Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites

Abstract

One sentence summary: A platform for generating trunk-like-structures with precursors of spinal cord, bone and muscle from stem cells in a dish Abstract: Post-implantation embryogenesis is a highly dynamic process comprising multiple lineage decisions and morphogenetic changes inaccessible to deep analysis in vivo. Mouse embryonic stem cells (mESCs) can form aggregates reflecting the post-occipital embryo (gastruloids), but lacking proper morphogenesis. Here we show that embedding of aggregates derived from mESCs in an extracellular matrix compound results in Trunk-Like-Structures (TLS) with a high level of organization comprising a neural tube and somites. Comparative single-cell RNA-seq analysis demonstrates that TLS execute gene-regulatory programs in an embryo-like order, and generate primordial germ cell like cells (PGCLCs). TLS lacking Tbx6 form ectopic neural tubes, mirroring the embryonic mutant phenotype. ESC-derived trunk-like structures thus constitute a novel powerful in vitro platform for investigating lineage decisions and morphogenetic processes shaping the post-implantation embryo.2 Vertebrate post-implantation development comprises a multitude of complex morphogenetic 1 processes, which result from self-organization of stem cells and their descendants shaping the 2 embryonic body plan 1 . Recently developed stem cell models represent powerful platforms for 3 deconstructing the dynamics of these processes that are inaccessible in the embryo 1, 2 . The 4 most advanced models in terms of developmental stage accomplished so far are gastruloids 2,3 , 5 aggregates of mESCs able to self-organize into the three body axes 3 . However, gastruloids lack 6 proper morphogenesis, such as a failure of neural tube formation from neural cells or somite 7 condensation from presomitic cells 3 . In vivo, the extracellular matrix (ECM) has a critical role in 8 tissue morphogenesis 4 . In vitro, matrigel can serve as ECM surrogate, and culture media 9 supplemented with a low percentage of matrigel have been shown to induce complex 10 morphogenesis in organoids 5 . 11We therefore explored if embryo-like morphogenetic features could be induced by 12 embedding 96h post aggregation gastruloids in 5% matrigel for an additional 24h period ( Fig. 13 1a). We also tested a combination of matrigel together with the WNT signaling activator 14 CHIR99021 (CHIR) and the BMP signaling inhibitor LDN193189 (LDN), that have been reported 15 to induce a (pre-)somitic mesoderm ((P)SM) fate in 2D and 3D differentiation protocols 6-8 . To 16 facilitate high-throughput characterization and quantification of our conditions, we generated 17 mESCs with T::H2B-mCherry (hereafter T mCH ) and Sox2::H2B-Venus (hereafter Sox2 VE ) reporters, 18 marking the mesodermal (ME) or neural (NE) lineage respectively (Extended Data Fig. 1a). 19 Strikingly, embedding in 5% matrigel alone was sufficient for segmentation in the T mCH+ ME 20 domain and formation of a Sox2 VE+ neural tube like structure ( Fig. 1b,d; Extended Data Fig. 21 1b,c). The vast majorit...

Cell-state transitions and collective cell movement generate an endoderm-like region in gastruloids

“…Upon exposure to external cues and morphogens, gastruloids show an intrinsic capability to polarize, elongate and pattern 11,12 . Strikingly, 3D gastruloids exhibit a multiaxial organization and spatiotemporal patterning of Hox genes 13 with signatures of somitogenesis 14, 15 . From in-vitro assays, it is evident that a combination of particular cell types is needed to generate lumen in the blastocyst 16 and establish an anterior-posterior axes prior gastrulation 17 .…”

Translating Embryogenesis to Generate Organoids: Novel Approaches to Personalized Medicine

“…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 ). The presence of an active segmentation clock in gastruloids similar to an in vivo embryo extends the power of this model system to characterize mammalian development ( van den Brink et al., 2020 ; Veenvliet et al, 2020 ). Nevertheless, the advent of blastoids and gastruloids from mESCs has opened new avenues to understand embryogenesis with wide application in biomedical discoveries especially for understanding developmental malformations.…”

“…datasets provide evidence on the emergence of neural tube and somites recapitulating early organogenesis ( van den Brink et al., 2020 ; Veenvliet et al, 2020 ). 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 ).…”

“… Generate neural tube, mesodermal and endodermal derivatives, and presence of segmentation clock. Beccari et al., 2018 ; van den Brink et al., 2020 ; Veenvliet et al, 2020 Human ESC Form derivatives of three germ layers and undergo elongation along an anteroposterior axis. Signature of somitogenesis resembling Carnegie-stage-9 embryos.…”

“…A collective understanding of how ESCs can be manipulated in vitro to mimic developmental stages of the embryo ( Shahbazi and Zernicka-Goetz, 2018 ) is influential to generate more complex organ type in vitro . For example, the presence of an extracellular matrix, similar to a developing embryo, led to the formation of somites in gastruloids ( van den Brink et al., 2020 ; Veenvliet et al, 2020 ). The role of the laminin-rich extracellular matrix (also called Matrigel) in the growth and proliferation of intestinal tubules and mammary glands ( Li et al., 1987 ; Montesano et al., 1991 ) led the foundation to initiate self-organizing structures from ESCs.…”