Extended Data Fig. 6: Development of region-specific µNS.
From: A patterned human neural tube model using microfluidic gradients
a. Protocol for generating region-specific µNS. Human PS cells are seeded into the central channel on day 0 using mTeSR (Step 1). After gel loading on Day 1, culture medium of the central channel is switched to NIM (Step 2). From Day 2 to Day 5, CHIR (3 µM), FGF8 (200 ng mL−1), and RA (500 nM) are added into the right reservoir of the central channel in addition to NIM, to induce caudalization of µNS (Step 3). From day 5 to day 7, all caudalizing factors are removed, and only NIM is added into the two medium reservoirs of the central channel (Step 4). Tissues are analyzed at different time points as indicated. b. Representative stitched brightfield (left) and confocal (right) micrographs showing a regular array of µNS in the patterning region on indicated days. µNS on day 2 and day 4 was stained for OCT4, SOX2, and ZO-1. On day 7, they were stained for OTX2, HOXB1, and HOXB4. Zoom-in views of boxed regions are shown on the right. Experiments were repeated three times with similar results. c. Representative confocal micrographs showing µNS on day 7 stained for PAX6, OTX2, HOXB1, HOXB4, and HOXC9 as indicated. Experiments were repeated three times with similar results. d. Pie charts showing percentages of different types of µNS at different locations of the patterning region on day 7. n = 3 experiments. In b and c, nuclei were counterstained with DAPI. Scale bars, 400 µm (b) and 100 µm (c).
