Controlled Self-assembly of Stem Cell Aggregates Instructs Pluripotency and Lineage Bias

TLDR

Stem cell‑derived organoids and 3D microtissues hold great promise for drug screening, disease modeling, and regenerative medicine, yet reproducibility is hampered by uncontrolled spontaneous aggregation during biomanufacturing. This study aims to elucidate how aggregation parameters affect cell behavior and thereby influence the production of organoids. We developed a bioengineered platform of labile substrate arrays that enable scalable, controllable 2D‑to‑3D self‑assembly of cell aggregates. Using this platform, we demonstrated that substrate‑controlled aggregation yields size‑ and shape‑defined embryoid bodies, that faster aggregation drives pluripotency loss and ectoderm bias while slower aggregation promotes mesoderm/endoderm, increases porosity and growth‑factor signaling, and overall suggests that tuning aggregation kinetics can enhance 3D microtissue models.

Abstract

Stem cell-derived organoids and other 3D microtissues offer enormous potential as models for drug screening, disease modeling, and regenerative medicine. Formation of stem/progenitor cell aggregates is common in biomanufacturing processes and critical to many organoid approaches. However, reproducibility of current protocols is limited by reliance on poorly controlled processes (e.g., spontaneous aggregation). Little is known about the effects of aggregation parameters on cell behavior, which may have implications for the production of cell aggregates and organoids. Here we introduce a bioengineered platform of labile substrate arrays that enable simple, scalable generation of cell aggregates via a controllable 2D-to-3D "self-assembly". As a proof-of-concept, we show that labile substrates generate size- and shape-controlled embryoid bodies (EBs) and can be easily modified to control EB self-assembly kinetics. We show that aggregation method instructs EB lineage bias, with faster aggregation promoting pluripotency loss and ectoderm, and slower aggregation favoring mesoderm and endoderm. We also find that aggregation kinetics of EBs markedly influence EB structure, with slower kinetics resulting in increased EB porosity and growth factor signaling. Our findings suggest that controlling internal structure of cell aggregates by modifying aggregation kinetics is a potential strategy for improving 3D microtissue models for research and translational applications.