Developmental biology

from organoids to gastruloids Last year was hailed as ‘the year of the ’ as news of mini- brains, livers and intestines grown in the laboratory hit the headlines. Here, Dr David Turner explains the latest in vitro systems for modelling human development and disease, including embryonic stem cells, organoids and gastruloids

ome of the most important these questions, traditionally using genetic shown to play an important role in experimentally) that is cheaper than Embryoid bodies fail patterning the early embryo, and it is this questions in biology relate experiments in embryos from animals development, are very difficult to assess; in vivo work, is ethically responsible, and well-grounded understanding that enables to how we are formed and such as the fruit fly, frog, chicken, zebrafish there is a great deal of expense involved in can be utilised to ask specific questions to produce structures us to guide ESCs towards specific tissue develop. For example, how is it and mouse. In terms of mammalian maintaining mouse lines (and generating regarding developmental processes and types and cell fates by applying signals or that a single fertilised egg is able, development, the mouse has proven new ones with specific traits), and it is differentiation, which can eventually be with any similarity modifying cells’ gene expression in culture. Sin a relatively short space of time, to exceptionally useful as a model system. difficult and technically challenging to targeted to understanding human For example, neural tissues can be to the embryo produce so many different cell types, Although many developmental processes experimentally manipulate the mouse development and disease. generated by applying retinoic acid or tissues and organs, and position them so and patterning events are conserved embryo at the early stages. inhibiting bone morphogenetic protein precisely within the body? Why does throughout diverse animal species, how There are also ethical considerations STEM CELLS, DISEASE (BMP) signalling, and beating human development occasionally go sure are we that what we see in the mouse to take into account when using mice AND DEVELOPMENT by Evans and Kaufmann in cardiomyocytes can be enriched through wrong, producing birth defects and (and other model systems) recapitulates and their embryos (although a number of Embryonic stem cells (ESCs) offer an the 1980s). application of BMP, Activin A and vascular pathological conditions? human development? Mice and other initiatives are underway to dramatically alternative and, arguably, parallel route The pluripotent, self-renewing trait endothelial growth factor. The answers to these questions are vital model systems have a number of reduce or replace the numbers of mice to dissecting the development of embryos, of ESCs is essential during development It is often the case that by using a tissue in uncovering the mechanisms behind limitations. For example: it is not always and other animals involved in experiments delineating the processes and mechanisms and useful experimentally, as it prevents culture-based approach, new insights can disease states, eventually instructing us in possible to discount the effects of and refine their use, such as the utilised physiologically. ESCs are a premature exhaustion of the cells as they be gained regarding the signals involved in ways to treat them through regenerative redundancy between genes when assessing National Centre for the Replacement, self-renewing, pluripotent population are cycled into different cell fates in vivo, cell fate specification. Examples of this medicine, or manage their severity through the effect of mutant phenotypes (i.e. Refinement and Reduction of Animals in of cells derived from the early mouse and enables us to culture them indefinitely include our recent work on the role of Wnt/ better medicines and pharmaceuticals. biological systems are buffered, and Research, NC3Rs). embryo which can give rise to all the in the laboratory. β-Catenin and fibroblast growth factor We can use a number of experimental multiple genes may have similar functions); What is needed is a fully tractable tissues and organs of the embryo proper Careful experimentation has determined signalling in generating a population of tools and techniques to tease apart some of mechanical forces, which have been system (one that’s easy to control (they were first isolated from mouse many of the genes and signals involved in cells that serves as a pool for generating

14 / The Biologist / Vol 64 No 5 Vol 64 No 5 / The Biologist / 15 Developmental biology Organoids

