Extended Data Fig. 1: Development of R-C patterned µNTLS.
From: A patterned human neural tube model using microfluidic gradients
a. (Left) Side view of a Carnegie Stage (CS) 12 human embryo, showing its head-to-tail length of about 4 mm. Image reproduced from ref. 42, Springer Nature Limited. During human embryogenesis, both rostral and caudal neuropores close at around CS12, leading to a completely closed NT structure at around CS1211. (Right) Transverse sectional image of a CS12 human embryo through somite 3, marked by the dashed red line shown on the left. Image reproduced from the Endowment for the Human Development website (https://www.ehd.org), with permission from R. F. Gasser (1975), all rights reserved. Height of NT along the D-V axis is about 200 µm. b. Schematic of microfluidic device, containing top, central, and bottom microchannels, with both ends of each channel connected with medium reservoirs. In the device center marked by a red rectangle, the three channels are separated by two linear arrays of circular support posts, which defines a patterning region in the central channel marked by a dashed red rectangle. Within the patterning region, stable gradients of chemical signals are established along the length of the central channel (R-C axis) by supplementing different concentrations of chemical factors in the two reservoirs of the central channel. Similarly, through passive diffusion from the top and bottom channels, stable gradients of dorsalizing and ventralizing factors are established perpendicular to the central channel (D-V axis). In the schematic, an array of rectangular colonies of human PS cells is formed in the patterning region. c. Microfluidic device design. All microchannels have a height of 150 µm. Central channel has a width of 4 mm. Circular support posts have a diameter of 100 µm and an edge-to-edge distance of 50 µm. Patterning region in the central channel is defined by a 4 mm × 4 mm square as indicated in b. d. Photograph showing microfluidic devices generated through batch fabrication. e. Schematics and brightfield and confocal images showing microcontact printing to generate rectangular Geltrex adhesive islands, microfluidic device assembly, cell and gel loading into the device, and lumenogenesis of human PS cell colonies to form µNTLS. Specifically, rectangular Geltrex adhesive islands (length: 4 mm; width: 100 µm) are printed onto a coverslip using microcontact printing with a polydimethylsiloxane (PDMS) stamp. A PDMS structural layer is then attached onto the coverslip with Geltrex islands aligned with the patterning region of the central channel. On day 0, dissociated single human PS cells are loaded into the central channel and allowed to adhere to Geltrex islands. One hour after cell seeding, floating human PS cells not attached to Geltrex islands are flushed away gently. On day 1, 100% Geltrex is loaded into the central channel, and a neural induction medium (NIM), comprising basal medium and dual SMAD inhibitors (DSi; see Methods), is added into the two medium reservoirs of the central channel. Colonies of human PS cells self-organize and undergo lumenogenesis, with small lumens, demarcated by ZO-1, emerging on day 2 (see Supplementary Video 1). These lumens grow over time and coalesce with each other. By day 3, human PS cell colonies, which still express OCT4, form an elongated tubular structure containing a single continuous, central apical lumen. Zoom-in views of some marked regions are provided. Arrowheads mark small apical lumens demarcated by ZO-1. f. Protocol for generating R-C 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, CHIR99021 (CHIR, 3 µM), FGF8 (200 ng mL−1) and retinoic acid (RA, 500 nM) are added into the right reservoir of the central channel in addition to NIM (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). g. Representative brightfield images showing a single µNTLS on different days as indicated. Zoom-in views of a marked region are provided. h. Representative stitched confocal images showing an array of R-C patterned µNTLS on day 7 from a single microfluidic device stained for HOXB1, HOXB4, and HOXC9. i. Intensity maps showing relative mean expression levels of indicated markers as a function of relative R-C position in R-C patterned µNTLS on day 7. nOTX2 = 12, nHOXB1 = 22, nHOXB4 = 22, nHOXC9 = 12, and nexperiment = 3. j. (Left) Protocol for generating µNTLS with a default dorsal forebrain identity. human PS cells are seeded into the central channel on day 0 using mTeSR (Step 1). After gel loading on Day 1, culture medium is switched to NIM from Day 1 onwards (Step 2). µNTLS are analyzed on Day 7. (Right) Representative stitched confocal micrographs showing µNTLS on Day 7 stained for ZO-1, PAX6, OTX2, and EdU, as indicated. Micrographs on the right show y-z planes of selected regions in µNTLS. k. Representative confocal micrographs showing x-y and y-z planes of selected regions in R-C patterned µNTLS on day 7 stained for ZO-1, ADP-ribosylation factor-like protein 13B (ARL 13B), EdU, and phospho-histone H3 (pH3), respectively. Arrowheads mark ARL 13B-enriched cilia on µNTLS apical surfaces. l. Schematic showing dissection of R-C patterned µNTLS on day 7 using a surgical scissor into four tissue segments of equal lengths for downstream RT-qPCR analysis. m. Dorsal view of human NT and expression pattern of HOX family genes in HB and SC. Color coding of HOX family genes represents their expression domains along the R-C axis of NT. n. Heatmaps showing normalized expression of HOX family genes as a function of the four segments of day 7 R-C patterned µNTLS. n = 3 experiments. In e, h, j and k, nuclei were counterstained with DAPI. In e, g, h, j and k, experiments are repeated three times with similar results. Scale bars, 1 mm (side view image in a), 200 µm (transverse section image in a), 15 mm (d), 1 mm (whole µNTLS array images in e), 50 µm (zoom-in images in e), 800 µm (g and h), 400 µm (j), and 150 µm (k).
