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

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.Human pluripotent stem cells (hPSCs) offer considerable promise as a cell source for regenerative medicine. Traditional 2-dimensional (2D) stem cell culture is suitable for basic research applications but lacks the scalability required for biomanufacturing and the biological complexity required to generate organoids for drug/toxin screening 1,2 . Alternatively, three-dimensional (3D) cell aggregates are an attractive cell culture format for such applications. Stem cell aggregates offer increased surface area for cell growth per media volume, which enables stem cell expansion at the scale required for cell therapies 3 . In addition, cell aggregates applied as implantable "scaffold-free" constructs show enhanced survival and function in vivo [4][5][6][7] . Stem cell aggregates also serve as starting materials for generating organoids, complex multicellular constructs that recapitulate structural and functional aspects of human organs and are useful as in vitro tissue models for predicting responses to drugs and toxins 8,9 . The process of cell aggregate formation, typically via reaggregation of singularized cells, is a critical initial step for the generation of many organoids. While several types of stem/progenitor cells have demonstrated an intrinsic capacity to "self-organize" into 3D tissue-specific organoids 10,11 , current approaches offer little control over parameters associated with the aggregation process (e.g., aggregate size, shape, formation kinetics), which limits optimization of stem cell expansion/differentiation processes and impedes identificat...

Challenges and Solutions for Commercial Scale Manufacturing of Allogeneic Pluripotent Stem Cell Products

“…Depending on a particular cell expansion and differentiation process, there is an optimal range of spherical cell aggregate sizes that can maximize the efficiency and production yield of expansion and differentiation processes in a bioreactor [7][8][9]. If a PSC aggregate becomes too large, nutrients and the growth or differentiation factors may be unable to evenly diffuse from the aggregate surface into its center, leading to unwanted cell death or heterogeneous cell populations during expansion or differentiation [10,11].…”

Thalidomide Inhibits Human iPSC Mesendoderm Differentiation by Modulating CRBN-dependent Degradation of SALL4

“…3D cultures of PSCs have historically been generated with forced aggregation. However, forced aggregation reduces hPSC pluripotency under maintenance conditions and may prime the resulting aggregates to spontaneous ectoderm differentiation 65 . Thus we employed a 3D culture system amenable to mesoderm differentiation.…”

Capturing Neonatal Cardiomyocyte – Endothelial interactions on-a-Chip

“…Some of the cells that wrapped around the large cell aggregates exhibited contractile activity typical of beating CMs (Video S3). The cell aggregates that were observed here, could be nascent vascular structures [46][47][48][49] that are seen in cardiac organoids [50][51][52] .…”