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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.
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.
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VCU Chemical and Life Science Engineering Publications
(A) Image of a Teflon stamp with micronipple arrays that was used to create the hydrogel microwells for the hEB formation; (B) Low-melting-point agarose was used to make microwells to form hEBs.
Table_S1.doc (67 kB)
LSMeansa and Tukeys post hoc comparisons for the cross sectional area of hEBs formed using approx. 15,000 BG01V/hOG hESC/well.
Table_S2.doc (68 kB)
LSMeansa and Tukeys post hoc comparisons for the cross sectional area of hEBs formed using approx. 25,000 H9 hESC/well.