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"
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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)
本数据集包含以下文件： 一、文中附图： 图1 细胞因子预处理后脐带间充质干细胞（WJ-MSCs，N=7）的转录组谱变化：A 未预处理（浅蓝色）与预处理组（深蓝色）转录组数据的主成分分析（Principal Component Analysis，PCA）。B 火山图展示人类分子特征数据库（Hallmark基因集、WikiPathways基因集、KEGG Medicus基因集、Reactome基因集及GO生物过程基因集）的通路水平富集分析结果，概览细胞因子预处理响应的主要通路。每个点代表一条独立通路，横轴为标准化富集得分（Normalized Enrichment Scores，NES），纵轴为校正后p值的-log₁₀转换值，颜色编码对应所用生物数据库。C 预处理组显著上调的两个Hallmark基因集的富集图：干扰素γ应答通路及经NFKB的TNFα信号通路。绿色曲线代表富集得分（Enrichment Score，ES），黑色条形标注基因在排序列表中的位置。 图2 基于基因本体（Gene Ontology，GO）术语分析，细胞因子预处理后与细胞分化相关的上调及下调基因的分布（log₂倍变化，FC）。展示以下GO术语：GO:0002062 软骨细胞分化、GO:0045445 成肌细胞分化、GO:0035914 骨骼肌细胞分化、GO:0051145 平滑肌细胞分化、GO:0045444 脂肪细胞分化、GO:0001649 成骨细胞分化、GO:0030182 神经元分化、GO:0030154 细胞分化。将鉴定得到的基因按倍变化范围手动分类（>1、0.5~1、<0.5、<-0.5、-0.5~-1及>-1），并以各类别的基因数量进行可视化呈现。 图3 未预处理及经细胞因子预处理的脐带间充质干细胞（WJ-MSCs，N=4，复孔为2）在诱导分化7天后的成骨及成脂基因表达情况。数据以诱导组相对于未诱导对照组细胞的相对表达均值±标准差展示。注：Ns 无统计学显著性；* p<0.05；** p<0.01。 图4 脐带间充质干细胞（WJ-MSCs，N=4，复孔为2）条件培养基中分泌型生长因子（A）及细胞因子（B）的含量。数据以均值±标准差展示。注：* p<0.05。 图5 采用Seahorse实验检测的未预处理及经细胞因子预处理的脐带间充质干细胞（WJ-MSCs，N=6，复孔为3）的氧消耗速率（OCR，A、B）及细胞外酸化速率（ECAR，D、E）。展示预处理组与未预处理（对照）组的代表性OCR（C）及ECAR（F）检测曲线。注：Ns 无统计学显著性；* p<0.05；** p<0.01；*** p<0.001。 图6 细胞因子预处理后脐带间充质干细胞（WJ-MSCs）的代谢组及脂质组谱（N=4，复孔为2）：A 未预处理（浅蓝色）与预处理组（深蓝色）WJ-MSCs样本代谢组数据的主成分分析（PCA）。B 火山图展示细胞因子预处理诱导的WJ-MSCs中主要代谢物类别的显著分离。每个点代表通路内富集程度最高的独立代谢物，颜色标注其所属的特定代谢物类别。缩写：三羧酸循环代谢物（Tricarboxylic Acid Cycle，TCA）、甘油三酯（Triglyceride，TG）、游离脂肪酸（Free Fatty Acids，FAs）、溶血磷脂酰胆碱（Lysophosphatidylcholine，LPC）、溶血磷脂酰乙醇胺（Lysophosphatidylethanolamine，LPE）、胆固醇酯（Cholesterol Ester，CE）及氨基酸（Amino Acids，AAs）。C 细胞因子预处理后WJ-MSCs中选定代谢物家族的变化。注：Ns 无统计学显著性；* p<0.05；** p<0.01。D 散点图展示细胞因子预处理组与未预处理对照组间代谢物水平的log₂倍变化。每个点代表一个代谢物，其位置表示倍变化幅度（log₂），颜色与大小代表统计学显著性：小黑点（p<0.05）、中灰点（p<0.01）、大白点（p<0.001）。 二、附加文件： 附加文件1.pdf - 表1：RNA测序结果中检测到的上调或下调最显著基因列表（log₂倍变化>1，p<0.001） 附加文件2.pdf - 表1：RNA测序结果中检测到的与间充质干细胞分化相关的基因列表，这些基因经细胞因子预处理后相较于未预处理细胞的表达log₂倍变化，p值阈值设为0.05 附加文件3.pdf 补充附图： 图1S 未预处理组（上排）与预处理组（下排）WJ-MSCs的成骨（碱性磷酸酶染色）及成脂（尼罗红染色）诱导的形态学特征与检测指标。图表展示通过尼罗红染色强度定量分析的成脂分化情况，结果以每100,000个细胞的染色强度标准化后呈现。 图2S 比较预处理组与未预处理组WJ-MSCs中HALLMARK_OXPHOS（氧化磷酸化）及HALLMARK_GLYCOLYSIS（糖酵解）相关基因集的火山图。横轴为log₂倍变化（FC），纵轴为校正后p值的-log₁₀转换值，每个点代表预处理后差异表达的基因。 图3S 短期细胞因子预处理后目标基因的表达变化。 图4S 经IDO抑制与未抑制的未预处理及经细胞因子预处理的WJ-MSCs的成骨及成脂基因表达情况（N=4，复孔为2）。 图5S 与成骨分化相关的基因表达变化热图。依据GO:0001649（成骨细胞分化）鉴定经细胞因子预处理后相较于未预处理细胞存在log₂倍表达变化的基因，p值阈值设为0.05。 图6S 与成脂分化相关的基因表达变化热图。依据GO:0045444（脂肪细胞分化）鉴定经细胞因子预处理后相较于未预处理细胞存在log₂倍表达变化的基因，p值阈值设为0.05。 三、其他数据文件： 3. RNA测序（原始数据） 4. 代谢组学分析（原始数据） 5. 展示跨多种生物数据库通路水平分析结果的标准化富集得分列表，包括Hallmark基因集、KEGG数据库、Reactome数据库、基因本体（GO）及WikiPathways数据库（文件名为GO_terms）
