Reproducible, Ultra High-Throughput Formation of Multicellular Organization from Single Cell Suspension-Derived Human Embryonic Stem Cell Aggregates
Figure 1
Conventional hESC differentiation protocols result in heterogeneous aggregates with inconsistent organization and structure: A.
Conventional differentiating hESC aggregates, formed by scraping colonies of hESC off the culture surface, are predominantly disordered. As hESC colonies differ widely in size and shape, this heterogeneity is passed on to the differentiating aggregate. Consequently the local microenvironment is neither consistent between nor within the aggregates. Scale bar represents 200 microns. Inset: Fusing aggregates exhibit expression of the transcription factor CDX2 (green, counterstain 7AAD, red) at points of contact, but not elsewhere, demonstrating the ability of microenvironmental cues to override macro-environmental conditions. Scale bar represents 200 microns. B–D. Rare hESC-derived aggregates exhibit self-organization. Within heterogeneous populations of scraped hESC-derived aggregates, a rare subpopulation of hEB can be observed. These hEB are characterized by the presence of two distinct domains, visible in phase contrast (B). An inner domain is positive for the pluripotency marker Oct4 (C/D – red), while the outer domain is positive for the endodermal marker FoxA2 (shown in green in panel C). A laminin-containing membrane at least partially defines the interface between these two domains (shown in green in panel D, counterstained with Hoechst in blue). Scale bars represent 100 microns.