Embryoid bodies, Fig. 2. An array of fluorescent light a) Colorectal organoid different organ micrograph types have been generated using organoid culture techniques. the body axis,1 or how cells resolve binary Examples include fate decisions depending on the signalling colorectal and environment they are exposed to and the gastric organoids time in which they see these signals.2 (a and b), mini- brains (c) and ESCs are, when compared with in vivo b) Gastric kidney organoids studies in the mouse, exceptionally easy to organoid (d) as well as manipulate (genetically and chemically), forebrains, optic reduce the necessity for animal cups and liver experimentation and are orders of organoids (not magnitude less expensive than keeping mice c) Mini-brain pictured). in the laboratory. However, there are a few Images courtesy of disadvantages that have the potential to a) Cellesce compromise our complete reliance on ESCs b) Kyle McCracken with respect to in vivo work. c) Jürgen Knoblich at One of these involves the topographical the Institute of differences between a 2D layer of cells Molecular grown on a plate and the 3D nature of Biotechnology, a whole embryo – the 3D spatial Vienna d) Atsuhiro Taguchi organisation in the embryo between cells and Ryuichi and tissues is not replicated on a tissue Nishinakamura via culture plate. This limitation can to some Taguchi et al. Cell extent be solved by culturing cells as 3D d) Kidney organoid 2014. aggregates known as embryoid bodies (Fig. 1). To generate these bodies, cells are typically plated in little droplets on the inside lid of a tissue culture dish and (Fig. 1), they spontaneously differentiate and development, from human induced between the observed outcomes, and the form spherical structures on the same inverted so they form aggregates at the form self-organised, stratified tissues. pluripotency stem cells (iPSCs; cells frequency of these outcomes is low.8 scale as the early mouse embryo at the bottom of a droplet through gravity (Fig. However, within the last decade, there has from adult organisms that have been If organoids are to be used as model stage. This is in contrast to 1). Cells grown in this way produce many as generating precursor populations for been something of a revolution in what can essentially reprogrammed to their systems for disease states or understanding most other organoid systems or embryoid cell types associated with the three further differentiation protocols. be accomplished in this field, with the rise embryonic state) and exposing them to development, as well as being used bodies where larger numbers of cells are embryonic germ layers over time, and can of organoids (Fig. 2). the Zika virus, they were able to determine clinically for drug screening or for typically used. even form spontaneously beating regions THE ORGANOID REVOLUTION Generally speaking, cellular material the neural cells the virus preferentially regenerative medicine, it is essential that Over time, and under appropriate as cardiac precursors are generated. Early studies using 3D structures, in parallel (such as mouse or human ESCs, tissue targeted and how microcephaly may be they are reliable, reproducible and signalling conditions, these spherical Unfortunately, embryoid bodies are highly with work on embryoid bodies, used fragments, primary cells and so on) that is established6 (Fig. 2). quantifiable. Advances need to be made so aggregates or, rather, embryonic disorganised3,4 and fail to produce artificial scaffolds and matrices to provide grown in 3D, and over time, can form As well as modelling the progression that organoid systems can be precisely organoids, begin to show something structures with any similarity to the embryo. support for growing tissues in what is structures very similar in the patterning of disease states, organoids may have controlled and manipulated with remarkable, undergoing many of the Their usefulness is therefore limited to known as mechanically supported air-liquid and, sometimes, function to their in vivo uses in regenerative medicine. Using minimal experimental variation. morphological and patterning events of broad questions on the signals required for interface culture. When skin or oesophageal counterpart5 following the same cellular material from patients through the early embryo. Furthermore, they differentiation of various cell types, as well primary keratinocytes are grown in this way developmental progression as in the generating iPSCs, a patient’s own cells can GASTRULOIDS – EMBRYONIC undertake a process similar to embryo. Brain (mini-brains), optic cup and be guided towards the required cellular ORGANOIDS in the embryo, generating cell types that gut organoids have been produced, to lineage or fate, and the organoids that are With this in mind, our laboratory in correspond to the three germ layers.9,11,13 Monolayer Culture Mechanically Supported Culture name just a few (Fig. 2). These have the formed from this tissue source can be Cambridge developed a new, highly It is the combination of these traits that very real potential to be powerful model transplanted back into the patient, such reproducible and tractable tissue-culture gave rise to the name of these embryonic Cell medium Primary tissues systems for probing both how organs as in the use of liver organoids to treat liver technique using mouse ESCs to study organoids: gastruloids. develop normally, and how pathologies and cirrhosis.7 This has the added benefit of early mammalian development and A striking aspect of gastruloids is the ∂t disease states can affect their development. avoiding tissue rejection associated with axial patterning5,9–11 (Fig. 2). Building on finding that they can form a structure One of the most important examples of transplants from other individuals and earlier observations with the P19 similar to one that forms only in Stratified Cell monolayer this came from the Hongjun Song and Guo-li abrogates the necessity for a lifetime of embryonal carcinoma cell line4 and the mammalian embryos: the node.11 The Ming’s research groups and modelled the immunosuppressant drugs to counteract suspension culture techniques used by node is a key signalling centre in effect of the Zika virus on human brain organ rejection. other groups,12 we aggregated small mammalian embryos and is important Embryoid Body Culture Gastruloid Culture development6 (Fig. 2). The Zika virus, Organoids have significant advantages numbers of mouse ESCs in suspension to for identifying which side of the embryo declared by the World Health Organization over 2D techniques in terms of studying is left or right, and the disruption of the Cell Suspension as a public health emergency of international the spatial organisation and development left-right patterning causes severe birth ∂t concern, is particularly problematic for of organs and tissues, significantly so when defects, usually leading to chronic heart Invert lid pregnant women, as it is passed on to compared with embryoid body culture. Gastruloids can form disease. Presently, the only way to study the developing foetus, resulting in However, with the exception of the Zika the effect of left-right axis disruption is developmental defects and neurological virus study mentioned above, there are a structure similar to through animal models, so the gastruloid Non-adherent culture; disorders such as microcephaly. key issues in their reproducibility: in other system offers an attractive window into Water/PBS Low cell number one that forms in By generating brain organoids, which words, under the same experimental studying this process cheaply and without

