c1 fluidigm chip 1 5  (fluidigm)

Journal: EBioMedicine

Article Title: Cholangiopathies – Towards a molecular understanding

doi: 10.1016/j.ebiom.2018.08.024

Figure Lengend Snippet: Single Cell RNA sequencing experiments of liver, or cells differentiated into liver cells.

Article Snippet: Human In vitro: 2D culture of iPSCs (TkDA3–4, University of Tokyo) undergoing hepatic differentiation and 3D culture of liver bud organoids derived from hepatic cells differentiated from the iPS cell line, cocultured with HUVECS (Lonza) and MSCs (Lonza) In vivo: Adult (three donors: donor 1, female, 55; donor 2, male, 65; donor 3, male, 21) and fetal (two donors, gestation weeks 10.5 and 17.5) Mouse E14.5, E15.5, and E16.5 , Liver bud organoid cells: Liver bud organoids, different constellations of cells: 177 cells dissociated, no selection. Isolation of adult human liver cells: 256 cells from human adult liver. Protocol of hepatocyte or other cell isolation from adult liver published in [ ]; liver is dissociated and cell types separated using centrifugation steps. Isolation of fetal human cells: 238 cells from fetal stages, dissociated and briefly cultured (12h) on laminin-coated plates to remove red blood cells, followed re-dissociation of cells. Isolation of mouse hepatoblasts: 92 cells from mouse liver, dissociated, erythrocytes were lysed, and magnetic bead sorted for Dlk1. , C1 Fluidigm chip 1–5 million reads per cell. Cells were excluded from further analyses if they had < 100,000 reads, < 1,000 expressed genes or failed to express housekeeping genes ACTB or GAPDH , This manuscript does not explicitly identify cholangiocytes, but provides valuable insight into which culture systems better support in vitro differentiation faithful to in vivo hepatoblast growth. , iPSC-derived hepatoblasts undergoing culture in liver bud organoids more closely resemble fetal liver hepatic cells than do 2D cultured iPSC-derived hepatoblasts. Ligand-receptor pair analyses of co-cultured cells in organoids showed a KDR/VEGFA signaling pair in which VEGFA secreted by immature hepatocytes stimulates KDR on endothelial cells, which in turn support hepatoblast growth. , [ ] .

Techniques: RNA Sequencing, Sequencing, Isolation, Expressing, Comparison, Marker, Lysis, Modification, In Vitro, Derivative Assay, In Vivo, Selection, Cell Isolation, Cell Culture, Control