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Early neurulation recapitulated in assemblies of embryonic and extraembryonic cells

Noémie M. L. P. Bérenger-Currias, Maria Mircea, Esmée Adegeest, Patrick R. van den Berg, Marleen Feliksik, Mazène Hochane, Timon Idema, Sander J. Tans, Stefan Semrau

Preprint posted on February 14, 2020 https://www.biorxiv.org/content/10.1101/2020.02.13.947655v1

ES + XEN = neuruloids! A great model to recapitulate neurulation in vitro with huge potential for studying development.

Selected by Monica Tambalo

Background

The mammalian embryo is generated by the interplay of embryonic and extraembryonic tissues: the epiblast gives rise to the embryo proper, while trophectoderm and the primitive endoderm generate the placenta and yolk sac, respectively. The extraembryonic tissues are a known source of signaling cues acting on early cell fate decisions, germ-layer specification, and tissue patterning [1], therefore they are essential for proper embryo development. The interplay of embryonic and extraembryonic tissues has been addressed only in part in in vitro organoids, mainly focusing on the first stages of development [2]. Bérenger-Currias and colleagues have advanced the organoid system by assembling mouse embryonic stem cells (mESCs) and extraembryonic endoderm (XEN) and assessed the organogenesis capacity of such ‘assembloids’ after gastrulation.

Key findings

The ‘neuruloid’ culture system

The authors have adapted a previously published gastruloid protocol [3] by co-aggregating XEN with mESCs. Strikingly, they observed structures resembling the neural tube without any need for external signaling sources (Figure 1); thus, creating a new in vitro model system for neural induction that was named ‘neuruloid’. Cell type diversity was extensively addressed by single-cell comparisons of neuruloids, gastruloids, and mouse in vivo data. The main finding was that neuruloids presented additional cell types compared to gastruloids, mainly derivatives of XEN cells, among them extraembryonic endoderm cells, nascent and pharyngeal mesoderm, and haematoendothelial progenitors.

Figure 1. Neural tube-like structures are induced by XEN cells. Gastruloid protocol (inset) gives rise to a primitive streak-like domain (Brachyury T+ in red) and neural progenitor-like cells (SOX2+ in green). Strikingly, in neuruloids the addition of XEN cells (eGFP+ in blue) triggers stratification of the epithelia with the formation of multiple lumina (SOX2+ in green). Scale bars 100 µm.

How does the neuronal lineage differ between neuruloids and gastruloids?

The authors found that neuruloids present significant differences in gene expression in their neural progenitor population compared to gastruloids. Neuruloid cells are most similar to dorsal neural progenitors and express genes likely downstream of BMP signaling, and components of this signaling pathway are secreted by XEN cells. Additionally, when developmental signaling pathways are perturbed the neural tube-like structures of neuruloids respond as the in vivo counterparts, suggesting that cells are competent to respond to neurogenic signals and have the potential to further differentiate into specific brain regions. Thus, to explore the neuruloids’ potential the authors cultured them for longer in the appropriate differentiating media [4]. Strikingly, they found that neuruloids formed layered cerebral cortex-like tissues surrounding ventricle-like structures (Figure 2), with the presence of vasculature presumptive precursors.

Figure 2. 8-day old neuruloids present layered cerebral cortex-like tissue. Section of neuruloid-derived cerebral cortex-like tissue with immunostaining of neural progenitors (SOX2 in green) and neurons (TUJ1 in red). Scale bars 100 µm.

How do XEN cells differentiate in neuruloid culture?

When looking at the XEN lineage in neuruloids, the authors always observed XEN cells forming the outermost layer of neuruloids, strikingly resembling how the extraembryonic endoderm encapsulates the developing embryo. Indeed, the transcriptome of XEN-derived cells in neuruloids mapped to in vivo extraembryonic endoderm profiles of both parietal endoderm (PE) and visceral endoderm (VE). The authors further demonstrated by single-molecule FISH that undifferentiated XEN cells have both PE and VE characteristics but later become more VE-like due to the influence of mESCs. This is reminiscent of the in vivo organization, where VE cells are in contact with the embryo while PE cells go on to contribute to the yolk sac.

How do XEN cells influence mESCs in neuruloid culture?

By observing a battery of neuruloids the authors made the observation that XEN cells may guide symmetry breaking on adjacent mESC-derived cells, resembling the well-known role of the VE in embryonic development. The authors speculate that this role is mediated by the presence of a basement membrane and diffusible factors, i.e. WNT signaling inhibition given its known role in vivo. In this version of the paper, the authors do not directly prove that the same mechanism is taking place in vitro.

