Additional file 1 of Characterisation of extraembryonic endoderm-like cells from mouse embryonic fibroblasts induced using chemicals alone

Additional file 1 : Figure S1. Small molecules induced mouse fibroblasts to transform into ciXEN cells. Expression of Epcam (a) and Cxcr4 (b) and mesenchymal markers (c) during chemical induction as measured by qPCR. d Western blot analysis for the expression of E-cadherin and Vimentin during chemical induction. e Morphological changes of MNFs induced by chemicals and bFGF (bar, 100 μm). f Numbers of cell clones from different numbers of initial cells: 3w, 4w, and 5w. g Co-immunostaining for the expression of Sox17 and Foxa2 in MNF-derived clone (bar, 50 μm). * p < 0.05, ** p < 0.01, *** p < 0.001. Mean ± SEM, t-test and two-way ANOVA analysis, n ≥ 3. Figure S2. Characteristics of ciXEN cells at different passages. qPCR results for the expression of Epcam, Pdgfra, (a) and Cxcr4 (b) in ciXEN cells at p5. c The ultrastructure of MEFs and ciXEN cells. The thin arrow shows the endoplasmic reticulum (white) in MEFs and the cilium (black) in ciXEN cells; the thick arrow shows the mitochondria in ciXEN cells. (bar, 2 μm and 1 μm). d Cell morphologies of ciXEN cells at p5, p10, p20, p30 (bar, 100 μm). e Karyotype analysis of MEFs and ciXEN cells at p5, p15, and p30. mRNA levels of fibroblast genes (Prrx1, Pdgfrb, Col1a1, and Thy1), epithelial genes (Ocln and Cdh1) (f), XEN markers (Sox17, Foxa2, Gata4, and Gata6) (g), and pluripotent genes (Oct4, Sox2, and Nanog) (h). p15: passage 15; p20: passage 20. * p < 0.05, ** p < 0.01, *** p < 0.001. Mean ± SEM, t-test and two-way ANOVA analysis, n = 3. Figure S3. The metabolic patterns of ciXEN cells. a OCR of MEFs and ciXEN cells. b Total ATP of MEFs and ciXEN cells. Expression of Glut1 (c) and Pfk1, Ldha, and Hk2 (d) during chemical induction as measured by qPCR. e Numbers of cell clones under different treatment conditions: MEFs + VPACRFE and MEFs + VPACRFE + PS48. * p < 0.05, ** p < 0.01, *** p < 0.001. Mean ± SEM, t-test, n = 3. Figure S4. PCR/qPCR analysis for the expression of hepatic genes during the induction of iHeps. * p < 0.05, ** p < 0.01, *** p < 0.001. Mean ± SEM, t-test, n = 3. Figure S5. The characteristics of iXEN cells and their potential to differentiate into iHeps. a Experimental procedures of ciXEN cells cultured in different conditions: 1% FBS and 10% FBS. b The morphologies of the cells cultured in the medium containing 1% FBS (left) and 10% FBS (right) (bar, 50 μm). c qPCR results for the expression of hepatic markers in the cells with 1% FBS and 10% FBS. d EdU assay for the proliferative ability of MEFs, ciXEN cells and iXEN cells (bar, 50 μm). e Morphology of iHeps induced from iXEN cells (bar, 100 μm). f mRNA levels of hepatic genes, XEN genes (Sox17, Foxa2, Gata4, and Gata6) and Sox2, as measured by qPCR. g Co-immunostaining for the expression of Afp and Hnf4a, Alb and Foxa3, Asgpr1 and E-cadherin (bar, 50 μm). h PAS staining of iXEN cell-derived iHeps (bar, 100 μm). * p < 0.05, ** p < 0.01, *** p < 0.001. Mean ± SEM, t-test, n = 3.

