Organoid Differentiation

Cerebral Organoid differentiation of ES or iPS cells

The protocol begins with the generation of EBs from human stem cells using AggreWell800 (Qian, X., et al., Brain -region-specific organoids using mini-bioreactors for modeling zikv exposure. Cell, 2016, 165, 1-17). The day on which EBs are made is day 0. On day 1, the EBs are transferred to a T-25 flask containing Dorsomorphin/A83 medium and are grown for 2-4 days. On day 5-6, the EBs were gradually switched to CS induction medium (with SB-431542, CHIR, and Heparin). On day 7, about 20 EBs are embedded in Matrigel and then grown in one well of a 6-well plate for 7 days in CS induction medium. On day 14, the embedded EBs are transferred to the spinning bioreactor and then grown in differentiation medium. One week later, organoids will be picked into 12 wells of 6 well plates for initial analysis. The remaining organoids will be cultured for another 2 weeks and 4 weeks prior to delivery to the investigator for immunostaining, RNAseq, qPCR, or imaging. The project should require 8-10 weeks, but time points longer than 8-10 weeks will extend the timetable and cost.