Biology:Gastruloid

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An example of a Gastruloid formed from Brachyury::GFP mouse ESCs, treated with a pulse of the Wnt/β-Catenin agonist CHIR99021 between 48 and 72h and imaged by wide-field fluorescence microscopy at 120h. Notice the polarised expression of Brachyury::GFP (Bra) at the elongating tip of the Gastruloid. Image from van den Brink et al. (2014), used with CC-BY licence.

Gastruloids are three dimensional aggregates of embryonic stem cells (ESCs) that, when cultured in specific conditions, exhibit an organization resembling that of an embryo. They develop with three orthogonal axes and contain the primordial cells for various tissues derived from the three germ layers, without the presence of extraembryonic tissues. Notably, they do not possess forebrain, midbrain, and hindbrain structures. Gastruloids serve as a valuable model system for studying mammalian development, including human development, as well as diseases associated with it. They are a model system an embryonic organoid for the study of mammalian development (including humans) and disease.[1][2][3]

Background

The Gastruloid model system draws its origins from work by Marikawa et al..[4] In that study, small numbers of mouse P19 embryonal carcinoma (EC) cells, were aggregated as embryoid bodies (EBs) and used to model and investigate the processes involved in anteroposterior polarity and the formation of a primitive streak region.[4] In this work, the EBs were able to organise themselves into structures with polarised gene expression, axial elongation/organisation and up-regulation of posterior mesodermal markers. This was in stark contrast to work using EBs from mouse ESCs, which had shown some polarisation of gene expression in a small number of cases but no further development of the multicellular system.[5][6]

Following this study, the Martinez Arias laboratory in the Department of Genetics at the University of Cambridge demonstrated how aggregates of mouse embryonic stem cells (ESCs) were able to generate structures that exhibited collective behaviours with striking similarity to those during early development such as symmetry-breaking (in terms of gene expression), axial elongation and germ-layer specification.[1][2][3] To quote from the original paper: "Altogether, these observations further emphasize the similarity between the processes that we have uncovered here and the events in the embryo. The movements are related to those of cells in gastrulating embryos and for this reason we term these aggregates ‘gastruloids’". As noted by the authors of this protocol, a crucial difference between this culture method and previous work with mouse EBs was the use of small numbers of cells which may be important for generating the correct length scale for patterning, and the use of culture conditions derived from directed differentiation of ESCs in adherent culture[1][7][3][2][8]

Brachyury (T/Bra), a gene which marks the primitive streak and the site of gastrulation, is up-regulated in the Gastruloids following a pulse of the Wnt/β-Catenin agonist CHIR99021[9] (Chi; other factors have also been tested[1]) and becomes regionalised to the elongating tip of the Gastruloid. From or near the region expressing T/Bra, cells expressing the mesodermal marker tbx6 are extruded from the similar to cells in the gastrulating embryo; it is for this reason that these structures are called Gastruloids.[1]

Further studies revealed that the events that specify T/Bra expression in gastruloids mimic those in the embryo.[2] After seven days gastruloids exhibit an organization very similar to a midgestation embryo with spatially organized primordia for all mesodermal (axial, paraxial, intermediate, cardiac, cranial and hematopoietic) and endodermal derivatives as well as the spinal cord.[10][11][3] They also implement Hox gene expression with the spatiotemporal coordinates as the embryo.[3] Gastruloids lack brain as well as extraembryonic tissues but characterisation of the cellular complexity of gastruloids at the level of single cell and spatial transcriptomics, reveals that they contain representatives of the three germ layers including neural crest, Primordial Germ cells and placodal primordia.[12][13]

A feature of gastruloids is a disconnect between the transcriptional programs and outlines and the morphogenesis. However, changes in the culture conditions can elicit morphogenesis, most significantly gastruloids have been shown to form somites[13][12] and early cardiac structures.[14] In addition, interactions between gastruloids and extraembryonic tissues promote an anterior, brain-like polarised tissue.[15]

Gastruloids have recently been obtained from human ESCs,[16] which gives developmental biologists the ability to study early human development without needing human embryos. Importantly though, the human gastruloid model is not able to form a human embryo, meaning that is a non-intact, non-viable and non-equivalent to in vivo human embryos.

The term Gastruloid has been expanded to include self-organised human embryonic stem cell arrangements on patterned (micro patterns) that mimic early patterning events in development;[17][18] these arrangements should be referred to as 2D gastruloids.

