Journal: Hepatology Communications

Article Title: Single-cell transcription reveals hepatocyte-to-cholangiocyte reprogramming and biliary gene profile in biliary atresia

doi: 10.1097/HC9.0000000000000710

Figure Lengend Snippet: Enriched pathways and genes in the cholangiocytes of BA. (A) Flowchart of isolating BA epithelial cells from the merged groups. (B) t-SNE visualization of hep, rep, and cho in BA. (C) Violin plots showing the expression levels of the marker genes of hep, rep, and cho in BA. (D) Heatmap displaying enriched pathways of cholangiocytes in BA by GSVA analysis. (E) Violin plots displayed the EMT score among the 3 types of epithelial cells in BA. (F) Heatmap displaying the average expression level of EMT markers, fibrotic markers, inflammatory genes, and m 6 A readers in BA hep, rep, and cho. (G) Representative immunohistochemistry sections of CDH2, smad2/3, FGFR2, PDGFA, CXCL6, and IGF2BP2 in a TMA. Dashed circles in PDGFA were recognized as cholangiocytes. H. Representative immunofluorescence staining of CDH2, Smad2/3, FGFR2, PDGFA, CXCL6, and IGF2BP2 in TMA of BA group. I. Box plots showing the expression levels of the indicated genes in TMA, colored by different groups. * p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001. Abbreviations: BA, biliary atresia; CHO, cholangiocytes; CS, cholestasis; EMT, epithelial-mesenchymal transition; HEP, hepatocytes; NC, normal control; REP, reprogrammed cells; TMA, tissue microarray.

Article Snippet: After antigen blocking with 5% BSA, the sections were incubated overnight at 4 °C with the following primary antibodies: CK19 (Abcam, ab52625, 1:600), HNF4A (Abcam, ab201460, 1:2000), SOX9 (CST, 82630, 1:600), CDH2 (CST, 13116, 1:200), Smad2/3 (CST, 8685, 1:400), FGFR2 (CST, 23328, 1:200), PDGFA (santa cruz, sc-9974, 1:200), CXCL6 (CST, 73201, 1:100), IGF2BP2 (Abcam, ab124930, 1:200), CFTR (Abcam, ab270238, 1:500), MMP7 (Abcam, ab207299, 1:4000), VTCN1 (Abcam, ab209242, 1:1000), LAMC2 (Abcam, ab210959, 1:500), ANKRD1 (Proteintech, 11427-1-AP, 1:200), ELF3 (Sigma, HPA003479, 1:200), KLF5 (Abcam, ab273672, 1:500), and HNF1B (Abcam, ab128912, 1:100).

Techniques: Expressing, Marker, Immunohistochemistry, Immunofluorescence, Staining, Control, Microarray

Journal: Biomaterials Research

Article Title: Derivation of Mesenchymal Stem Cells through Sequential Presentation of Growth Factors via Gelatin Microparticles in Pluripotent Stem Cell Spheroids

doi: 10.34133/bmr.0184

Figure Lengend Snippet: Bone morphogenetic protein-4 (BMP4)- and fibroblast growth factor 2 (FGF2)-induced differentiation of induced pluripotent stem cells (iPSCs) to mesenchymal stem cells (MSCs) in 2-dimensional (2D) conditions. (A) Schematic diagram depicting iPSC-to-induced MSC (iMSC) differentiation via mesoderm induction. (B) Images of iPSCs, mesoderm-induced cells, and iMSCs. Scale bar = 200 μm. (C) Quantitative real-time gene analysis (qPCR; top panel) and western blot (WB) analysis (bottom panel) of the mesoderm markers Brachyury and SNAI1 (Snail1), along with the pluripotency marker SOX2, at 4 d postdifferentiation. (D) qPCR (top panel) and WB analysis (bottom panel) of the MSC markers CD73, CD90, CD105, and CD44 at 11 d postdifferentiation. (E) Flow cytometry (fluorescence-activated cell sorting [FACS]) analysis of the MSC markers CD73, CD90, and CD44 in cells treated with either BMP4 only or BMP4 + FGF2. The qPCR data were normalized to 18S. The WB data were normalized to β-actin. All data represent results from 3 independent experiments, each conducted in triplicate. The data are presented as mean ± SD (ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). Individual data points and significance levels are indicated in graphs. DMEM, Dulbecco’s modified Eagle medium; SR, serum replacement; hiPSCs, human iPSCs; mRNA, messenger RNA; GF, growth factor.

Article Snippet: The slow- and fast-degrading GelMPs were incubated with primary antibodies against FGF2 (Santa Cruz Biotechnology, USA) and BMP4 (PeproTech, USA) overnight and labeled with Alexa Fluor 488 (Invitrogen, Thermo Fisher Scientific, USA) and Texas Red (Invitrogen, Thermo Fisher Scientific, USA) at 1 μl/100 μl of PBS, respectively.

Techniques: Western Blot, Marker, Flow Cytometry, Fluorescence, FACS, Modification

Journal: Biomaterials Research

Article Title: Derivation of Mesenchymal Stem Cells through Sequential Presentation of Growth Factors via Gelatin Microparticles in Pluripotent Stem Cell Spheroids

doi: 10.34133/bmr.0184

Figure Lengend Snippet: Gelatin microparticle (GelMP) fabrication, characterization, GF conjugation, and the release profiles of BMP4 and FGF2. (A) Scanning electron microscopy (SEM) images of fast-degrading (4 mM glutaraldehyde) and slow-degrading (12 mM glutaraldehyde) GelMPs. Scale bar = 100 μm. Size distributions of fast- and slow-degrading GelMPs. (B) Physical properties of fast- and slow-degrading GelMPs. (C) Representative immunofluorescent images of BMP4-conjugated fast-degrading GelMPs and FGF2-conjugated slow-degrading GelMPs. Scale bar = 100 μm. (D) Degradation rate of 5 mg of microparticles in collagenase (5 μg/ml) solution. (E) Cumulative release (%) of 1 μg of BMP4 from fast-degrading GelMPs and 1 μg of FGF2 from slow-degrading GelMPs incubated for 20 d in collagenase (5 μg/ml) solution. The obtained data represent results from 3 independent experiments, each conducted in triplicate. PBS, phosphate-buffered saline.

Article Snippet: The slow- and fast-degrading GelMPs were incubated with primary antibodies against FGF2 (Santa Cruz Biotechnology, USA) and BMP4 (PeproTech, USA) overnight and labeled with Alexa Fluor 488 (Invitrogen, Thermo Fisher Scientific, USA) and Texas Red (Invitrogen, Thermo Fisher Scientific, USA) at 1 μl/100 μl of PBS, respectively.

Techniques: Conjugation Assay, Electron Microscopy, Incubation, Saline