Micropatterned, clickable culture substrates enable in situ spatiotemporal control of human PSC-derived neural tissue morphology

Abstract

We describe a modular culture platform that enables spatiotemporal control of the morphology of 2D neural tissues derived from human pluripotent stem cells (hPSCs) by simply adding clickable peptides to the media. It should be widely applicable for elucidating how spatiotemporal changes in morphology and substrate biochemistry regulate tissue morphogenesis.

Scheme 1. Azide-functionalized PEGMA-grafted substrates undergo 1,3-dipolar cycloaddition reaction with DBCO conjugated RGD–peptides.

Fig. 1. (A) Fluorescent images of micropatterned PEGMA brushes conjugated with RGD–DBCO at varying reaction concentrations, 150 μm scale bars. (B) Surface density of RGD–DBCO on micropatterned substrates across all reaction concentrations, *p < 0.05.

Fig. 2. (A) Schematic of neural tissue outgrowth onto RGD–DBCO-modified micropatterend substrates. (B) Bright field images of neural tissue outgrowth onto PEGMA brushes following addition of RGD–DBCO at time = 0, 300 μm scale bars. (C) Fluorescent images of polarized NSCs on micropatterned substrates, 50 μm scale bars.

Fig. 3. (A) Fluorescent images of neural tissues with a polarized NSC core and radial expansion due to migrating progeny at 24 (B) and 96 h after supplementation with 10.0 μM RGD–DBCO, 250 μm scale bars.