Why I chose the paper

Organoid culture protocols are reductionist models of development and often rely on well-defined differentiating media to drive specific tissue/organ differentiation. A great challenge remains to fully recapitulate organogenesis in vitro, mimicking as much as possible embryonic development. Bérenger-Currias and colleagues with this preprint address this need by developing a new in vitro system that better resembles early stages of embryogenesis where multiple lineages (ESCs and XEN cells) are in close contact and mutually influence their fates. Strikingly, neuruloids form neural tube-like structures a feature not frequently seen in other organoid culture protocols and new cell types are produced in this model system.

How this work moves the field forward

Given the reductionist nature of the neuruloid system, I believe it will be instrumental in understanding the minimal requirements for proper mammalian neurulation, help pinpoint key mechanisms of extraembryonic endoderm development and heterotypic cell-cell interactions. Following this work other preprints recently came available, one, in particular, describes how the embedding of mESCs-organoids in extracellular matrix is able to improve tissue organization with the formation of neural tube-like structures and somites [5]. However, Bérenger-Currias and colleagues found that the consequences of adding XEN cells to organoids had a greater impact on morphogenesis than extracellular matrix alone. Furthermore, what I consider the greatest potential of the neuruloid system is its great cellular complexity that includes cells from all embryonic germ layers and extraembryonic endoderm. This has the potential of improving existing organoid culture conditions; one possibility is to perfect this method in order to allow vascularization of the cerebral-cortex organoids. This would help the further growth of cerebral organoids, avoiding the issue of necrosis in the core of the tissue due to poor oxygenation. I believe this work, by inspiring new advances in organoid cultures, has the potential of improving our understanding of cross-tissue interactions, also in the human system, in a more holistic view.

Questions to the authors

  1. Would it be possible to replicate this work with human-derived cells? If so, can the authors speculate which similarities and differences they may expect?
  2. Have the authors tested additional differentiating protocols or only the cerebral cortex development?
  3. To test the potential of this protocol would it be possible to grow even further the neuruloids and check the complexity and maturation of cortical neuronal cell types?

References

  1. Tam, P. P. L. & Loebel, D. A. F. Gene function in mouse embryogenesis: get set for Nat. Rev. Genet. 8, 368–381 (2007).
  2. Sozen, B. et al. Self-assembly of embryonic and two extra-embryonic stem cell types into gastrulating embryo-like structures. Cell Biol. 20, 979–989 (2018).
  3. Van Den Brink, S. C. et al. Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells. 141, 4231–4242 (2014).
  4. Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013).
  5. Veenvliet, J. V, Bolondi, A., Kretzmer, H., Haut, L. & Scholze-wittler, M. Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites. bioRxiv 1–72 (2020).

Tags: development, embryogenesis, embryonic stem cells, extraembryonic endoderm cells, mouse, neural tube, neuruloids, organoids

Posted on: 24th April 2020

doi: https://doi.org/10.1242/prelights.19229

Read preprint (1 votes)




Author's response

Stefan Semrau shared

Q1 – Would it be possible to replicate this work with human-derived cells? If so, can the authors speculate which similarities and differences they may expect?

To our knowledge, XEN cells have not been derived from human embryos, yet. However, XEN-like cells were differentiated from human embryonic stem cells (hESCs) [1]. It would certainly be interesting to tester whether those human XEN-like cells would have a similar effect as the mouse XEN cells in a gastruloid context. We might also be able to combine mouse XEN with hESCs. One significant difference between early mouse and human embryos is their shape. Compared to mouse, human embryos have a more ellipsoid shape. Several human in vitro systems with such a shape were developed successfully [2,3] and could be used as a basis for a neuruloid system including XEN. We would be very curious to see where the XEN cells would position themselves in this geometry and if they have a similar behavior as in our neuruloids.

Q2 – Have the authors tested additional differentiating protocols or only the cerebral cortex development?

As highlighted in your post, we observed some cells with vascular markers in the cerebral organoids. They could be the starting point for a proper vasculature, so we are currently trying to stimulate the proliferation and further differentiation of these cells.

Q3 – To test the potential of this protocol would it be possible to grow even further the neuruloids and check the complexity and maturation of cortical neuronal cell types?

It has been shown that gastruloids can be grown for a couple of weeks and cerebral organoids for several months. We would expect that the cells in neuruloids continue to mature but additional patterning will probably require external inputs, given that important sources of signaling molecules do not form properly in gastruloids and neuruloids.

References

  1. Linneberg-Agerholm, M. et al. Naïve human pluripotent stem cells respond to Wnt, Nodal and LIF signalling to produce expandable naïve extra-embryonic endoderm. Development 146, (2019).
  2. Warmflash, A., Sorre, B., Etoc, F., Siggia, E. D. & Brivanlou, A. H. A method to recapitulate early embryonic spatial patterning in human embryonic stem cells. Nature Methods 11, 847–854 (2014).
  3. Haremaki, T. et al. Self-organizing neuruloids model developmental aspects of Huntington’s disease in the ectodermal compartment. Nature Biotechnology 37, 1198–1208 (2019).

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