The blastocyst is an early preimplantation developmental stage in embryogenesis. A blastocyst, upon implantation, gives rise to the embryo and the placenta. Despite the key role this structure plays in maintaining pregnancy, the study of human blastocysts has been challenged by ethical and technical concerns. Now, two independent teams have developed methods to generate human blastocyst-like structures in vitro to enable studies of early human development.

iBlastoids immunostained for markers CDX2 and NANOG, representing trophectoderm-like and inner cell mass–like cells, respectively. Scale bar, 50 μm. (Adapted with permission from X. Liu et al. Nature 591, 627–632, 2021).

Researchers at the University of Texas Southwestern Medical Center have generated blastocyst-like structures called ‘human blastoids’ by successive lineage differentiation of human embryonic and induced pluripotent stem cells (Yu et al., 2021).

“There is a lack of access to human embryos as we try to understand human development. And we have to rely a lot on animal models, mostly mice, especially during these early stages of preimplantation or early postimplantation. That has severely hindered human research. So the goal of our study was to see whether we can use stem cells to model early human embryos in vitro,” explains lead researcher Jun Wu.

The team first demonstrated that the WIBR3 human embryonic stem cell line could be independently differentiated into cells mimicking all three blastocyst cell types: the trophoblast, hypoblast and epiblast. To develop these structures together, they used a three-dimensional (3D) culture system and stimulated the cells sequentially with growth factor combinations promoting hypoblast and trophoblast development. Blastoids developed that contained inner cell mass–like and trophectoderm-like compartments, had a cavity, and were morphologically similar to blastocysts.

Next, using immunofluorescence and single-cell RNA sequencing (scRNA-seq), they found that the blastoids resembled human preimplantation embryos. Gene ontology analysis also revealed enrichment of lineage-specific genes. For example, genes associated with placenta development were upregulated in the trophoblast-like cells, while the epiblast-like cells and hypoblast-like cells were enriched in pluripotency- and morphogenesis-related genes, respectively.

In a parallel study, researchers at Monash University used a different protocol to generate blastocyst-like structures called iBlastoids in vitro (Liu et al., 2021). The team, led by Jose Polo, reprogrammed human fibroblasts to induce pluripotent stem cells using a well-established transcription factor cocktail. They observed that, when reprogrammed in a 3D culture system, the cells aggregated and developed a cavity, consistent with a blastocyst-like structure.

“In nature, pluripotency doesn’t exist in isolation. It’s always together with the trophectoderm and hypoblast, or primitive endoderm. So, since the reprogrammed cells presented those signatures, we decided to put them together to study how the networks were going to evolve. In an unanticipated way, not only did the cells interact, but they aggregated and self-organized,” says Polo.

Immunofluorescence and scRNA-seq analyses revealed that the iBlastoids resembled preimplantation embryos. Here too, the epiblast-, trophoblast- and hypoblast-like cells from the culture clustered with their in vivo counterparts in transcriptomic analyses, denoting a strong overlap.

Curiously, both studies also found a small subset of cells that appeared unique to the in vitro models. These cells were uncommitted to any of the lineages and were of undetermined identity. This artifact was likely a result of the in vitro culture conditions or of the original cell line. “Cell lines are typically heterogeneous. They don’t synchronously differentiate, and they may also have different propensities towards differentiation into different cell types. So that may in part explain the noise observed in the single cell transcriptome,” says Wu.

The two studies also demonstrated that the blastoids could give rise to stem cell lines. The trophoblast stem cell lines could be further differentiated into extravillous cytotrophoblasts and syncytiotrophoblasts, cells that go on to form the placenta. Upon prolonged culture, both blastoids types could self-organize into a peri-implantation embryo-like structure with the presence of an epiblast-like cell–derived pro-amniotic cavity and an increase in the production of human chorionic gonadotropin, a hormone used to detect pregnancy.

These studies present the first in vitro models for human blastocysts and open up possibilities for research into the causes of pregnancy failure. “The advantage is that you can do modern biochemistry. You can obtain hundreds of these structures from one experiment, which allow you now to do all experiments that require more than one cell or two cells,” says Polo. The teams will now focus on fine-tuning the differentiation protocol to make generation of blastoids simpler and more efficient. The reproducible and scalable nature of these models will also help answer key questions involving crosstalk between these cells and the modulation of the transcriptome and epigenome.