附加文件1：图S1。小分子诱导小鼠成纤维细胞（mouse embryonic fibroblasts, MEF）转化为ciXEN细胞。化学诱导过程中，通过实时定量聚合酶链反应（quantitative real-time polymerase chain reaction, qPCR）检测Epcam（a）、Cxcr4（b）以及间充质标志物（c）的表达。d：蛋白质印迹（Western blot）分析化学诱导过程中E-钙粘蛋白（E-cadherin）与波形蛋白（Vimentin）的表达情况。e：化学试剂与碱性成纤维细胞生长因子（basic fibroblast growth factor, bFGF）诱导的小鼠成纤维细胞（MNFs）形态变化（标尺：100 μm）。f：不同初始接种细胞数对应的细胞克隆数：3周、4周及5周组。g：MNFs来源的克隆中Sox17与Foxa2表达的免疫荧光共染色（标尺：50 μm）。*p < 0.05，**p < 0.01，***p < 0.001。数据以平均值±标准误（standard error of the mean, SEM）表示，采用t检验与双因素方差分析（two-way ANOVA），n≥3。
图S2。不同传代次数ciXEN细胞的特征。a：第5代ciXEN细胞中Epcam与Pdgfra表达的qPCR检测结果，b：Cxcr4表达的qPCR检测结果。c：小鼠胚胎成纤维细胞与ciXEN细胞的超微结构：细箭头指示小鼠胚胎成纤维细胞内的内质网（白色）以及ciXEN细胞内的纤毛（黑色）；粗箭头指示ciXEN细胞内的线粒体（标尺：2 μm与1 μm）。d：第5、10、20、30代ciXEN细胞的细胞形态（标尺：100 μm）。e：第5、15、30代小鼠胚胎成纤维细胞与ciXEN细胞的核型分析。f：成纤维细胞基因（Prrx1、Pdgfrb、Col1a1及Thy1）的mRNA水平；g：上皮细胞基因（Ocln与Cdh1）的mRNA水平；h：XEN细胞标志物（Sox17、Foxa2、Gata4及Gata6）以及多能基因（Oct4、Sox2及Nanog）的mRNA水平。注：p15为第15代，p20为第20代。*p < 0.05，**p < 0.01，***p < 0.001。数据以平均值±SEM表示，采用t检验与双因素方差分析，n=3。
图S3。ciXEN细胞的代谢模式。a：小鼠胚胎成纤维细胞与ciXEN细胞的氧耗率（oxygen consumption rate, OCR）。b：小鼠胚胎成纤维细胞与ciXEN细胞的总三磷酸腺苷（adenosine triphosphate, ATP）含量。c：化学诱导过程中Glut1表达的qPCR检测结果；d：Pfk1、Ldha及Hk2表达的qPCR检测结果。e：不同处理条件下的细胞克隆数：小鼠胚胎成纤维细胞+VPACRFE组，以及小鼠胚胎成纤维细胞+VPACRFE+PS48组。*p < 0.05，**p < 0.01，***p < 0.001。数据以平均值±SEM表示，采用t检验，n=3。
图S4。诱导肝细胞（induced hepatic cells, iHeps）诱导过程中肝脏相关基因表达的PCR/qPCR分析。*p < 0.05，**p < 0.01，***p < 0.001。数据以平均值±SEM表示，采用t检验，n=3。
图S5。iXEN细胞的特征及其向iHeps分化的潜能。a：ciXEN细胞在不同胎牛血清（fetal bovine serum, FBS）浓度培养基（1% FBS与10% FBS）中培养的实验流程。b：1% FBS（左）与10% FBS（右）培养基中培养的细胞形态（标尺：50 μm）。c：不同血清浓度培养的细胞中肝脏标志物表达的qPCR检测结果。d：小鼠胚胎成纤维细胞、ciXEN细胞与iXEN细胞增殖能力的EdU检测（标尺：50 μm）。e：由iXEN细胞诱导得到的iHeps的形态（标尺：100 μm）。f：肝脏相关基因、XEN细胞标志物基因（Sox17、Foxa2、Gata4及Gata6）以及Sox2的mRNA水平的qPCR检测结果。g：Afp与Hnf4a、Alb与Foxa3、Asgpr1与E-钙粘蛋白表达的免疫荧光共染色（标尺：50 μm）。h：iXEN细胞来源的iHeps的过碘酸雪夫染色（Periodic Acid-Schiff staining, PAS染色，标尺：100 μm）。*p < 0.05，**p < 0.01，***p < 0.001。数据以平均值±SEM表示，采用t检验，n=3。