References

  1. ↑ 1.0 1.1 1.2 1.3 1.4 Brink, Susanne C. van den; Baillie-Johnson, Peter; Balayo, Tina; Hadjantonakis, Anna-Katerina; Nowotschin, Sonja; Turner, David A.; Arias, Alfonso Martinez (2014-11-15). "Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells" (in en). Development 141 (22): 4231–4242. doi:10.1242/dev.113001. ISSN 0950-1991. PMID 25371360. 
  2. ↑ 2.0 2.1 2.2 2.3 Turner, David A.; Girgin, Mehmet; Alonso-Crisostomo, Luz; Trivedi, Vikas; Baillie-Johnson, Peter; Glodowski, Cherise R.; Hayward, Penelope C.; Collignon, Jérôme et al. (2017-11-01). "Anteroposterior polarity and elongation in the absence of extra-embryonic tissues and of spatially localised signalling in gastruloids: mammalian embryonic organoids" (in en). Development 144 (21): 3894–3906. doi:10.1242/dev.150391. ISSN 0950-1991. PMID 28951435. 
  3. ↑ 3.0 3.1 3.2 3.3 3.4 Beccari, Leonardo; Moris, Naomi; Girgin, Mehmet; Turner, David A.; Baillie-Johnson, Peter; Cossy, Anne-Catherine; Lutolf, Matthias P.; Duboule, Denis et al. (October 2018). "Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids" (in En). Nature 562 (7726): 272–276. doi:10.1038/s41586-018-0578-0. ISSN 0028-0836. PMID 30283134. Bibcode: 2018Natur.562..272B. https://www.repository.cam.ac.uk/handle/1810/285960. 
  4. ↑ 4.0 4.1 Marikawa, Yusuke; Tamashiro, Dana Ann A.; Fujita, Toko C.; Alarcón, Vernadeth B. (2009-02-01). "Aggregated P19 mouse embryonal carcinoma cells as a simple in vitro model to study the molecular regulations of mesoderm formation and axial elongation morphogenesis" (in en). Genesis 47 (2): 93–106. doi:10.1002/dvg.20473. ISSN 1526-968X. PMID 19115346. 
  5. ↑ Leahy, Amy; Xiong, Jing-Wei; Kuhnert, Frank; Stuhlmann, Heidi (1999). "Use of developmental marker genes to define temporal and spatial patterns of differentiation during embryoid body formation" (in en). Journal of Experimental Zoology 284 (1): 67–81. doi:10.1002/(SICI)1097-010X(19990615)284:1<67::AID-JEZ10>3.0.CO;2-O. ISSN 1097-010X. PMID 10368935. https://onlinelibrary.wiley.com/doi/abs/10.1002/%28SICI%291097-010X%2819990615%29284%3A1%3C67%3A%3AAID-JEZ10%3E3.0.CO%3B2-O. 
  6. ↑ ten Berge, Derk; Koole, Wouter; Fuerer, Christophe; Fish, Matt; Eroglu, Elif; Nusse, Roel (November 2008). "Wnt Signaling Mediates Self-Organization and Axis Formation in Embryoid Bodies" (in en). Cell Stem Cell 3 (5): 508–518. doi:10.1016/j.stem.2008.09.013. PMID 18983966. 
  7. ↑ Baillie-Johnson, Peter; Brink, Susanne Carina van den; Balayo, Tina; Turner, David Andrew; Arias, Alfonso Martinez (2015). "Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro". Journal of Visualized Experiments (105): e53252. doi:10.3791/53252. PMID 26650833. PMC 4692741. http://www.jove.com/video/53252/generation-aggregates-mouse-embryonic-stem-cells-that-show-symmetry. 
  8. ↑ Girgin, Mehmet; Turner, David Andrew; Baillie-Johnson, Peter; Cossy, Anne-Catherine; Beccari, Leonardo; Moris, Naomi; Lutolf, Matthias; Duboule, Denis et al. (2018-10-12). "Generating Gastruloids from Mouse Embryonic Stem Cells" (in en). Protocol Exchange. doi:10.1038/protex.2018.094. ISSN 2043-0116. http://www.nature.com/protocolexchange/protocols/6977. 
