Journal: International Journal of Molecular Sciences

Article Title: Direct On-Chip Differentiation of Intestinal Tubules from Induced Pluripotent Stem Cells

doi: 10.3390/ijms21144964

Figure Lengend Snippet: Characterization of hiPSC-derived human intestinal-like tubules. ( A ) 3D reconstruction image of an iPSC-derived tubule at Day 4 stained with antibodies for SOX17 (green), FOXA2 (red) or DAPI for DNA (blue). Cells attach and form tubule at the DE stage. ( B ) Representative images of iPSC tubules stained for Definitive endoderm markers SOX17 and FOXA2 at day 4. ( C ) Hindgut marker CDX2 at day 7. ( D ) Gene expression was measured using TaqMan qRT-PCR from hiPSC tubules at different differentiation stages. The following genes were analysed: POU class 5 homeobox 1 (POU5F1, indicating pluripotency); Nanog homeobox (NANOG, indicating Primitive Streak):, forkhead box a2 (FOXA2, Definitive Endoderm), SRY (sex determining region Y)-box 17 (SOX17, Definitive Endoderm), and Homeobox protein CDX-2 (Posterior gut). ( E ) Representative images of iPSC tubules stained for intestinal markers Lysozyme (LYZ), Villin (VIL) and Chromogranin A (CHGA) at day 28 (green). Nuclei were stained with DAPI (blue) to visualize the overall morphology. Scale bars = 100 µm. ( F ) Relative gene expression analysis of Intestinal markers: Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), Mucin-2 (MUC2), Lysozyme (LYZ), Villin-1 (VIL1), Chromagranin A (CHGA) and sucrase-isomaltase (SI). ( G ) Relative mRNA expression of MDR1 (P-gp) and CYP3A4 in iPSC-derived intestine-like tubules on day 14, day 28 and day 31, human adult colon organoids and Caco-2 cells. Expression levels were normalized to beta-actin (ACTB), data represented as mean ± SD relative to the expression in undifferentiated miFF1 hiPSC ( n = 2, n ≥ 2–3). The Y-axis represents the LOG10 relative quantification (RQ). Caco-2 cells and primary colon organoid were also included to compare gene expression and to follow the differentiation of our model during the different stages. Data are presented as the average of two independent experiments +/− SD ( n = 3).

Article Snippet: The antibodies used: SOX17 (R&D Systems, MAB19241 1/100), FOXA2 (R&D Systems AF2400.

Techniques: Derivative Assay, Staining, Marker, Gene Expression, Quantitative RT-PCR, Expressing, Quantitative Proteomics

Journal: Stem Cell Reports

Article Title: Low Cell-Matrix Adhesion Reveals Two Subtypes of Human Pluripotent Stem Cells

doi: 10.1016/j.stemcr.2018.06.003

Figure Lengend Snippet: The Two Types of Cells Are Both Pluripotent (A) The images indicated that both MCoG and DCoG cells formed colonies and expressed AP. (B) NANOG and OCT4 were expressed both in MCoG and DCoG cells after 26 passages. (C) RT-PCR analysis of expression of pluripotency genes ( OCT4 and NANOG ) and differentiation genes ( PAX6 , ectoderm; BRACHYURY , mesoderm; and AFP , endoderm) in MCoG and DCoG cells. (D) Flow cytometric analysis of pluripotency markers in MCoG and DCoG cells. The 50,000 cells analyzed express high levels of hPSC-specific cell surface markers (TRA-1-60, SSEA-4) and low levels of differentiation-specific cell surface markers (SSEA-1). (E) Embryoid bodies formed by MCoG and DCoG cells differentiated into the three germ layers: ectoderm (β-tubulin), mesoderm (α-SMA), and endoderm (SOX17 and AFP). (F) Teratomas formed by MCoG and DCoG cells in severe combined immunodeficiency mice, containing tissues that are representative of all three embryonic germ layers. (G) Normal karyotype exhibited in both MCoG and DCoG cells. See also Figure S3 .

Article Snippet: Samples were blocked with blocking buffer (5%, v/v, normal goat serum; 5%, v/v, normal donkey serum; 3%, v/v, BSA; and 0.1%, v/v, Tween 20 in PBS) for 1 hr and incubated with the following primary antibodies diluted in blocking buffer at room temperature for 2 hr or at 4°C overnight: anti-human OCT4 (2 μg/mL, sc-5279, Santa Cruz Biotechnology, USA), anti-human NANOG (9.4 μg/mL, 4903S, Cell Signaling Technology, UK), anti-human β-tubulin III (6 μg/mL, 5568, Cell Signaling Technology), anti-human α-SMA (2 μg/mL, ab5694, Abcam, UK), anti-human SOX17 (20 μg/mL, MAB1924, R&D Systems, USA), anti-E-cadherin (1/300 dilution, 3195S, Cell Signaling Technology), and anti-MKL1 (3 μg/mL, HPA030782, Sigma-Aldrich).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing