DOI: 10.5281/zenodo.15221802
title: Revised data supporting the article "The role of cytokine licensing in shaping the therapeutic potential of Wharton's jelly MSCs: metabolic shift towards immunomodulation at the expense of differentiation"

description: Data set includes the following files:





Figures described in the article:

Figure 1. Changes in the transcriptomic profile of WJ-MSCs (N=7) following the cytokine licensing: A – Principal component analysis (PCA) of transcriptome data from unprimed (light blue) and primed (dark blue) samples. B – Volcano plot depicting pathway-level enrichment analysis across the Human collections of Molecular Signatures Database (Hallmark gene sets, WikiPathways gene sets, KEGG Medicus gene sets, Reactome gene sets, and GO biological process gene sets) representing the overview of the major pathways responded to the cytokine priming. Each point represents a distinct pathway, with the x-axis showing the normalized enrichment scores (NES) and the y-axis displaying the -log10 adjusted p-value. The colour coding corresponds to the biological database. C – Enrichment plots for two hallmark gene sets significantly upregulated in primed samples: Interferon Gamma Response and TNFA signalling via NFKB pathways. Green curves represent the enrichment score (ES), while black bars indicate the position of genes within the ranked list.

 Figure 2. Distribution of up- and downregulated genes (log 2 fold changes, FC) referred to cell differentiation following cytokine priming based on analysis of the Gene ontology (GO) terms. The following GO terms are presented: GO:0002062 Chondrocyte differentiation, GO:0045445 Myoblast differentiation, GO:0035914 Skeletal muscle cell differentiation, GO:0051145 Smooth muscle cell differentiation, GO:0045444 Fat cell differentiation, GO:0001649 Osteoblast differentiation, GO:0030182 Neuron differentiation, GO:0030154 Cell differentiation. The identified genes were then manually categorized based on fold change ranges (>1, 0.5–1, <0.5, <-0.5, -0.5 to -1, and >-1), and the number of genes in each category was used for the representation.  

 Figure 3. The expression of osteogenic and adipogenic genes of unprimed and cytokine licensed WJ-MSCs (N=4, in duplicates) following the 7 days of the differentiation induction. Data is presented as Mean ± SD relative expression of induced cells to control cells (not subjected to differentiation induction). Note: Ns – non-significant; * - p<0.05; ** - p<0.01.  

 Figure 4. The content of secreted growth factors (A) and cytokines (B) in the conditioned medium of WJ-MSCs (N=4, in duplicates). Data is presented as Mean ± SD. Note: * - p<0.05.

 Figure 5. The oxygen consumption rate (OCR) (A, B) and extracellular acidification rate (ECAR) (D, E) of the unprimed and cytokine licensed WJ-MSCs (N=6, in triplicates) assessed using Seahorse assay. Representative traces of OCR (C) and ECAR (F) were obtained and shown for primed and unprimed (control) cells. Note: Ns – non-significant; * - p<0.05; ** - p<0.01; *** - p<0.001.  

Figure 6. Metabolomic and lipidomic profile of WJ-MSCs following the cytokine licensing (N=4, in duplicates): A - Principal component analysis (PCA) of metabolomic data from unprimed (light blue) and primed (dark blue) WJ-MSCs samples. B – Volcano plot depicting the clear separation of the main metabolite classes in WJ-MSCs induced by the cytokine priming. Each point represents a distinct most enriched metabolite within the pathway, while its colour denotes the specific metabolite class it belongs to. Abbreviations: Tricarboxylic acid cycle metabolites (TCA), triglycerides (TG), free fatty acids (FAs), lysophosphatidylcholines (LPC), lysophosphatidylethanolamines (LPE), cholesterol esters (CE), and amino acids (AAs). C – The changes in selected metabolite families in WJ-MSCs following the cytokine priming Note: Ns – non-significant; * - p<0.05; ** - p<0.01. D – Dot plot depicting log2 fold changes in metabolite levels between cytokine-primed WJ-MSCs and unprimed controls. Each dot represents a metabolite, with its position indicating the magnitude of fold change (log2) and its color/size denoting statistical significance: small black dot (p<0.05), medium grey dot (p<0.01), big white dot (p<0.001).

 2. Additional files: 

Additional file 1.pdf - Table 1. The list of the most up- or down-regulated genes (log2fold >1, p<0.001), detected in RNA-seq results

Additional file 2.pdf - Table 1. The list of the genes related to MSC differentiation with log2 fold change in expression after cytokine priming, compared to unprimed cells, using a p-value threshold of 0.05, detected in RNA-seq results

Additional file 3.pdf

Figure 1S. Morphological properties and indicators of osteogenic (alkaline phosphatase staining) and adipogenic (Nile red staining) induction of unprimed (upper row) and primed (lower row) WJ-MSCs. The graph shows the quantitative analysis of adipogenic differentiation assessed by the intensity of Nile red staining, normalized to the number of cells (100,000 cells). 

Figure 2S. The volcano plot comparing gene sets related to HALLMARK_OXPHOS (oxidative phosphorylation) and HALLMARK_GLYCOLYSIS (glycolysis) in primed and unprimed WJ-MSCs. The x-axis represents the log2 fold change (FC), while the y-axis shows the -log10 p-adjusted values. Each dot indicates a differentially expressed gene after priming.

Figure 3S. Changes in the expression of genes of interest after short-term cytokine licensing. 

Figure 4S. The expression of osteogenic and adipogenic genes of unprimed and cytokine-licensed WJ-MSCs (N=4 in duplicates) with and without IDO inhibition. 

Figure 5S. Heatmap of changes occurred in the expression of genes related to osteogenic differentiation. The genes with a log2 fold change in expression after cytokine priming, compared to unprimed cells, were identified according to GO:0001649 (osteoblast differentiation), using a p-value threshold of 0.05.

Figure 6S. Heatmap of changes occurred in the expression of genes related to adipogenic differentiation. The genes with a log2 fold change in expression after cytokine priming, compared to unprimed cells, were identified according to GO:0045444 (fat cell differentiation), using a p-value threshold of 0.05.

3. RNA sequencing (initial data)

4. Metabolomic analysis (initial data)

5. List of normalized enrichment scores depicting pathway-level analysis across various biological databases, including Hallmark, KEGG, REACTOME, Gene Ontology, and WikiPathways (*file GO_terms)