Effect of Cell Spreading on Rosette Formation by Human Pluripotent Stem Cell-Derived Neural Progenitor Cells.

Ryan F Townshend,Sajedeh Nasr Esfahani,Kenichiro Taniguchi,Jianping Fu,Deborah L Gumucio,Chari L Cortez,Sicong Wang,Yue Shao,Jason R Spence,K Sue O’Shea

doi:10.3389/fcell.2020.588941

Abstract

Neural rosettes (NPC rosettes) are radially arranged groups of cells surrounding a central lumen that arise stochastically in monolayer cultures of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPC). Since NPC rosette formation is thought to mimic cell behavior in the early neural tube, these rosettes represent important in vitro models for the study of neural tube morphogenesis. However, using current protocols, NPC rosette formation is not synchronized and results are inconsistent among different hPSC lines, hindering quantitative mechanistic analyses and challenging live cell imaging. Here, we report a rapid and robust protocol to induce rosette formation within 6 h after evenly-sized “colonies” of NPC are generated through physical cutting of uniformly polarized NESTIN+/PAX6+/PAX3+/DACH1+ NPC monolayers. These NPC rosettes show apically polarized lumens studded with primary cilia. Using this assay, we demonstrate reduced lumenal size in the absence of PODXL, an important apical determinant recently identified as a candidate gene for juvenile Parkinsonism. Interestingly, time lapse imaging reveals that, in addition to radial organization and apical lumen formation, cells within cut NPC colonies initiate rapid basally-driven spreading. Further, using chemical, genetic and biomechanical tools, we show that NPC rosette morphogenesis requires this basal spreading activity and that spreading is tightly regulated by Rho/ROCK signaling. This robust and quantitative NPC rosette platform provides a sensitive system for the further investigation of cellular and molecular mechanisms underlying NPC rosette morphogenesis.

Highlights

The radial organization of cells into rosette-like structures containing a central apical lumen is a fundamental developmental hallmark of neuroepithelial tissue (Tomooka et al, 1993; Deglincerti et al, 2016)
We report a simple and robust protocol that triggers the formation of neural progenitor cells (NPC) rosettes from human pluripotent stem cell (hPSC)-derived NPC monolayers within 6 h after induction
Using this system as a platform for highly efficient quantitative and mechanistic analyses, we demonstrate that Rho/ROCK-dependent basal NPC spreading promotes NPC-rosette initiation, and that NPCrosette organization is modulated by the efficiency of basal spreading which controls the size of NPC colonies

Summary

Introduction

The radial organization of cells into rosette-like structures containing a central apical lumen is a fundamental developmental hallmark of neuroepithelial tissue (Tomooka et al, 1993; Deglincerti et al, 2016). In some neural differentiation culture systems, NPC monolayers undergo spontaneous radial morphogenesis and apical constriction, forming NPC rosettes, or neural rosettes, which are apico-basally polarized pseudostratified structures similar to the embryonic neural tube (Haigo et al, 2003; Curchoe et al, 2012; Christodoulou and Skourides, 2015; Deglincerti et al, 2016; Nikolopoulou et al, 2017). Deletion of Crb, a cell polarity protein, in mouse NPC results in defective neural rosette formation, even though Crb2−/− NPC monolayers show normal polarization (Boroviak and Rashbass, 2011), emphasizing that rosette structures have apico-basal properties that are distinct from those of polarized monolayers. PODXL has been well studied in early mouse embryogenesis and in kidney for its role in apical polarization and lumen formation (Takeda et al, 2000; Bryant et al, 2014; Yang et al, 2016; Shahbazi et al, 2017; Christodoulou et al, 2018), but the role of this protein in rosette formation has not been reported

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Conclusion