Abstract
Generating properly differentiated embryonic structures in vitro from pluripotent stem cells remains a challenge. Here we show that instruction of aggregates of mouse embryonic stem cells with an experimentally engineered morphogen signalling centre, that functions as an organizer, results in the development of embryo-like entities (embryoids). In situ hybridization, immunolabelling, cell tracking and transcriptomic analyses show that these embryoids form the three germ layers through a gastrulation process and that they exhibit a wide range of developmental structures, highly similar to neurula-stage mouse embryos. Embryoids are organized around an axial chordamesoderm, with a dorsal neural plate that displays histological properties similar to the murine embryo neuroepithelium and that folds into a neural tube patterned antero-posteriorly from the posterior midbrain to the tip of the tail. Lateral to the chordamesoderm, embryoids display somitic and intermediate mesoderm, with beating cardiac tissue anteriorly and formation of a vasculature network. Ventrally, embryoids differentiate a primitive gut tube, which is patterned both antero-posteriorly and dorso-ventrally. Altogether, embryoids provide an in vitro model of mammalian embryo that displays extensive development of germ layer derivatives and that promises to be a powerful tool for in vitro studies and disease modelling.
Highlights
Generating properly differentiated embryonic structures in vitro from pluripotent stem cells remains a challenge
The secretion of BMP4 by the extraembryonic ectoderm promotes expression of WNT and NODAL in the embryonic epiblast[25]. Antagonists of these morphogens (DKK1, CER-1 and LEFTY1) secreted by the anterior visceral endoderm restrict their expression and activity to the posterior domain of the embryo where they induce the formation of the primitive streak, a structure in which epiblast cells undergo an epithelial-to-mesenchymal transition giving rise to mesoderm and endoderm[26]
We observed that incubation of the ESC aggregates with BMP4 resulted in inducing expression of WNT3 and NODAL that in turn induced expression of their downstream target genes such as Eomesodermin (Eomes) and Brachyury (Bra) (Supplementary Fig. 1)
Summary
Generating properly differentiated embryonic structures in vitro from pluripotent stem cells remains a challenge. To achieve a much higher degree of organization, organs need to be vascularized and innervated and contain the full spectrum of cells and tissues type derived from the three germ layers that can be patterned and organized along the three main body axes Toward this goal, recent experimental strategies have allowed the development of in vitro models of embryos[3,4]. Combining ESC and TSC with extra-embryonic endoderm stem cells (XEN), resulted in embryo-like structures that exhibited an epithelial–mesenchymal transition, leading to mesoderm and definitive endoderm specification, similar to an embryo at mid-gastrulation[8] This may explain why gastruloids and TLS can only generate post-occipital structures
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