Journal: Advanced Science

Article Title: Expanded Hepatic Progenitor Cells Featured with Aggregation of α‐Synuclein Contribute to Pathologic Bile Duct Regeneration in Biliary Atresia

doi: 10.1002/advs.76054

Figure Lengend Snippet: Characterization of NCAM1 + EpCAM + HPCs in the expanded biliary clusters in BA. (a) Enrichment score of HPCs gene set in each biliary cluster. Comparisons among multiple clusters were performed using the Kruskal‐Wallis test, with a significance level of p < 0.001. (b) Venn diagram of DEGs showing higher expression in cluster 17 than other three biliary clusters (avg_log 2 FC>1), the DEGs showing higher expression in BA than the control group across all four biliary clusters (avg_log 2 FC>1), and the membrane protein database. (c) Gene density plot of NCAM1 in biliary clusters. (d): The proportion of NCAM1 positive cells of BA, Ctrl and external single‐cell dataset. P value was calculated by Chi‐squared test. (e): The proportion of NCAM1 + cells in EpCAM + cells of BA, Ctrl and normal cholangiocyte dataset ( GSE185477 contains four healthy human liver caudate lobes). P value was calculated by Chi‐squared test. (f): The expression of NCAM1 in BA compared with normal liver tissues in GSE122340 (171 BA and 7 normal liver tissues) calculated by wilcoxon rank‐sum test. (g): The expression of NCAM1 in BA (n = 38) and Ctrl liver tissues ( n = 21). P value was calculated by unpaired t test. (h) Immunohistochemical staining of NCAM1 on paraffin sections of livers from Ctrl, BA with mild liver fibrosis (BA‐FM) and BA with severe liver fibrosis (BA‐FS). Scale bar: 100 µm. (i) Comparison of H‐scores for NCAM1 in portal areas of liver tissues between BA ( n = 42) and Ctrl (n = 10). P value was calculated by unpaired t test. (j) Comparison of H‐scores for NCAM1 in portal areas of liver tissues between BA‐FM ( n = 30) and BA‐FS ( n = 12). P value was calculated by unpaired t test. (k) Morphology of NCAM1 + EpCAM + cells derived organoids and NCAM1 − EpCAM + cells derived organoids in BA (The left side of each group, scale bar: 200 µm). Regions highlighted with dotted boxes were magnified and shown on the right (scale bar: 50 µm). Calcein AM staining (Green) of the organoids were shown. (l) Volcano plot of DEGs of organoids derived from NCAM1 + EpCAM + cells ( n = 3) and NCAM1 − EpCAM + cells ( n = 4). (m) Bar chart showing transcriptional activity of TFs implicated in biliary and hepatic lineage. Red color indicated increased activity, while blue color indicated decreased activity in NCAM1 + EpCAM + cells derived organoids.

Article Snippet: After single cells (viability>80%) were obtained, NCAM1 (CD56)‐positive and ‐negative expressing cells were isolated according to the REAlease CD56 MicroBead kit manual (130‐117‐033; Miltenyi, USA).

Techniques: Expressing, Control, Membrane, Single Cell, Immunohistochemical staining, Staining, Comparison, Derivative Assay, Activity Assay

Journal: Advanced Science

Article Title: Expanded Hepatic Progenitor Cells Featured with Aggregation of α‐Synuclein Contribute to Pathologic Bile Duct Regeneration in Biliary Atresia

doi: 10.1002/advs.76054

Figure Lengend Snippet: NCAM1 + EpCAM + HPCs displayed transcriptomics associated with PD and α‐synuclein pathway. (a) Clinical variation analysis of the top 20 genes in cluster 17 calculated by Hypergeometric test. (b) Gene Set Variation Analysis of α‐synuclein pathway in each biliary cluster (cluster 7, 10, 15 and 17). The Kruskal‐Wallis test was used for multi‐group comparisons. (c) Gene Set Enrichment Analysis of α‐synuclein pathway in DEGs from organoids derived from NCAM1 + EpCAM + and NCAM1 − EpCAM + cells. p value was calculated by Permutation test. (d): The expression of α‐synuclein in BA compared with normal liver tissues in GSE122340 (171 BA and 7 normal liver tissues) calculated by wilcoxon rank‐sum test. (e): The expression of α‐synuclein in BA ( n = 38) and Ctrl liver tissues ( n = 17). P value was calculated by unpaired t test. (f) Representative immunofluorescence images of NCAM1, EpCAM and α‐synuclein in BA and control sample. Scale bar: 20 µm. (g) Immunofluorescence images of Thioflavin‐S and α‐synuclein co‐staining in 3 patients with BA. The white triangle points to the co‐localization region. Scale bar: 50 µm. (h) Histogram showing serum total α‐synuclein in 40 BAs and 20 controls. p value was calculated by unpaired t test. (i) ROC of serum total α‐synuclein combined with or without GGT as a predictor for BA. (j) The mRNA expression of Snca and Ncam1 in RRV ( n = 7) and Control liver tissues ( n = 7) after log10 conversion. P value was calculated by unpaired t test. (k) Representative immunofluorescence images of NCAM1, EpCAM and α‐synuclein in RRV and control liver tissues. Scale bar: 100 µm. (l) Representative morphology and whole‐mount immunofluorescent staining of KRT19 and α‐synuclein in PFFs‐treated (PFFs) and untreated (Normal) mouse single cell derived CLC organoids. Scale bar of bright field images: 200 µm. Scale bar of immunofluorescent staining image: 100 µm. (m) Relative expression of hepatic and biliary markers in PFFs treated mouse single cell derived CLC organoids ( n = 3) and normal single cell derived CLC organoids ( n = 3). P value was calculated by unpaired t test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: After single cells (viability>80%) were obtained, NCAM1 (CD56)‐positive and ‐negative expressing cells were isolated according to the REAlease CD56 MicroBead kit manual (130‐117‐033; Miltenyi, USA).

Techniques: Transcriptomics, Derivative Assay, Expressing, Immunofluorescence, Control, Staining, Single Cell