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

Jesse V Veenvliet, Adriano Bolondi, Helene Kretzmer, Leah Haut, Manuela Scholze-Wittler, Dennis Schifferl, Frederic Koch, Milena Pustet, Simon Heimann, Rene Buschow, Lars Wittler, Bernd Timmermann, Alexander Meissner, Bernhard G Herrmann

Preprint posted on March 04, 2020 https://www.biorxiv.org/content/10.1101/2020.03.04.974949v1

Article now published in Science at http://dx.doi.org/10.1126/science.aba4937

What would happen if you culture in vitro self-aggregated gastruloids embedded in Matrigel? Believe it or not, they differentiate into trunk-like structures containing neural tube and somites.

Selected by Martin Estermann

Background:

During evolution, mammalian pregnancy was key to protect the offspring during embryonic development. In utero development creates inconveniencies when studying mammalian embryonic development because of its difficult accessibility. In the recent years, new in vitro models were developed to overcome this issue, such as organoids, embryoids and gastruloids, which largely improved our understanding of organ development and fate commitment and highlighted the ability of cells to self-organize into more complicated structures.

When embryonic stem cells are cultured under appropriate conditions, they aggregate into tridimensional embryo-like structures called gastruloids.  These aggregates undergo similar morphological and patterning events of the early embryo, generating cell types that correspond to the three germ layers, but fail to develop other complex early embryonic structures such neural tube or somites. This preprint demonstrates that complex structures can be achieved when modifying the culture conditions, being able to generate embryonic trunk-like-structures by simply growing them in Matrigel scaffolds. Gastruloids constitute a novel tool to study early embryonic development and provide a starting point to study other complex developmental events.

 

Key findings:

 1) Growing gastruloids in matrigel was sufficient to induce mesodermal segmentation and neural tube-like structures

The authors generated mouse embryonic stem cells (mESCs) with neural (green) and mesodermal (pink) reporters (Fig. 1a) to study the differentiation of gastruloids grown in matrigel scaffolds. These structures developed Trunk-Like-Structures (TLS), which are characterised by segmentation of the mesoderm in somite-like structures as well as the formation of a neural tube-like structure (Fig. 1b). The supplementation of the growing conditions with a WNT activator (TLSc) and a BMP inhibitor (TLSCL) improved the separation of the segments and resulted in a disorganization of the neural tissue. This experiment demonstrates the flexibility and the potential of gastruloid differentiation and highlights the importance of the extracellular matrix during development.

Fig. 1. Trunk-like structures. (a) Double reporter mouse embryonic stem cell line generated and in vivo validation in E9 mouse embryo (Preprint Extended Data Fig. 1a). (b) Gastruloid and TLS generated under different growing conditions (Preprint Fig. 1b)

2) Trunk-Like-Structures can recapitulate somitogenesis in vitro

Using brightfield live imaging, the authors were able to demonstrate the mesoderm segmentation process in real time, which remarkably seems to occur in a similarly in vivo-like configuration. This suggests that the segmentation clock oscillator could also be active in the pre-somite mesoderm of trunk like structures. I totally recommend you see supplementary video of this mechanism, but for now you can appreciate these time series images.

Fig. 2. In vivo brightfield live imaging of the segmentation process in TLS (Preprint Extended Data Fig. 14)

3) Trunk-Like-Structures can recapitulate embryonic mutant phenotype

In vivo, loss of Tbx6 results in an expansion of neural lineage and the formation of ectopic neural tubes at the expense of presomitic mesoderm and somites, which makes this mutant the perfect candidate to screen in these TLS (Fig. 2e). This mutation, in vitro, failed to form segmented structures / somites (pink) in TLS, but instead, generated ectopic neural tubes (green), consistent with the in vivo phenotype (Fig. 2f-g).

Fig. 3. Trunk-Like-Structures can recapitulate embryonic Tbx6 knock-out phenotype (preprint Fig. 4e-f). (e) Schematic Tbx6 knock-out (KO) embryonic mouse, and schematic view of the mESC line used to generate the KO gastruloids. (f) Quantification of TLS segmentation phenotype. (e) Tbx6 KO TLS are characterized by ectopic neural tubes and reduced segmentation.

Why I choose this paper:

This paper will mark a before and after in in vitro early developmental studies. Combining gastruloid culture with matrigel, a common reagent used to generate organoids, the authors were able to show that they can form complex structures that resemble an embryonic trunk. They also demonstrated the plasticity of these structures and how different conditions can affect their fate.

Surprisingly, these trunk-like structures were highly similar to embryonic trunks, sharing their cell type composition, transcriptomic information and morphological process. This paper reaffirms these self-organizing structures’ research potential and invites to experiment with gastruloids in novel in vitro growing conditions. It also demonstrates that even a simple idea could lead to an outstanding discovery.

 

Future directions / questions for the authors:

  • WNT activator and the BMP inhibitor treatment shifted mesoderm differentiation process into pre-somitic mesoderm rather than intermediate mesoderm or lateral plate mesoderm. Would it be possible to generate intermediate mesoderm derivative structures like mesonephros or lateral plate mesoderm by adding BMPs to the culture media? How would these structures look like?
  • How would the different composition of the extracellular matrix affect the gastruloid differentiation? Would the usage of decellularized scaffold from different organs modify the fate of the different gastruloids?
  • The ability to generate primordial germ cells from gastruloids, which also migrate in contact with gut like structures is a novel discovery. Are this PGCs able to colonize gonadal ridges in vitro or in vivo? Are they capable to differentiate into oocytes or sperm cells or even enter in meiosis?
  • Somitogenesis live imaging time series suggests that the segmentation oscillatory clock is working in this TLC. Is the clock-and-wavefront model working in vitro? Are you considering in using reporters for different genes involved in this process? Increasing or decreasing the speed of the segmentation clock would also affect the number of somites? Would snake TLC generate somites faster than mouse TLC?

Tags: development, embryo, gastruloid, in vitro, matrigel, mesc, mouse, neural tube, reporter, rna-seq, scaffold, single cell rna seq, somites, somitogenesis, stem cells, transgenics

Posted on: 19th April 2020 , updated on: 5th May 2020

doi: https://doi.org/10.1242/prelights.18906

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