Effect of Cell Spreading on Rosette Formation by Human Pluripotent Stem Cell-Derived Neural Progenitor Cells
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 neu...
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doaj-9375d16dd074417da9596bd468b05bc62020-11-25T03:28:55ZengFrontiers Media S.A.Frontiers in Cell and Developmental Biology2296-634X2020-10-01810.3389/fcell.2020.588941588941Effect of Cell Spreading on Rosette Formation by Human Pluripotent Stem Cell-Derived Neural Progenitor CellsRyan F. Townshend0Yue Shao1Sicong Wang2Chari L. Cortez3Sajedeh Nasr Esfahani4Jason R. Spence5K. Sue O’Shea6Jianping Fu7Deborah L. Gumucio8Kenichiro Taniguchi9Kenichiro Taniguchi10Kenichiro Taniguchi11Department of Cell & Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, United StatesDepartment of Mechanical Engineering, University of Michigan, Ann Arbor, MI, United StatesDepartment of Mechanical Engineering, University of Michigan, Ann Arbor, MI, United StatesDepartment of Cell & Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, United StatesDepartment of Mechanical Engineering, University of Michigan, Ann Arbor, MI, United StatesDepartment of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, United StatesDepartment of Cell & Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, United StatesDepartment of Mechanical Engineering, University of Michigan, Ann Arbor, MI, United StatesDepartment of Cell & Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, United StatesDepartment of Cell & Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, United StatesDepartment of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, WI, United StatesDepartment of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, United StatesNeural 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.https://www.frontiersin.org/article/10.3389/fcell.2020.588941/fullneural rosettehuman pluripotent stem cellscell spreadingneural progenitor cellsactin cytoskeletal networkRhoA |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Ryan F. Townshend Yue Shao Sicong Wang Chari L. Cortez Sajedeh Nasr Esfahani Jason R. Spence K. Sue O’Shea Jianping Fu Deborah L. Gumucio Kenichiro Taniguchi Kenichiro Taniguchi Kenichiro Taniguchi |
spellingShingle |
Ryan F. Townshend Yue Shao Sicong Wang Chari L. Cortez Sajedeh Nasr Esfahani Jason R. Spence K. Sue O’Shea Jianping Fu Deborah L. Gumucio Kenichiro Taniguchi Kenichiro Taniguchi Kenichiro Taniguchi Effect of Cell Spreading on Rosette Formation by Human Pluripotent Stem Cell-Derived Neural Progenitor Cells Frontiers in Cell and Developmental Biology neural rosette human pluripotent stem cells cell spreading neural progenitor cells actin cytoskeletal network RhoA |
author_facet |
Ryan F. Townshend Yue Shao Sicong Wang Chari L. Cortez Sajedeh Nasr Esfahani Jason R. Spence K. Sue O’Shea Jianping Fu Deborah L. Gumucio Kenichiro Taniguchi Kenichiro Taniguchi Kenichiro Taniguchi |
author_sort |
Ryan F. Townshend |
title |
Effect of Cell Spreading on Rosette Formation by Human Pluripotent Stem Cell-Derived Neural Progenitor Cells |
title_short |
Effect of Cell Spreading on Rosette Formation by Human Pluripotent Stem Cell-Derived Neural Progenitor Cells |
title_full |
Effect of Cell Spreading on Rosette Formation by Human Pluripotent Stem Cell-Derived Neural Progenitor Cells |
title_fullStr |
Effect of Cell Spreading on Rosette Formation by Human Pluripotent Stem Cell-Derived Neural Progenitor Cells |
title_full_unstemmed |
Effect of Cell Spreading on Rosette Formation by Human Pluripotent Stem Cell-Derived Neural Progenitor Cells |
title_sort |
effect of cell spreading on rosette formation by human pluripotent stem cell-derived neural progenitor cells |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Cell and Developmental Biology |
issn |
2296-634X |
publishDate |
2020-10-01 |
description |
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. |
topic |
neural rosette human pluripotent stem cells cell spreading neural progenitor cells actin cytoskeletal network RhoA |
url |
https://www.frontiersin.org/article/10.3389/fcell.2020.588941/full |
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