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THE STEM CELL NICHE

Control of Human Embryonic Stem Cell Colony and Aggregate Size Heterogeneity Influences Differentiation Trajectories

^aDepartment of Chemical Engineering and Applied Chemistry,
^bInstitute of Biomaterials and Biomedical Engineering,
^cDepartment of Chemistry,
^dDepartment of Medicine, and
^eMcEwen Centre for Regenerative Medicine, University Health Network and Heart & Stroke Richard Lewar Centre of Excellence, University of Toronto, Toronto, Ontario, Canada

Key Words. Human pluripotent cells • Embryoid bodies • Embryoid body size • Cardiac induction • Micropatterning • Endogenous lineage specification • Differentiation mechanisms

Correspondence: Correspondence: Peter W. Zandstra, Ph.D., Terence Donnelly CCBR, 160 College Street, Office 1116, Toronto, Ontario, Canada M5S 3G9. Telephone: 416-978-8888; Fax: 416-978-2666; e-mail: peter.zandstra{at}utoronto.ca

Received on February 26, 2008; accepted for publication on June 19, 2008.

First published online in STEM CELLS EXPRESS  June 26, 2008.

To better understand endogenous parameters that influence pluripotent cell differentiation we used human embryonic stem cells (hESCs) as a model system. We demonstrate that differentiation trajectories in aggregate (embryoid body [EB])-induced differentiation, a common approach to mimic some of the spatial and temporal aspects of in vivo development, are affected by three factors: input hESC composition, input hESC colony size, and EB size. Using a microcontact printing approach, size-specified hESC colonies were formed by plating single-cell suspensions onto micropatterned (MP) extracellular matrix islands. Subsequently, size-controlled EBs were formed by transferring entire colonies into suspension culture enabling the independent investigation of colony and aggregate size effects on differentiation induction. Gene and protein expression analysis of MP-hESC populations revealed that the ratio of Gata6 (endoderm-associated marker) to Pax6 (neural-associated marker) expression increased with decreasing colony size. Moreover, upon forming EBs from these MP-hESCs, we observed that differentiation trajectories were affected by both colony and EB size-influenced parameters. In MP-EBs generated from endoderm-biased (high Gata6/Pax6) input hESCs, higher mesoderm and cardiac induction was observed at larger EB sizes. Conversely, neural-biased (low Gata6/Pax6) input hESCs generated MP-EBs that exhibited higher cardiac induction in smaller EBs. Our analysis demonstrates that heterogeneity in hESC colony and aggregate size, typical in most differentiation strategies, produces subsets of appropriate conditions for differentiation into specific cell types. Moreover, our findings suggest that the local microenvironment modulates endogenous parameters that can be used to influence pluripotent cell differentiation trajectories.

Disclosure of potential conflicts of interest is found at the end of this article.

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