Naomi Moris1 and Alfonso Martinez Arias2
Department of Genetics
University of Cambridge
Cambridge, CB2 3EH
What is a gastruloid?
A gastruloid is a three-dimensional structure made up of Embryonic Stem Cells (ESCs), that models certain features of early embryo development. In particular, gastruloids transition from an initial rounded to an elongated shape (a transformation similar to the early shape changes of the developing embryo), and generate cells associated with the three primary cell layers of early embryos (the germ layers) that are necessary for development of the different tissues and organs. Over time, these cells become organized with reference to a coordinate system, much as they do in the embryo. Gastruloids do not contain any brain cells or the cells necessary for the embryo to interact with the maternal environment.
Why can’t we study human embryos at this stage?
Since the development of IVF in the 1970s, discussions have arisen around the use of human embryos for research. This led to a proposal called the Warnock report, a 1978 internationally agreed set of rules that regulate research with human embryos. The report established what is known as ‘the day 14 rule’, a line that defines a time-limit for the observation of human embryos in a dish.
A human embryo at 14 days old will start the process of gastrulation. This is a process that transforms a group of cells into an individual organism: the embryo organises its cells into germ layers that will eventually give rise to the different tissues and organs. Before this time, the embryo can be split into two and will give rise to two individuals, or twins. Our knowledge of the process of gastrulation in human embryos is therefore based on anatomical studies of embryos collected by various academic and medical institutions over the 20th century. One example of such a collection is the Carnegie Collection of human embryos. However, there are very few specimens at early stages of development in these collections, which leads to a knowledge gap in our understanding of the process of gastrulation in humans. Human gastruloids provide a model to enable us to research this stage of development with a human cell-based model.
How is this work new?
Work with human Embryonic Stem Cells (ESCs) usually involves growing them in flat, adherent colonies on plastic dishes and controlling their behaviour by the addition of specific factors. This type of research has taught us a lot about the signals that are needed to convert cells towards different cell types, and their dynamics during this process.
This new work to generate human gastruloids, which builds on our previous research with mouse gastruloids, allows cells to interact with each other in three-dimensional aggregates. In this arrangement, and under specific culture conditions, cells assume multiple identities in an ordered manner and coordinate this process to form an organised pattern. This is the first time that a three-dimensional structure made of human ESCs has been shown to so-closely model the developing human embryo. The finding is particularly important because we have very little information about the early stages of development in human embryos and therefore, human gastruloids may provide a new model that we can use to find out more about normal human development and what happens when the process goes wrong.
What is the point of this research?
Understanding human development is an important endeavour that will provide knowledge about ourselves, a platform to understand disease and, in the future, a basis for regenerative medicine, the ability to produce tissues and organs for transplant. At the moment, these studies would require human embryos at early stages of development, which presents ethical and moral challenges.
One solution to this, is to look for models which mirror the events of a human embryo. Over the years, animal models have provided a large amount of information due to their similarity to humans in terms of the course of development and the genes associated with these processes. In particular the mouse has been an extremely useful tool to provide insight into mammalian development. However, there are differences between mouse and human embryos which suggest that if we want to understand human development, we need to use human models.
Gastruloids derived from human Embryonic Stem Cells (ESCs) are a reproducible and experimentally tractable model system that we believe provide an opportunity to explore the early stages of human development without having to use human embryos.
Is a gastruloid an embryo?
No, gastruloids are not embryos, nor do they have the potential to become embryos. Gastruloids lack the potential to make a brain and therefore they are incomplete structures. They also lack the cells that are necessary to allow the embryo to implant into the uterus, meaning they could not be implanted, and would not proceed much further in development. As models, gastruloids are simplified systems that mimic some aspects of embryo development, in a manner that facilitates further study. Models are simplified representations of reality that allow for the exploration of the properties and behaviour of otherwise complicated systems. For example, a flight simulator is a very sophisticated model of an aeroplane but it is not an aeroplane; it will not fly and will not carry people or cargo. Still, it allows us to learn a great deal about how to fly a plane and how to solve many of the problems that can arise in a real flight by simulating them. In the same manner gastruloids have many features of an embryo but, because they lack other features and potential, they allow us to study the mechanisms underlying the emergence of tissues and organs without using embryos.
Are human gastruloids ethical?
We believe they are. Human gastruloids do not show any evidence of cell types associated with the brain, nor do they form the cells required for implantation. Significantly, they lack the morphology (shape) of an early human embryo, and therefore do not manifest human organismal form. As such, they are non-intact, non-autonomous, and non-equivalent to human embryos, and do not have human organismal potential.
Our research was subject to review and approval from the Human Biology Research Ethics Committee of the University of Cambridge, in compliance with the ISSCR 2016 guidelines.
What is the future of gastruloids?
