Matrigel droplets

Small intestine mouse organoids were established and maintained as described^{42 (link)} from isolated crypts collected from the entire length of the small intestine. The basic culture medium (advanced DMEM/F12, with penicillin/streptomycin, 10 mM HEPES, 1× Glutamax, 1× B27 [all from Life Technologies] and 1 mM N-acetylcysteine (Sigma Aldrich) was supplemented with 50 ng/mL murine recombinant EGF (Peprotech), Noggin-CM (1% v/v) and R-spondin1-CM (2.5% final volume, if not indicated otherwise) to obtain ENR medium. Wnt3a-CM was used at 50% (v/v) to obtain WENR. The Rnf43/Znrf3 double knock-out (R/Z dKO) organoid line was a gift of BK Koo, Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, UK^{62 (link)} and grown in EN medium (ENR without Rspo1). Organoids were cultured in matrigel droplets (Corning).

Angiogenic properties of miPS-Huh7cmP cells and miPS-BT549cmP cells were tested in vivo with a chorioallantoic membrane assay (CAM). Fertilized chicken eggs (Gallus gallus) were incubated in a humidified atmosphere at 37 °C for three days. At day 4 of embryonic age, albumin was removed to detach the developing CAM from the eggshell. At day 8, eggs with exposed CAM were incubated with plastic discs containing 9 × 10⁵ cells pretreated or untreated dissolved in growth factor reduced Matrigel™ droplets (Corning Inc., Corning, NY, USA). Plastic discs containing PBS droplets were used as a negative control. Eggs were incubated for four consecutive days and at day 13, the test was stopped. Images were taken with a Sony HDR-XR350VE Handycam camera (Sony corporation, Tokyo, Japan).

The human cerebral organoids were generated from H9 hESCs using a previously reported protocol with minor modification [20 (link)]. Detailed composition of cerebral organoid differentiation medium was described in the Table 1. Briefly, Accutase (A1110501, Gibco, CA, USA) was used to disassociate the hESCs into single cells. A total number of 2000 cells were then plated into the hanging drop culture plates (InSphero AG, Schlieren, Switzerland) to form the single embryoid body (EB) in the first 24 h, and then the EBs were transferred to the ultra-low-attachment 96-well plates (7007, Corning, New York, NY, USA) in the EB formation medium with 4 ng/mL bFGF (100-18B, Peprotech, NJ, USA) and 50 mM ROCK inhibitor Y27632. The EBs were fed every other day for 6 days then transferred to the low adhesion 24-well plates in the induction media. On day 12, the EBs were embedded in the seated Matrigel droplets (356231, Corning, New York, NY, USA), and these droplets were solidified at 37 °C. Embedded EBs were subsequently cultured in the neural expansion medium. The embedded EBs were cultured in the stationary condition in 6-well plates for 4 days. Then the plates were transferred to an orbital shaker (HS-25A, MIULAB, Hangzhou, China) rotating continuously at 75 rpm. The culture medium in the dishes was replaced with the neural maturation medium.