title: Control of human embryonic stem cell colony and aggregate size heterogeneity influences differentiation trajectories.
abstract: 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.
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University Health Network and Heart & Stroke Richard Lewar Centre of Excellence, University of Toronto,Toronto, Ontario, Canada.
authors.forename: Céline Liu
authors.initials: CL
authors.lastname: Bauwens
authors.forename: Raheem
authors.initials: R
authors.lastname: Peerani
authors.forename: Sylvia
authors.initials: S
authors.lastname: Niebruegge
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Woodhouse
authors.forename: Eugenia
authors.initials: E
authors.lastname: Kumacheva
authors.forename: Mansoor
authors.initials: M
authors.lastname: Husain
authors.forename: Peter W
authors.initials: PW
authors.lastname: Zandstra
mesh_terms: D000943:Antigens, Differentiation / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology; D002454:Cell Differentiation; D019070:Cell Lineage; D049109:Cell Proliferation; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000502:physiology; D006801:Humans; D018874:Spheroids, Cellular / Q000166:cytology* / Q000502:physiology
chemical_list: D000943:Antigens, Differentiation