An in vitro model of early anteroposterior organization during human development

Abstract

The body plan of the mammalian embryo is shaped through the process of gastrulation, an early developmental event that transforms an isotropic group of cells into an ensemble of tissues ordered with reference to three orthogonal axes 1 . While model organisms have provided much insight into this process, we know very little about gastrulation in humans due to the difficulty of obtaining embryos at such early stages of development, as well as to the ethical and technical restrictions that limit the feasibility of observing gastrulation ex vivo 2 . Here we show that human embryonic stem cells can be used to generate gastruloids: three dimensional multicellular aggregates that differentiate to derivatives of the three germ layers organised spatiotemporally, without additional extra-embryonic tissues. Human gastruloids undergo elongation along an anteroposterior axis and, using spatial transcriptomics, we show that they exhibit patterned gene expression. This includes a somitogenesis signature that suggests that 72 hour human gastruloids exhibit features of Carnegie Stage 9 embryos 3 . Our study represents a new, experimentally tractable model system to reveal and probe human-specific regulatory processes occurring during axial organisation in early development.The body plan of mammalian embryos emerges through interactions of sequential cell fate decisions and morphogenetic events, which have hitherto been difficult to observe in humans.Human Embryonic Stem Cells (hESCs) 4 have opened up opportunities for studying early fate decisions, and have hinted at the existence of regulatory mechanisms specific to humans 5,6 . But, in contrast to the embryo, where proportionate populations interact with one another to generate tissues and organs, differentiation in adherent culture is heterogeneous and favours a limited number of cell types 7 . Seeding hESCs on micropatterned surfaces yields coordinated patterns of gene expression, but without the axial organization characteristic of embryos 8 . However, when mouse ESCs are aggregated in suspension under defined conditions, they generate 'gastruloids': a three-dimensional, in vitro model of mammalian development, which exhibits an embryo-like spatiotemporal organization of gene expression 9,10 . We hypothesised that similar human gastruloids could be derived from hESCs. Generation of human gastruloidsWhen hESCs in 2D culture were treated with Chiron, a Wnt agonist, for one day before seeding defined numbers in low-adherence plates in the presence of Chiron, they formed compact, spherical aggregates within a few hours (Fig. 1a, Extended Data Fig. 1a-c). These aggregates progressively broke symmetry and formed elongated structures, with maximal elongation at 72-96h (Fig. 1a-d). On average, ~66% of aggregates from each experiment displayed an elongated morphology at 72h. Although some of the structures remained TOP award (NWO-CW 714.016.001), and the Foundation for Fundamental Research on Matter, financially supported by NWO (FOM-14NOISE01) to S.C.v.d.B., A.A. and A.v.O.. This w...

A spatially resolved single cell atlas of human gastrulation

“…Therefore, efforts to understand human gastrulation have predominantly focused on in vitro models such as monolayers of human Embryonic Stem Cells (hESCs) cultured on circular micropatterns (Warmflash et al 2014). More recently, these have been extended to hESC colonies engrafted into chick embryos (Martyn et al 2018) or 3D cellular models derived from hESC (Simunovic et al 2019;Moris et al 2020). While these powerful approaches provide valuable insights, currently there is no in vivo data on the molecular control of human gastrulation to compare them against or further refine them.…”

Translating Embryogenesis to Generate Organoids: Novel Approaches to Personalized Medicine

“…Culturing mouse gastruloids with an underlying extracellular matrix led to the formation of structures resembling somites and neural tube ( van den Brink et al., 2020 ; Veenvliet et al, 2020 ), whereas another report suggested that mixing gastruloids and extra-endodermal cells can lead to the formation of the neural tube ( Bérenger-Currias et al, 2020 ). Recently, gastruloids have been generated from self-organizing hESCs, which can also differentiate to three germ layers ( Moris et al., 2020 ). Somite formation is a rhythmic process that involves molecular oscillation and the expression of certain genes that pattern the embryo, collectively known as the molecular segmentation clock that has been widely characterized in several model systems and PSCs ( Diaz-Cuadros et al., 2020 ; Matsuda et al., 2020 ; Pourquie, 2003 ).…”

Human Developmental Cell Atlas: milestones achieved and the roadmap ahead

“…Self-organization of human embryonic tissue can be captured from the earliest moments in vitro , and extended to gastrulation, anterior-posterior embryonic patterning, and early phases of somitogenesis 102 . The recent human gastrulation embryo dataset will be informative as a benchmark to further refine in vitro directed differentiation of human cells, including gastruloid models 102 , to more faithfully recapitulate early in vivo differentiated cell types and gastrulation.…”