Extended Data Fig. 7: Development and characterization of R-C and D-V patterned µNTLS.
From: A patterned human neural tube model using microfluidic gradients
a. Protocol for generating R-C and D-V patterned µNTLS. Human PS cells are seeded into the central channel on day 0 using mTeSR (Step 1). After gel loading on day 1, culture medium in 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 and R-C patterning of µNTLS (Step 3). From day 5 to day 9, BMP4 (25 ng mL−1) and RA (500 nM) / smoothened agonist (SAG, 500 nM), unless otherwise specified, are added into the top and bottom channels, respectively, to induce D-V patterning of µNTLS. b. Representative brightfield images showing R-C and D-V patterned µNTLS on day 9 and day 21 as indicated. Experiments were repeated five times with similar results. c. Representative stitched confocal micrographs showing R-C and D-V patterned µNTLS on day 9 stained for OTX2, HOXB1, HOXB4, and HOXC9. Zoom-in views of boxed regions are shown on the bottom. Experiments were repeated five times with similar results. d. Representative confocal micrographs showing transverse sections of rostral (d’) and caudal (d”) SC regions of day 9 R-C and D-V patterned µNTLS as indicated, stained for HOXB1, HOXB4, HOXC9, SOX10, PAX3, OLIG2, NKX2.2, and FOXA2. Zoom-in views of boxed regions are included. Intensity maps depict relative mean values of indicated markers as a function of relative D-V position in µNTLS. For rostral SC regions, nPAX3 = 20, nOLIG2 = 24, and nexperiment = 5. For caudal SC regions, nPAX3 = 22, nOLIG2 = 38, and nexperiment = 5. e. Plot showing percentages of µNTLS containing a single lumen in their rostral and caudal regions on day 9 and day 21 as indicated. Error bars represent mean ± SEM. nexperiment = 3. f. Representative confocal micrographs showing transverse sections of rostral (f’) and caudal (f”) regions of day 21 R-C and D-V patterned µNTLS cultured under different conditions as indicated, stained for ZO-1, PAX6, DLX2, NKX2-1, SOX10, PAX3, OLIG2, NKX2-2, and FOXA2. 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 to induce R-C patterning of µNTLS. From day 5 to day 9, BMP4 (25 ng mL−1) and RA (500 nM or 2,000 nM) / smoothened agonist (SAG, 500 nM or 2,000 nM) are added into the top and bottom channels, respectively, to induce D-V patterning of µNTLS. Zoom-in views of boxed regions are included. Intensity maps depict relative mean values of indicated markers as a function of relative D-V position in µNTLS. For 2,000 nM RA / 2,000 nM SAG condition, nPAX6-rostral = 20, nDLX2 = 20, nNKX2-1 = 20, nPAX6-caudal = 21, nOLIG2 = 40, nNKX2-2 = 19, nFOXA2 = 19, and nexperiment = 5; for 500 nM RA / 500 nM SAG condition, nPAX3 = 20, nOLIG2 = 41, nNKX2-2 = 21, nFOXA2 = 21, and nexperiment = 5. In c, d, and f, nuclei were counterstained with DAPI. Scale bars, 400 µm (b and c) and 100 µm (d and f).
