Journal: Reproduction

Article Title: Culture conditions antagonize lineage-promoting signaling in the mouse blastocyst

doi: 10.1530/rep-20-0107

Figure Lengend Snippet: Figure 1 BSA antagonizes signaling by exogenous FGF4. (A) Experimental design for embryos shown and analyzed in this figure. Red = NANOG indicative of epiblast cell fate, green = SOX17 indicative of primitive endoderm cell fate. (B) Immunostaining for SOX17 to identify primitive endoderm (PE) cells and NANOG to identify epiblast (EPI) cells in embryos cultured for 66 hours starting from two-cell stage in KSOM with 1 mg/mL BSA or without BSA (Millipore-Sigma MR-121-D and MR-107-D, respectively), but with 1 mg/mL added PVA (Sigma 360627). n = number of embryos examined. P = Student’s t-test. n.s. = not significant (P > 0.2). (C) Quantification of the total inner cell mass (ICM) and trophectoderm (TE) cells in embryos cultured in indicated conditions and proportion of ICM cells contributing to PE and EPI in these embryos. Each symbol represents a single embryo, column = mean, and error bars = standard deviations. P = Student’s t-test. n.s. = not significant (P > 0.2). (D) Immunostaining for SOX17 and NANOG in embryos cultured in KSOM + 150 ng/mL FGF4 (R&D Systems 235-F4-025) and 1 µg/mL heparin (Sigma H3149) in the presence or absence of 1 mg/mL BSA. Each symbol represents a single embryo, column = mean, and error bars = s.d. P = Student’s t-test. n.s. = not significant (P > 0.2). (E) Quantification of lineage specification in embryos cultured in conditions indicated. Each symbol represents a single embryo, column = mean, and error bars = s.d. (F) Response of embryos cultured in KSOM without 1 mg/mL BSA to low dose of exogenous FGF4 and 1 µg/mL heparin. Each symbol represents a single embryo, column = mean, and error bars = s.d. P = Student’s t-test.

Article Snippet: = not significant (P > 0.2). (D) Immunostaining for SOX17 and NANOG in embryos cultured in KSOM + 150 ng/mL FGF4 (R&D Systems 235-F4-025) and 1 μg/mL heparin (Sigma H3149) in the presence or absence of 1 mg/mL BSA.

Techniques: Immunostaining, Cell Culture

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