Transcriptome analysis and characterization of differential pathways of hMSCs in response to BiFP containing integrin or DDR2 binding motifs creates signals

To demonstrate the capacity of BiFP with specific integrin or receptor binding motifs for signal creation, three individual hMSCs from distinct sources, such as bone marrow (BM-hMSCs) and dental pulp (DP-hMSCs), were cultured in complete growth medium with BiFP containing integrin a2ß1 (GFOGER) or discoidin domain receptors 2 (DDR2, GVMGFO) binding motifs for 48 h, followed by RNA-seq analysis. Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) revealed that BiFP containing a2ß1 or DDR2 binding motifs enriched different gene sets compared to the same cells treated with peptides not containing the binding motifs (Fig. S6A). These data suggest that BiFP containing integrin or receptor binding motifs creates signals that may play a role in instructing cell fate determination. Overall design: Three individual hMSCs from distinct sources, such as bone marrow (BM-hMSCs) and dental pulp (DP-hMSCs), were cultured in complete growth medium with 40 uM BiFP containing integrin a2ß1 (GFOGER) or discoidin domain receptors 2 (DDR2, GVMGFO) binding motifs for 48 h, followed by RNA-seq analysis. (A) Venn diagram showing overlap (pathway numbers) among gene sets associated with three individual hMSCs. (B) Heat map of top 10 significantly shared signaling pathways and bio-functions by gene set enrichment analysis (GSEA) of RNA-seq data associated with integrin or DDR2 binding motifs creates signals that may play a role in instructing cell fate determination. (C) PI3K/AKT activation pathway in the top 10 significantly reactome pathways from overlap integrin-response genes of DP-hMSCs.

为验证携带有特定整合素或受体结合基序的双功能荧光蛋白（BiFP）的信号构建能力，我们选取来自不同组织来源的3株独立人间充质干细胞（hMSCs），包括骨髓来源BM-hMSCs与牙髓来源DP-hMSCs，将其置于添加了携带有整合素α2β1（GFOGER）或盘状结构域受体2（DDR2，GVMGFO）结合基序的BiFP的完全生长培养基中培养48小时，随后进行RNA测序（RNA-seq）分析。基因本体（Gene Ontology，GO）与基因集富集分析（Gene Set Enrichment Analysis，GSEA）结果显示，与经不含结合基序的肽段处理的同组细胞相比，携带有整合素α2β1或DDR2结合基序的BiFP可富集得到不同的基因集（图S6A）。上述数据表明，携带有整合素或受体结合基序的BiFP可构建可参与调控细胞命运决定的信号通路。实验整体设计：将3株独立的来自不同组织来源的人间充质干细胞（hMSCs），包括骨髓来源BM-hMSCs与牙髓来源DP-hMSCs，置于添加了40 μM携带有整合素α2β1（GFOGER）或盘状结构域受体2（DDR2，GVMGFO）结合基序的BiFP的完全生长培养基中培养48小时，随后进行RNA测序分析。(A) 韦恩图展示3株独立hMSCs相关基因集之间的重叠通路数量；(B) 对整合素或DDR2结合基序相关的RNA测序数据进行基因集富集分析（GSEA）后，得到的前10个显著共有的信号通路与生物功能的热图，这类信号可能参与调控细胞命运决定；(C) 牙髓来源DP-hMSCs的整合素应答重叠基因所对应的前10个显著Reactome通路中，PI3K/AKT激活通路的相关情况。