Pluripotent stem cells offer promise for new therapeutic interventions, particularly where there has been limited progress in understanding disease mechanisms as well as limited cell or tissue resources to apply to treatments. Refinement of differentiation strategies used to manipulate stem cells will limit variation, enhance reproducibility and unify the field. In pluripotent stem cell differentiation, a three-dimensional (3D) multicellular aggregate precursor that is an embryoid body (EB) is frequently used to mimic aspects of the early embryonic environment for tissue and organoid development. Methods of embryoid body formation have remained little changed until recently as groups try alternate methods to scale up production of embryoid bodies for clinical applications. Multiple questions still remain surrounding the use of embryoid bodies, including what size is optimal, whether there is a preferred method of formation, and whether embryoid body formed by different methods are comparable. Here we address these fundamental questions applying photolithography to bring uniformity to embryoid body formation in a high throughput platform. We recently further extended this work to include evaluation of the transcriptomes of EBs. We anticipate that our studies on embryoid body will be broadly useful to the stem cell community and accelerate the promise of stem cell research in biomedical therapies to improve the quality of human life.
Tomov ML1, Olmsted ZT1, Paluh JL2.[expand title=”Show Affiliations”]
- SUNY Polytechnic Institute, Colleges of Nanoscale Science and Engineering, Nanobioscience, Nanofab East, 257 Fuller Road, Albany, New York, 12203, USA.
- SUNY Polytechnic Institute, Colleges of Nanoscale Science and Engineering, Nanobioscience, Nanofab East, 257 Fuller Road, Albany, New York, 12203, USA. [email protected], [email protected]
In pluripotent stem cell differentiation, embryoid bodies (EBs) provide a three-dimensional [3D] multicellular precursor in lineage specification. The internal structure of EBs is not well characterized yet is predicted to be an important parameter to differentiation. Here, we use custom SU-8 molds to generate transparent lithography-templated arrays of polydimethylsiloxane (LTA-PDMS) for high throughput analysis of human embryonic stem cell (hESC) embryoid body formation and internal architecture. embryoid bodies formed in 200 and 500 μm diameter microarray wells by use of single cells, 2D clusters, or 3D early aggregates were compared. We observe that 200 μm embryoid bodies are monocystic versus 500 μm multicystic embryoid bodies that contain macro, meso and microsized cysts. In adherent differentiation of 500 μm embryoid body, the multicystic character impairs the 3D to 2D transition creating non-uniform monolayers. Our findings reveal that embryoid body core structure has a size-dependent character that influences its architecture and cell population uniformity during early differentiation.
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