Fig. 1. Strategies for growing 2D and 3D layers of cells mimic key aspects of human cortical LIBRARY PHOTO NIBSC/SCIENCE conditions, there is significant variation mammalian embryos using animals.

16 / The Biologist / Vol 64 No 5 Vol 64 No 5 / The Biologist / 17 Developmental biology Organoids

It is important for us to the former) and also take starting material directly from patients to study their disease keep in mind potential in a genetic background identical to the patient’s or provide source material for problems that may possible regenerative medicine (in the case arise in generating of the latter). On a final note, it is important for us to embryonic structures keep in mind potential problems that may arise in generating embryonic structures in culture from human ESCs/iPSCs, as recently pointed out by Aach et al (2017). Interestingly, the differences between As organoids have the potential to form gastruloids and the embryo have later embryonic structures without already helped provide an alternative progressing through a primitive streak, it interpretation of the function of certain may arise that we have created a structure embryonic tissues.11 that has neurulated, generating something that has the potential to feel pain or ETHICAL MINEFIELDS develop sentience14 (hence this is one of Rat cortical Despite the above examples, a burning the reasons for stopping human embryo Kidney organoid spheroid Gastruloids issue still lingers: organoids don’t fully work at the 14-day/primitive streak stage). HOFFMAN-KIM LAB, BROWN UNIVERSITY BROWN LAB, HOFFMAN-KIM MINORU TAKASATO equate to human development. Even though many signalling pathways and Current guidelines regarding the use References patterning mechanisms have been This image of a mini- of human embryos cannot apply in this brain organoid shows conserved throughout evolution, other case since the primitive streak hasn’t 1) Turner, D. A. et al. Wnt/β-catenin and FGF signalling a lobe of cortical animals may do things differently to the formed. Research and bioethical direct the specification and maintenance of a tissue surrounding a neuromesodermal axial progenitor in ensembles of human embryo, and disease-modelling fluid-filled ventricle. communities may need to find new ways to mouse embryonic stem cells. Development 141, using material from tissues, organoids The red areas show address these ethical problems with their 4243–4253 (2014). and embryos that are different to that of Nestin, a marker of findings disseminated and communicated 2) Turner, D. A. et al. An interplay between extracellular signalling and the dynamics of the exit from pluripotency neural stem cells human origin may give rise to errors in with the wider public. It may be the case drives cell fate decisions in mouse ES cells. our understanding that will impact the that new legislation should be drawn up to Biology Open 3, 614–626 (2014). effectiveness of treatment. reflect both our new scientific 3) ten Berge, D. et al. Wnt signaling mediates self- organization and axis formation in embryoid bodies. An approach more indicative of human understanding based on what our Cell Stem Cell 3, 508–518 (2008). development will eventually be required experiments tell us about development, 4) Marikawa, Y. et al. Aggregated P19 mouse embryonal to fully appreciate how we develop. and our changing perspectives on what is carcinoma cells as a simple in vitro model to study the 14 molecular regulations of formation and axial However, there are limits on what is ethically acceptable. elongation . Genesis 47, 93–106 (2009). permissible regarding experimentation There are many methods used to 5) Turner, D. A. et al. Organoids and the genetically on human embryos and for how long study development, generating structures encoded self-assembly of embryonic stem cells. scientists can culture human embryos. that can be used both as a substitute for Bioessays 38, 181–191 (2016). 