Mechanical Actuation of Organoids in Synthetic Microenvironments.

Organoids are a powerful model system to explore the role of mechanical forces in sculpting emergent tissue cytoarchitecture. The modulation of the mechanical microenvironment is most readily performed using synthetic extracellular matrices (ECM); however, such materials provide passive, rather than active force modulation. Actuation technologies enable the active tuning of mechanical forces in both time and magnitude. Using such instruments, our group has shown that extrinsically imposed stretching on human neural tube organoids (hNTOs) enhanced patterning of the floor plate domain. Here, we provide a detailed protocol on the implementation of mechanical actuation of organoids embedded in synthetic 3D microenvironments, with additional details on methods to characterize organoid fate and behavior. Our protocol is easy to reproduce and is expected to be broadly applicable to investigate the role of active mechanics with in vitro model systems.

A New Approach to Generate Gastruloids to Develop Anterior Neural Tissues.

Embryonic development is a complex process integrating cell fate decisions and morphogenesis in a spatiotemporally controlled manner. Previous studies with model organisms laid the foundation of our knowledge on post-implantation development; however, studying mammalian embryos at this stage is a difficult and laborious process. Early attempts to recapitulate mammalian development in vitro begun with embryoid bodies (EBs), in which aggregates of mouse embryonic stem cells (mESCs) were shown to differentiate into spatially arranged germ layers. A more revised version of EBs, gastruloids, improved the germ layer differentiation efficiency and demonstrated cell fate patterning on multiple axes. However, gastruloids lack anterior neural progenitors that give rise to brain tissues in the embryo. Here, we report a novel culture protocol to coax mESCs into post-implantation epiblast-like (EPI) aggregates in high throughput on bioengineered microwell arrays. We show that upon inhibition of the Wnt signaling pathway, EPI aggregates establish an extended axial patterning, leading to co-derivation of anterior neural progenitors and posterior tissues. Our approach is amenable to large-scale studies aimed at identifying novel regulators of gastrulation and anterior neural development that is currently out of reach with existing embryoid models. This work should contribute to the advancement of the nascent field of synthetic embryology, opening up exciting perspectives for various applications of pluripotent stem cells in disease modeling and tissue engineering. Key features A new gastruloid culture system to model post-implantation mouse embryonic development in vitro High-throughput formation of epiblast-like aggregates on hydrogel microwells Builds upon conventional gastruloid cultures and provides insight into the role of Wnt signaling for the formation of anterior neural tissues Graphical overview.

Gastruloids generated without exogenous Wnt activation develop anterior neural tissues.

When stimulated with a pulse from an exogenous WNT pathway activator, small aggregates of mouse embryonic stem cells (ESCs) can undergo embryo-like axial morphogenesis and patterning along the three major body axes. However, these structures, called gastruloids, currently lack the anterior embryonic regions, such as those belonging to the brain. Here, we describe an approach to generate gastruloids that have a more complete antero-posterior development. We used hydrogel microwell arrays to promote the robust derivation of mouse ESCs into post-implantation epiblast-like (EPI) aggregates in a reproducible and scalable manner. These EPI aggregates break symmetry and axially elongate without external chemical stimulation. Inhibition of WNT signaling in early stages of development leads to the formation of gastruloids with anterior neural tissues. Thus, we provide a new tool to study the development of the mouse after implantation in vitro, especially the formation of anterior neural regions.