Murine egf

Rat tail type I collagen, Transwell inserts (0.9 cm² cell culture area, 1.6 × 10⁶ pores/cm²), 12-well polystyrene tissue culture dishes, 12-well polycarbonate plates and Transwell inserts (1.12 cm² cell culture area, 1.6 × 10⁸ pores/cm²) were purchased from Corning (Corning, NY). SB202190 was from Selleckchem (Houston, TX). Collagenase (type IV) was purchased from Worthington Biochemicals (Lakewood, NJ). Y-27632 and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) were purchased from ApexBio Technology (Houston, TX). Human collagen (type I) was obtained from Advanced Biomatrix (Carlsbad, CA). The ALP assay kit was from AbCam (Cambridge, MA). Gastrin was from Anaspec (Freemont, CA). UGT-Glo™ Assay and P450-Glo™ CYP3A4 Assay were obtained from Promega (Madison, WI). N-acetyl cysteine was purchased from MP Biomedicals (Santa Ana, CA). Murine EGF was procured from Peprotech (Rock Hill, NJ). Primocin was purchased from InvivoGen (San Diego, CA). Nicotinamide, L-alanine p-nitroanilide, fluorescein diacetate, loperamide, zafirlukast, cobalt chloride, potassium phosphate buffer, bovine serum albumin, and A83–01 were acquired from Sigma-Aldrich (St. Louis, MO). All other reagents and Bicinchoninic Acid (BCA) Protein Assay Kit were purchased from Thermo Fisher Scientific (Waltham, MA).

All animal procedures were performed according to the National Institute of Biological Sciences Guide for the care and use of laboratory animals. neural stem cells (NSCs) were isolated from a newborn all-iPS mouse, which was generated from an iPSC line through tetraploid complementation [26] (link), [27] . Plasmid preparation, and the procedure of iPS derivation were performed according to the methods described previously [26] (link), [28] (link).
Single cells were picked from digested NSCs and plated individually on a 96-well dish with media of DMEM/F12 (Life Technologies) supplemented with 1 × B27 (Life Technologies), 20 ng/ml murine EGF (Peprotech), and 20 ng/ml bFGF (Peprotech). Neurospheres, that were formed approximately 5–6 days later, were then digested and Tet-on induced on the feeder cells with regular embryonic stem cell (ESC) media supplemented with 1 μg/ml doxycycline and 10 ng/ml ascorbic acid. Approximately 15–18 days later, the ESC-like spheroids were mechanically picked up and further cultured into iPSC lines. IPSC culture medium contained DMEM (Life Technologies) supplemented with 15% fetal bovine serum (FBS), 1 mM L-glutamine, 0.1 mM mercaptoethanol, 1% nonessential amino acids, and 1000 U/ml leukocyte inhibitory factor (LIF) (all from Chemicon). Culture dishes were kept at 37 °C in a humidified atmosphere of 5% CO₂ in air.

To generate intestinal organoids (IOs), intestinal crypts were isolated from the mouse small intestine as previously described [53 (link),54 (link)]. In brief, the harvested mouse small intestine was longitudinally opened and villi were removed by gentle scraping with a glass coverslip. The tissue was cut into small pieces (2–3 mm) then incubated in gentle cell dissociation solution (Stemcell Technologies, Vancouver, BC, Canada) for 20 min at room temperature followed by vigorous shaking to dislocate the crypts. After the removal of the intestinal segment, the supernatant containing crypts was washed with PBS at 400× g for 5 min at 4 °C to pellet the crypts. A total of 300 crypts mixed with 25 μL Matrigel (at 1:1 ratio) were seeded onto one well of 24 well plates. Organoids were kept in growth media composed of DMEM/F-12 (Gibco, Grand Island, NY, USA), N2 (Gibco), B27 (Gibco), 1 mM N-acetylcysteine (Sigma-Aldrich), 100 ng/mL Noggin (Peprotech, Rocky Hill, NJ, USA), 100 ng/mL murine EGF (Peprotech) and 10% of R-spondin conditioned media. Media change was done every 2–3 days and the subculture of organoids was conducted every 5–7 days.