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 MPEBs 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. STEM CELLS

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation

“…EBs generated from hES cells and human iPSCs efficiently differentiate into neural rosettes when cultured in specific selective culture media with growth factor supplements, 15 but EBs can have variable differentiation outcomes based on factors such as their initial size. 16 To enable more homogenous differentiation, microwell arrays have been specifically developed to allow the formation of EBs with uniform size (StemCell Technologies, Inc. Inc.).…”

Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of WNT5a and WNT11

“…The size of EBs is considered as an important parameter that influences ES cell differentiation (31). However, the complex series of interactions within a differentiating cell aggregate is difficult to analyze and is further increased in complexity in that most methods to form EB cultures are either too cumbersome or unable to uniformly control the size of EBs.…”

Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions

“…We consider several possibilities to explain these observations. Optimal spatial pattern of UV treatment may (i) establish favorable gradients of soluble factors guiding preferential migration toward other hESCs/hiPSCs (31), (ii) improve crosstalk between soluble factors and mechanical signal transduction (33), and (iii) facilitate the previously noted preference for hESCs/hiPSCs to be in cell-cell contact (22,23,34). Constraining cells to spots of small diameters prevented large multicellular aggregates from forming after seeding ( Fig.…”