  9. ↑ Ring, David B.; Johnson, Kirk W.; Henriksen, Erik J.; Nuss, John M.; Goff, Dane; Kinnick, Tyson R.; Ma, Sylvia T.; Reeder, John W. et al. (2003-03-01). "Selective glycogen synthase kinase 3 inhibitors potentiate insulin activation of glucose transport and utilization in vitro and in vivo". Diabetes 52 (3): 588–595. doi:10.2337/diabetes.52.3.588. ISSN 0012-1797. PMID 12606497. 
  10. ↑ Hashmi, Ali; Tlili, Sham; Perrin, Pierre; Martinez-Arias, Alfonso; Lenne, Pierre-François (2020-05-24) (in en). Cell-state transitions and collective cell movement generate an endoderm-like region in gastruloids. doi:10.1101/2020.05.21.105551. 
  11. ↑ Vianello, Stefano; Lutolf, Matthias P. (2020-06-09) (in en). In vitro endoderm emergence and self-organisation in the absence of extraembryonic tissues and embryonic architecture. doi:10.1101/2020.06.07.138883. http://biorxiv.org/lookup/doi/10.1101/2020.06.07.138883. 
  12. ↑ 12.0 12.1 Veenvliet, Jesse V; Bolondi, Adriano; Kretzmer, Helene; Haut, Leah; Scholze-Wittler, Manuela; Schifferl, Dennis; Koch, Frederic; Pustet, Milena et al. (2020-03-04) (in en). Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites. doi:10.1101/2020.03.04.974949. http://biorxiv.org/lookup/doi/10.1101/2020.03.04.974949. 
  13. ↑ 13.0 13.1 van den Brink, Susanne C.; Alemany, Anna; van Batenburg, Vincent; Moris, Naomi; Blotenburg, Marloes; Vivié, Judith; Baillie-Johnson, Peter; Nichols, Jennifer et al. (June 2020). "Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids" (in en). Nature 582 (7812): 405–409. doi:10.1038/s41586-020-2024-3. ISSN 1476-4687. PMID 32076263. Bibcode: 2020Natur.582..405V. https://www.nature.com/articles/s41586-020-2024-3. 
  14. ↑ Rossi, Giuliana; Boni, Andrea; Guiet, Romain; Girgin, Mehmet; Kelly, Robert G.; Lutolf, Matthias P. (2019-10-14) (in en). Embryonic organoids recapitulate early heart organogenesis. p. 802181. doi:10.1101/802181. 
  15. ↑ Bérenger-Currias, Noémie M. L. P.; Mircea, Maria; Adegeest, Esmée; van den Berg, Patrick R.; Feliksik, Marleen; Hochane, Mazène; Idema, Timon; Tans, Sander J. et al. (2020-02-14) (in en). Early neurulation recapitulated in assemblies of embryonic and extraembryonic cells. doi:10.1101/2020.02.13.947655. http://biorxiv.org/lookup/doi/10.1101/2020.02.13.947655. 
  16. ↑ Moris, Naomi; Anlas, Kerim; van den Brink, Susanne C.; Alemany, Anna; Schröder, Julia; Ghimire, Sabitri; Balayo, Tina; van Oudenaarden, Alexander et al. (June 2020). "An in vitro model of early anteroposterior organization during human development" (in en). Nature 582 (7812): 410–415. doi:10.1038/s41586-020-2383-9. ISSN 0028-0836. PMID 32528178. Bibcode: 2020Natur.582..410M. http://www.nature.com/articles/s41586-020-2383-9. 
  17. ↑ Etoc, Fred; Metzger, Jakob; Ruzo, Albert; Kirst, Christoph; Yoney, Anna; Ozair, M. Zeeshan; Brivanlou, Ali H.; Siggia, Eric D. (2016). "A Balance between Secreted Inhibitors and Edge Sensing Controls Gastruloid Self-Organization". Developmental Cell 39 (3): 302–315. doi:10.1016/j.devcel.2016.09.016. PMID 27746044. 
  18. ↑ Warmflash, Aryeh; Sorre, Benoit; Etoc, Fred; Siggia, Eric D; Brivanlou, Ali H (2014). "A method to recapitulate early embryonic spatial patterning in human embryonic stem cells". Nature Methods 11 (8): 847–854. doi:10.1038/nmeth.3016. PMID 24973948. 



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