Journal: Nature protocols
Article Title: Engineering human hepato-biliary-pancreatic organoids from pluripotent stem cells
doi: 10.1038/s41596-020-00441-w
Figure Lengend Snippet: a, Typical morphology of PSC colonies cultured on Matrigel and mTeSR1 medium before passage at day −2. Images are representative of six independent PSC lines with similar results. b, Bright-field images of PSCs from day 0 to day 3 at the definitive endoderm stage. c, Optimal differentiation of PSCs into definitive endoderm displays ~90% of GATA4, E-cadherin positive expression and ~80% SOX17 and FOXA2 double-positive expression by day 3. d, Bright-field image of PSCs from day 3 to day 6. e, Low-magnification images of posterior foregut cells at day 7. The left images are representative of an ideal differentiation; the right images are an example of insufficient differentiation. f, Bright-field images of anterior and posterior gut cells at day 7. g, Flow cytometry analysis of anterior or posterior gut cells. 70–90% of cells were EpCAM positive epithelial cells. h, Confirmation of reproducibility by using various PSC lines. Scale bars, 200 μm (a,b,d,f and h), 100 μm (c) and 500 μm (e).
Article Snippet: Donkey anti-goat IgG Alexa Fluor 647 (Thermo Fisher Scientific, cat. no. A-21447; RRID: AB_2535864) Donkey anti-rabbit IgG Alexa Fluor 555 (Thermo Fisher Scientific, cat. no. A-31572; RRID: AB_162543) Donkey anti-mouse IgG Alexa Fluor 488 (Thermo Fisher Scientific, cat. no. A-21202; RRID: AB_141607) Donkey anti-guinea pig IgG Alexa Fluor 647 (Jackson ImmunoResearch Labs, cat. no. 706–605-148; RRID: AB_2340476) DAPI mounting solution (Sigma, cat. no. F6057–20ML) Rabbit anti-SOX2 (Seven Hills Bioreagents, cat. no. WRAB-1236; RRID: AB_2715498) Mouse anti-CDX2 (BioGenex, cat. no. AM392; RRID: AB_2650531) Guinea pig anti-PDX1 (Abcam, cat. no. ab47308; RRID: AB_777178) Rabbit anti-HHEX (R & D Systems, cat. no. MAB83771) Rabbit anti-PROX1 (Abcam, cat. no. ab38692; RRID: AB_2170708) Mouse anti-GATA4 (Santa Cruz Biotechnology, cat. no. sc-25310; RRID: AB_627667) Mouse anti-SOX17 (R & D Systems, cat. no. MAB1924; RRID: AB_2195646) Goat anti-SOX17 (R & D Systems, cat. no. AF1924; RRID: AB_355060) Goat anti-epithelial cell adhesion molecule (EpCAM) (R & D Systems, cat. no. AF960; RRID: AB_355745) Goat anti-E-cadherin (R & D Systems, cat. no. AF648; RRID: AB_355504) Mouse anti-alpha-fetoprotein (Thermo Fisher Scientific, cat. no. 14–9499-82; RRID: AB_2572949) Rabbit anti-HNF4 (Abcam, cat. no. ab200142) Mouse anti-FOXA2 (Thermo Fisher Scientific, cat. no. 14–4778-82; RRID: AB_10714832) Rabbit anti-RFP (Rockland, cat. no. 600–401-379S; RRID: AB_11182807) Mouse anti-albumin (Sigma-Aldrich, cat. no. A6684; RRID: AB_258309) Fluorescein Labeled Dolichos Biflorus Agglutinin (DBA) (Vector Laboratories, cat. no. FL-1031; RRID: AB_2336394)
Techniques: Cell Culture, Expressing, Flow Cytometry