokine pathway list was taken from PathCards v5.7.551.0. Size node represents p value as indicated in E. (G) Heatmap of cytokine pathways genes that are differentially expressed in LongHS vs. LongNT.