A proper understanding of early human development is crucial if we are to improve reproductive technologies and prevent pregnancy loss and birth defects. However, studying early development is a challenge – few human embryos are available, and research is subject to significant ethical and legal constraints. The emergence of techniques that make cells cultured in vitro constructing models of mammalian embryos therefore offers exciting opportunities1. Two papers in Nature is now making significant progress in this area, showing that human embryonic stem cells2 or cells reprogrammed from adult tissues2,3 can be induced to organize themselves in a dish and form structures resembling early human embryos. It is the first integrated human embryo model containing cell types associated with all the basic cell lines of the fetus and its supporting tissues.
In mammals, a fertilized egg undergoes a series of cell divisions during the first days of development, leading to the formation of a structure called the blastocyst. The blastocyst contains an outer cell layer called the trophectoderm, which surrounds a cavity that contains a cell group called the inner cell mass (ICM). As the blastocyst develops, the ICM is separated into two adjacent cell populations – the epiblast and the hypoblast (known as the primitive endoderm in mouse embryos). The blastocyst is then implanted in the uterine tissue, which forms the stage for an event called gastrulation, in which epiblast cells give rise to the three basic cell layers that will form the entire fetus. The trophectoderm forms the largest part of the placenta, and the hypoblast forms a few layers of a structure called the yolk sac, which is needed for early fetal blood supply.
The first in vitro models to recapitulate blastocyst formation using cultured cells (structures known as blastoids) used mouse stem cells corresponding to the stem cells found in the epiblast, trophoblast, and primitive endoderm in the mouse blastosis.4–6. However, the generation of similar blastoids from human cells has so far not been achieved.1. Previous models of early human development have evolved human stem cells similar to epiblastic cells after implantation, prior to gastrulation7–9. Although they were able to recapitulate some stages of human development after implantation, they did not have sexes related to the trophectoderm, hypoblast, or both.
In current newspapers, Yu et al.2 in Liu et al.3 describe human blastoids. The key to these technological breakthroughs appears to be twofold: first, the use of cells representative of sex lines in the human blastocyst; and secondly, the optimization of cultural protocols.
Yu et al. begins with either human embryonic stem cells, which are derived from human blastocysts, or induced pluripotent stem cells, which are generated from adult cells. Importantly, both of these stem cell types are developmentally similar to epiblast cells in blastosis, and may also give rise to lineages associated with the trophectoderm and hypoblast. In contrast, Liu et al. reprogrammed adult skin cells called fibroblasts to form a mixed cell population that contains cells with gene expression profiles similar to those of the cells of the epiblast, trophectoderm, and hypoblast. As in mouse blastoid protocols4–6Both approaches involved the cells being seeded in 3D culture dishes called Aggrewell plates, and treated with liquid growth medium containing chemical factors to control the signaling activities necessary for blastocyst development (Fig. 1). Yu and colleagues treated the cells with two different types of culture media sequentially to promote the differentiation of the cells into sex lines representing the trophectoderm and hypoblast.
Figure 1 | The generation of human blastoids. Blastocysts are structures formed early in mammalian development and consist of three cell types that will give rise to the embryo, placenta and a supporting tissue called the yolk sac. Two groups report in vitro methods of generating human blastoids, which accurately form blastocysts. a, Yu et al.2 used human pluripotent stem cells (hPSCs), which can give rise to sex lines associated with all cell types in the blastocyst. The hPSCs were isolated from human blastocysts or generated by reprogramming adult human cells. The authors placed hPSCs in 3D culture dishes called Aggrewell plates, and used a two-step culture process to induce the formation of human blastoids. b, On the other hand, Liu et al.3 adult human cells reprogrammed into cell types with gene expression profiles corresponding to the three cell types found in blastocysts (some cells of unknown types were also generated, indicated by different colors). They used a single-step 3D culture process to generate the blastoid. (In both protocols, the blastoids contain a number of cells of an unknown type, not shown).
Both groups found that human blastoids developed after 6-8 days of culture, with a formation efficiency of up to almost 20%, comparable to the efficacy of the mouse blastoid protocols.4–6. The human blastoids were of the same size and shape as natural blastocysts, with a similar total number of cells. They contained a cavity and an ICM-like group.
Detailed characterization of the blastoids (including genome-wide expression analysis and comparisons with human embryo data) showed that their cell lines have molecular similarities to those of the human blastocyst before implantation. The spatial organization of the epiblast, trophectoderm, and hypoblast-related lineages is similar to that found in human embryos before implantation. The groups also showed that the blastoid cells have the most important properties of blastocyst lines – cells isolated from the blastoids can be used to generate different stem cell types. Yu et al. showed that, if these stem cells were transplanted into mouse blastocysts, gave rise to cells that could integrate with the corresponding mouse lines in the mouse embryo.
Next, the researchers analyzed the further development of the blastoids using an established test that mimics implantation in the uterus in culture dishes. Like blastocysts, when blastoids were cultured for four to five days, some attached to the culture dish and developed further. In a portion of these attached blastoids, the cell group representative of the epiblast reorganized into a structure enclosing a central cavity – reminiscent of the pro-amniotic cavity, which forms in the epiblast of blastocysts after implantation. And in some blastoids, the trophectoderm-related cell lines have spread and shown signs of differentiation into specialized placental cell types. Yu et al. also a second cavity in the hypoblast-related cell lines observed in some blastoids, similar to the yolk sac cavity.
The group’s data together show that human blastoids are promising in vitro models of pre-implantation and early post-implantation blastocyst development. However, there are notable limitations to overcome. The development of the blastoids, for example, is inefficient and varies between cell lines produced by different donors, and between experimental groups. In addition, the three sex lines against single blastoids appear to differ slightly, and the development of blastoids in the same dish does not appear to be synchronized. Spatial organization of the hypoblast-related genus in blastoids has yet to be improved. Furthermore, the blastoids contain unidentified cell populations that do not have counterparts in natural human blastocysts.
Another challenge is that the development of the blastoids is limited in stages after implantation, unlike in mouse blastoids.4–6. Further optimization of culture and experimental conditions is necessary to improve the culture of human blastoids after implantation in vitro, up to the equivalent of 14 days in vivo. Strict ethical rules prevent the cultivation of human embryos after this stage when structures related to gastrulation begin to appear. Three-dimensional systems for the cultivation of human blastocysts10, which effectively promotes the development of post-implantation, can help to improve our ability to grow blastoids, to this limit by maintaining the normal 3D tissue architecture and spatial relationships between the different cell lines in the blastoids.
Human blastoids are the first human embryo models derived from cultured cells in vitro and which has all the basic cell lines of the fetus and its supporting tissues. As protocols are optimized, these blastoids will closely mimic human blastocysts. This will inevitably lead to bioethical questions. What should be the ethical status of human blastoids, and how should they be regulated? Should the 14-day rule apply? These questions must be answered before research on human blastoids can proceed with the necessary caution. For many people, the study of human blastoids will be less ethically challenging than the study of natural human blastocysts. Others, however, may view research on human blastoids as a pathway to the development of human embryos. The ongoing development of human embryo models, including human blastoids, therefore calls for public debate on the scientific importance of such research, as well as on the societal and ethical issues it raises.