The research published so far provides a protocol for generating human gastruloids, and a characterisation of their properties. The details of this technique can, and will, be improved to increase the utility of the system, and further details characterising their properties will improve our understanding of the early steps in human development. It is probable that in the future human gastruloids could be made from induced Pluripotent Stem Cells (iPSCs) rather than Embryonic Stem Cells. We believe that in the future, it will be possible to use human gastruloids to improve our understanding of normal human development, and to examine what happens when things go wrong – including modelling diseases caused by genetic mutations and exposure to environmental perturbations. Because they can be grown in large numbers, it is possible that they could be used for drug screening purposes and to develop additional assays for a variety of uses.
What are embryonic stem cells (ESCs)?
Embryonic Stem Cells (ESCs) are a special type of cell, derived from mammalian embryos, before they implant. In humans, they typically come from IVF-derived embryos, and their derivation is regulated by the HFEA and UK Stem Cell Bank.
Induced Pluripotent Stem Cells (iPSCs) are a type of cell obtained by reprogramming adult differentiated cells, which has the properties of ESCs. These cells can be used in many experiments instead of ESCs thus reducing the use of embryos and, importantly, can be derived from individuals with specific pathologies.
ESCs and iPSCs they have two important properties: they have the potential to differentiate into all the cells of an organism, and they can keep dividing maintaining this potential in laboratory conditions indefinitely. This means that human ESCs do not need to be continually derived but can be grown by many researchers for many different questions over very long periods of time, reducing the use of human embryos in research.
Over the last few years, human ESCs and iPSCs have been used as a means to probe the instructions that guide the development of heart, muscle and gut cells and they have been coaxed into three-dimensional structures, or organoids, that resemble specific organs like the kidney, the pancreas, the intestine or even the brain.
What is gastrulation?
Gastrulation is the process that generates cells of the three germ layers (mesoderm, endoderm and ectoderm) of the embryo and organises them relative to one another. These germ layers are the precursors of the different cell types that will organize into tissues and organs. An important feature of gastrulation involves the emergence of a system of coordinates that orients the cells into two main axes: head/tail (anterior-posterior) and back/belly (dorsal-ventral).
Is a gastruloid just a new type of “organoid”?
No. Although organoids are similar to gastruloids in that they are three-dimensional structures with multiple cell types organised spatially, organoids only recapitulate a single organ (or part thereof). Gastruloids, by comparison, model the organisation of the embryo, which includes precursor cells for multiple tissues and early primordia of many organs. In gastruloids, these precursors organize relative to each other in a manner similar to that of the embryo.
Is a gastruloid just a new type of “embryoid body”?
No. Embryoid Bodies (EBs) have been used for many years to promote the differentiation of pluripotent stem cells towards multiple lineages. However, the different cell types that emerge in EBs are disorganized and often do not represent the organisation of the embryo.
One potential reason for these differences is that EBs are generated from thousands of cells and under different culture conditions, while gastruloids are generated from a few hundred cells under specific conditions that favour the spatial and temporal patterning of the group of cells.
How does the age of a gastruloid relate to the age of an embryo?
As the fertilized egg begins to divide, it first generates two groups of cells (extraembryonic tissues) that surround the embryo and will mediate its interaction with the mother. These processes take a few days.
By day 13/14 the embryo is ready to begin the process of gastrulation.
Embryonic stem cells are derived from embryos before gastrulation but after the segregation of some extraembryonic tissues. For this reason, researchers can differentiate human embryonic stem cells towards various cell types in the laboratory.
Therefore, the reason why the gastruloid protocol takes several days (3-5 days) to reach a state that is comparable to a human embryo at ~day 18-20 is because they start from a more advanced state than a fertilised egg cell.
How do we know that gastruloids model embryo-like processes?
In the case of the mouse gastruloids, comparisons between the organization of the gastruloid and that of embryos have established a good correspondence in the organization of different cell types and organ precursors, as well as in their temporal emergence. The basic organization revealed by these studies conforms to a basic pattern that is characteristic of vertebrate embryos.
Similarly, a comparison between mouse and human gastruloids reveals a good correspondence with this conserved outline and supports the conclusion that, at the global level, what we see corresponds to the organization of a human embryo. Importantly, differences between mouse and human gastruloids might reflect differences between mouse and human embryos.
How do we know the corresponding embryonic stage of a gastruloid?
When we look at the organization of gene expression in human gastruloids, we notice that the order of certain genes along the top-tail axis (anterior-posterior) is reminiscent of a process called ‘somitogenesis’ in mammalian embryos.
Somitogenesis generates the length of our body axis through the sequential production of blocks of cells, called ‘somites’, that will produce the ribs, vertebrae and thoracic muscles. The number of somites along the body axis is related to the age of the embryo. The Carnegie embryo collection shows that the first somites appear in the human embryo around day 16-17 and that the process is ongoing by day 19-20.
Therefore, the gene expression within human gastruloids suggests that somitogenesis is ongoing, which is why we suggest that they display some of the features of a human embryo at around day 19-20.