hes cell line h9  (WiCell Research Institute Inc)

Journal: Nature materials

Article Title: Mechanics-guided embryonic patterning of neuroectoderm tissue from human pluripotent stem cells

doi: 10.1038/s41563-018-0082-9

Figure Lengend Snippet: Self-organized neuroectoderm patterning in circular hPS cell colonies. (a) Schematic of neural induction in vivo and in vitro. During neural induction in vivo, embryonic cells in the ectoderm form the neural plate (NP, or neuroectoderm). Embryonic cells at the neural plate border (NPB) separate the neuroectoderm from the epidermal ectoderm. Neural induction of circular hPS cell colonies leads to autonomously patterned neuroectoderm tissues, with NP cells at colony central region and NPB cells at colony periphery. (b) Representative phase contrast and fluorescence images showing cell morphology and nuclei (stained by DAPI), respectively, at different days. White dashed lines mark colony periphery. Experiments were repeated three times with similar results. Bottom average intensity maps show spatial distributions of DAPI intensity. Number of colonies analyzed were pooled from n = 3 independent experiments. Data were plotted as the mean. (c) Representative immunofluorescence micrographs and average intensity maps showing colonies at day 9 stained for neuroectoderm marker PAX6 and NPB markers PAX3, ZIC1 and MSX1. White dashed lines mark colony periphery. Experiments were repeated three times with similar results. Relative intensity maps were normalized to DAPI signals. Number of colonies analyzed were pooled from n = 3 independent experiments. Data were plotted as the mean. (d) Representative phase contrast and fluorescence images and average intensity maps from live cell assays using SOX10:EGFP hES cells. White dashed lines mark colony periphery. White arrowheads mark GFP+ cells at colony border on day 6. Experiments were repeated three times with similar results. Number of colonies analyzed were pooled from n = 3 independent experiments. Data were plotted as the mean. Scale bars in b-d, 100 μm.

Article Snippet: Both H1 hES cell line (WA01, WiCell; NIH registration number: 0043) and SOX10-EGFP bacterial artificial chromosome hES cell reporter line (H9; WA09, WiCell; NIH registration number: 0062) were cultured on mitotically inactive mouse embryonic fibroblasts (MEFs; GlobalStem) in growth medium.

Techniques: In Vivo, In Vitro, Fluorescence, Staining, Immunofluorescence, Marker

Journal: Nature materials

Article Title: Mechanics-guided embryonic patterning of neuroectoderm tissue from human pluripotent stem cells

doi: 10.1038/s41563-018-0082-9

Figure Lengend Snippet: BMP-SMAD signaling is required for mechanically guided neuroectoderm patterning. (a) Representative immunofluorescence images showing colonies at day 4 stained for phosphorylated SMAD 1/5 (p-SMAD 1/5). The default neural induction condition (control) was modified as following: - LDN / + BMP4, LDN was replaced with BMP4 (BMP-HI); - KSR / + E6: KSR was replaced with E6 (BMP-LO). CHIR was kept in culture protocols for both BMP-HI and BMP-LO conditions. Zoomed-in image shows a magnified view of colony central area. Scale bar, 100 μm. Experiments were repeated three times with similar results. (b) Percentage of cells with nuclear p-SMAD 1/5 as a function of distance from colony centroid. Number of colonies analyzed were pooled from n = 3 independent experiments. Data represent the mean ± s.e.m. P values were calculated between BMP-HI vs. control and BMP-LO vs. control using unpaired, two-sided Student’s t-tests. (c) Representative fluorescence micrographs and average intensity maps showing colonies at day 9 stained for PAX6 and PAX3. Scale bar, 100 μm. Experiments were repeated three times with similar results. Number of colonies analyzed were pooled from n = 3 independent experiments. Data in intensity maps represent the mean. (d) Geometrical confinement leads to self-organization of morphogenetic factors. Increased cell shape and contractile force at colony periphery result in nuclear accumulation and transcriptional activation of p-SMAD 1/5, which in turn up-regulates NPB specifier genes including PAX3, SOX9 and SOX10. Confined cell shape with limited contractile force at colony center leads to nuclear exclusion of p-SMAD 1/5 and NE differentiation. Modulating cell shape by mechanical stretching or geometrical confinement can thus mediate BMP signaling to regulate neuroectoderm patterning.

Article Snippet: Both H1 hES cell line (WA01, WiCell; NIH registration number: 0043) and SOX10-EGFP bacterial artificial chromosome hES cell reporter line (H9; WA09, WiCell; NIH registration number: 0062) were cultured on mitotically inactive mouse embryonic fibroblasts (MEFs; GlobalStem) in growth medium.

Techniques: Immunofluorescence, Staining, Modification, Fluorescence, Activation Assay