Generation of Budoids: 3D Multilineage Limb Models From Mouse Embryonic Stem Cells

Limb development requires the coordination of multiple cell types, including the limb bud mesoderm and surface ectoderm, by the apical ectodermal ridge (AER), a specialized signaling center secreting numerous morphogens. Characterizing these cell-cell interactions is crucial for understanding limb morphogenesis, but they are challenging to study in vivo. Furthermore, existing in vitro models do not capture the multilineage complexity of the limb. We recently developed a robust 7-day differentiation protocol using mouse embryonic stem cells (mESCs) to generate heterogeneous cultures containing cells with characteristics of the limb bud mesoderm, surface ectoderm, and AER. Dissociating and reaggregating these cultures in low attachment 96-well plates forms budoids, organoids that display certain limb bud-like features. Budoids undergo chondrogenesis-mediated symmetry breaking and elongation within 5 days of culture. Altogether, our protocols have enabled the study of cell-cell interactions in limb development and provide an easily scalable model adaptable for various applications, including drug testing and congenital disorder modeling. Key features • Induction of heterogeneous 2D cultures containing three main cell types of the developing limb bud from mESCs. • Free-floating budoid cultures achieve 3D morphogenesis without manual manipulation or embedding. • Highly scalable protocol, per differentiation experiment yielding > 9 × 10⁶ cells, sufficient for generating >1,000 budoids.