f4 recombinant human fgf10 r d systems  (R&D Systems)

Journal: Cell stem cell

Article Title: Functional human gastrointestinal organoids can be engineered from three primary germ layers derived separately from pluripotent stem cells.

doi: 10.1016/j.stem.2021.10.010

Figure Lengend Snippet: Figure 5. ENS cells promote in vitro growth and patterning of gastric mesenchyme (A) Schematic illustrating the method of recombining hAGOs with ENCCs at day 6 and day 9 of the hAGO protocol. (B) Representative images of 4-week in vitro hAGOs with (bottom) and without (top) ENCCs recombined on day 6 of the hAGO protocol, stained with TUJ1 neurons (green) and FOXF1 mesenchyme (red) and epithelial ECAD (white). Higher-magnification images are shown on the right. (C) Representative images of 4-week in vitro hAGOs with (bottom) and without (top) ENCCs recombined on day 6 (left) or day 9 (right) of the hAGO protocol, demonstrating an increase in FOXF1+ mesenchyme (red). (D) Quantification of FOXF1+ mesenchyme contribution (n = 16–24 fields from at least 3 organoids from one differentiation; the same trend is seen across at least two individually seeded differentiations; *p < 0.05, ***p < 0.001, Student’s t test). Scale bars represent S.D. (E) Relative expression of key gastric mesenchymal genes (BARX1, NKX3-2, FGF10, ISL1, and SIX2) in hAGOs+ENCCs compared with control hAGOs without ENCCs (n = 4–12 wells with a minimum of 3 organoids per well from 5 individual differentiations, *p < 0.05, Student’s t test). Scale bars represent S.D. See also Figure S4, Video S2, and Table S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Biological samples Human Stomach and Duodenum Samples Wells Lab CCHMC GI-1B-9952 A B Human Antral and Fundic Samples Helmrath Lab CCHMC 14-241, 14-242, 14-243, 14-244 Human Stomach and Duodenum Samples CCHMC Pathology Core N/A Chemicals, peptides, and recombinant proteins 1x DMEM Invitrogen Cat# 11965-084 50x B27 supplement Thermo Fisher Scientific Cat# 17504044 50x B27 supplement w/o Vitamin A Thermo Fisher Scientific Cat# 12587-010 A8301 Torcis Cat# A83-01 Activin A Cell Guidance Systems Cat# GFH6 Accutase Thermo Fisher Scientific Cat# A11105-01 Advanced DMEM:F12 Thermo Fisher Scientfic Cat# 12634-010 Atropine sulfate salt monohydrate Sigma-Aldrich Cat# A0257 Bethanechol Sigma-Aldrich Cat# 1071009 C59 Cellagen Technology Cat# C7641-2s CHIR99021 ReproCell Cat# 04-0004-10 Collagen Type IV Thermo Fisher Scientific Cat# 17104019 Defined fetal bovine serum (dFBS) Hyclone Cat# SH30070.02 Dispase Thermo Fisher Scientific Cat# 17105-041 DMEM/F12 Thermo Fisher Scientific Cat# 11320033 DMEM/F-12, GlutaMAX Thermo Fisher Scientific Cat# 10565018 EGF Sigma-Aldrich Cat# 1257-0.1MG F12 Media Invitrogen Cat# 11765-054 Fetal Bovine Serum (FBS) HyClone Cat# SH30071.03 Fibronectin Corning Cat# 354008 Geltrex Thermo Fisher Scientific Cat# A1413201 HEPES Buffer Thermo Fisher Scientific Cat# 15630080 hESC-qualified Matrigel BD Biosciences Cat# 354277 Insulin from bovine pancreas Sigma-Aldrich Cat# I4011-50 L-glutamine (100x) Thermo Fisher Scientific Cat# 25030-081 L-NAME Sigma-Aldrich Cat# N5751 Matrigel Matrix Basement Membrane BD Biosciences Cat# 354234 mTeSR1 media Stem Cell Technologies Cat# 5850 N2 Supplement Thermo Fisher Scientific Cat# 17502-048 Non-essential Amino Acids (100x) Thermo Fisher Scientific Cat# 11140050 Normal donkey serum Jackson Immunoresearch Labs Cat# 017-000-121 Neurobasal Medium Thermo Fisher Scientific Cat# 21103049 PD0325901 Stem Cell Technologies Cat# 72184 Pen/Strep (100x) Thermo Fisher Scientific Cat# 15140-122 PIK90 EMD Millipore Cat# 528117 PMA Torcis Cat# 1201 recombinant human BMP4 R&D Systems Cat# 314-BP recombinant human EGF R&D Systems Cat# 236-EGF recombinant human FGF2 R&D Systems Cat# 233-FB recombinant human FGF4 R&D Systems Cat# 235-F4 recombinant human FGF10 R&D Systems Cat# 345-FG recombinant human Noggin R&D Systems Cat# 6057-NG recombinant human WNT3A R&D Systems Cat# 5036-WN (Continued on next page) Cell Stem Cell 29, 36–51.e1–e6, January 6, 2022 e2

Techniques: In Vitro, Staining, Expressing, Control