6) Qian, X. et al. Brain-region-specific organoids using Presently, human embryos can only be the embryo in research and for mini-bioreactors for modeling ZIKV exposure. Cell 165, cultured for a limited period of time, either regenerative medicine and modelling 1238–1254 (2016). for up to 14 days or until the ‘primitive disease progression. However, it is 7) Willenbring, H. & Soto-Gutierrez, A. Transplantable liver organoids made from only three ingredients. Stem streak’ structure forms, which signifies essential that we bridge the gap between Cell 13, 139–140 (2013). the start of gastrulation.14 what we are able to accomplish through 8) Huch, M. et al. The hope and the hype of organoid Many of the questions regarding the our experiments and the translation of our research. Development 144, 938–941 (2017). 9) van den Brink, S. C. et al. Symmetry breaking, germ development of our organs and how knowledge to the public, without layer specification and axial organisation in aggregates human embryos are patterned occur after overselling what we hope to achieve or of mouse embryonic stem cells. Development 141, this point. There are many ethical what is actually possible.8 4231–4242 (2014). 10) Baillie-Johnson, P. et al. Generation of aggregates of questions surrounding the use of ESCs, Still, as great progress has been achieved mouse embryonic stem cells that show symmetry which must be obtained from human in a relatively short space of time, it will be breaking, polarization and emergent collective behaviour embryos that are then destroyed. interesting to see what new developments in vitro. J .Vis. Exp. (2015). doi:10.3791/53252 11) Turner, D. A. et al. Gastruloids develop the three body It is here that organoids have the will arise and how this highly evolving field axes in the absence of extraembryonic tissues and potential to overcome this barrier in an can translate its findings for regenerative spatially localised signalling. BioRxiv (2017). ethically acceptable manner and (in terms medicine and modelling diseases. doi:10.1101/104539 of regenerative medicine and tailored 12) Eiraku, M. et al. Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by treatments) in ways more tailored to extrinsic signals. Cell Stem Cell 3, 519–532 (2008). 13) Turner, D. A. et al. Interactions between nodal and specific disease states in patients. Dr David A Turner CBiol MRSB is an wnt signalling drive robust symmetry-breaking and axial Generating organoids from current stocks NC3Rs David Sainsbury Research organisation in gastruloids (embryonic organoids). Fellow working in Alfonso Martinez of human ESCs or from human iPSCs, BioRxiv 1–31 (2016). doi:10.1101/051722 we will be able to limit the requirement Arias’ laboratory at the University of 14) Aach, J. et al. Addressing the ethical issues raised Cambridge. He uses the gastruloid for further human embryos to study by synthetic human entities with embryo-like features. model system to study development Elife 6, e20674 (2017). early human development (in the case of MADELINELANCASTER, LABORATORYMRC MOLECULAROF BIOLOGY in the mammalian embryo.

18 / The Biologist / Vol 64 No 5 Vol 64 No 5 / The Biologist / 19