ROCK Inhibitor Is Not Required for Embryoid Body Formation from Singularized Human Embryonic Stem Cells

Authors

Giuseppe Pettinato, Virginia Commonwealth University, Clemson University
Wendy S. Vanden Berg-Foels, Virginia Commonwealth University, Clemson University, Medical University of South Carolina
Ning Zhang, Virginia Commonwealth University, Clemson UniversityFollow
Xuejun Wen, Virginia Commonwealth University, Clemson University, Tongji UniversityFollow

Document Type

Article

Original Publication Date

2014

Journal/Book/Conference Title

PLoS ONE

Volume

9

Issue

11

DOI

10.1371/journal.pone.0100742

Comments

Originally published at http://dx.doi.org/10.1371/journal.pone.0100742

Date of Submission

November 2015

Abstract

We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation, elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment, and low-cost scalability, which will directly support automated, large-scale production of hEBs and hESC-derived cells needed for clinical, research, or therapeutic applications.

Rights

Copyright: © 2014 Pettinato et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Is Part Of

VCU Chemical and Life Science Engineering Publications