Bovine blastocyst like structures derived from stem cell cultures
Posted on: 15 February 2023 , updated on: 16 February 2024
Preprint posted on 21 December 2022
Article now published in Cell Stem Cell at http://dx.doi.org/10.1016/j.stem.2023.04.003
Categories: cell biology, developmental biology
Background
The blastocyst, a hollow ball of cells formed prior to implantation of the mammalian embryo, is comprised of the trophoblast, which develops into the placenta, and the inner cell mass (ICM). As development progresses, the ICM gives rise to the hypoblast, which forms the extraembryonic tissues, and the epiblast, which is the source of all cells that comprise the developing fetus. The capacity of these cells to give rise to any cell type of the body is referred to as pluripotency. Pluripotency is a phenomenon that only exists transiently in vivo, being lost once the epiblast differentiates into the three embryonic germ layers and the germline. However, the derivation and in vitro maintenance of embryonic stem cells (ESC) from blastocysts captures and indefinitely extends cellular pluripotency, since ESC lines can be maintained perpetually while self-renewing and retaining their characteristics.
After years of concerted efforts from several groups, the first culture condition to support long-term stable bovine embryonic stem cells (bESC) was published in 2018. Since then, further progress has been made toward establishing conditions that support bESC in alternative states of pluripotency, such as bovine expanded potential stem cells (bEPSC) in 2021. In addition, the first condition to support the derivation and culture of trophoblast stem cells (bTSC) from bovine blastocysts was reported in 2022.
Objectives
The objective of this preprint was to determine if it would be possible to assemble bEPSC with bTSC in order to generate bovine blastocyst-like structures, termed blastoids. Furthermore, the authors sought to compare the features and developmental competence of bovine blastoids to in vitro fertilization (IVF)-derived blastocysts.
Key findings
(1) Bovine blastoids can be assembled in vitro. To design a condition that would support bovine blastoid formation, the authors adapted a reported condition that supports differentiation of hypoblast-like cells from naïve human pluripotent stem cells (FGF2, Activin A, CHIR99021), integrating findings from a previous report on bovine embryo culture (+LIF) and optimizing signaling molecules to support bEPSC differentiation into both epiblast and hypoblast lineages (decreased [FGF2], +PD0325901). This optimized condition supported self-assembly of bovine blastoids with high efficiency (64.2±7.6%) within 4 days of bEPSC and bTSC aggregation.
(2) Bovine blastoids closely resemble IVF blastocysts. Day-4 blastoids had blastocele and ICM sizes equivalent to day-8 bovine blastocysts. Moreover, immunofluorescence localization of known markers of blastocyst cell populations demonstrated that blastoids had distinct epiblast (SOX2), hypoblast (SOX17), and trophoblast (CDX2) populations (Fig. 1); however, differences in the intensity of fluorescent signal, i.e., protein expression level, were observed. Single-cell RNA-sequencing of bovine blastoids was performed and compared against existing datasets from zygote-blastocyst stage IVF bovine embryos. Blastoid-derived cells clustered with respective cell populations of blastocyst-staged embryos following joint uniform manifold approximation and projection (UMAP) and pseudotime analysis, indicating similarity between the cell populations from both structures in terms of global transcriptome and developmental progress.
(3) Bovine blastoids undergo continued development in extended culture and signal maternal pregnancy recognition upon transfer. Using a 3D rotating culture method for extended culture of IVF blastocysts and blastoids, trophoblast cell and ICM proliferation as well as blastocele cavity expansion continued in both structures over more than two weeks of culture. However, conceptus elongation did not occur in blastoids or IVF blastocysts, as would be seen in vivo at comparable stages of development (which is a common limitation of extended culture systems). Following transfer into recipient dams, blastoids stimulated maternal recognition of pregnancy, as shown by interferon-tau (IFNτ; an anti-luteolytic signal for maternal pregnancy recognition in ruminant animals, secreted by the trophectoderm during conceptus elongation) being detected in the blood of recipient dams seven days later at comparable concentrations to those following transfer of IVF blastocysts.
Importance
This preprint is the first demonstration of blastoid formation in a livestock species. Previous achievements in mice and humans have taken decades to accomplish in bovine (e.g., stable ESC), so it is especially remarkable to see this accomplishment so soon after the initial reports in these species. Bovine blastoids represent an alternative model for early embryonic development, which could be useful in cases of limited access to biological materials for in vitro embryo production. Moreover, further studies may demonstrate the potential to use bovine blastoids for new assisted reproductive technologies, which could revolutionize cattle breeding approaches.
Questions for the authors
- How did the lineage composition of blastoids (Fig. 1F) compare to that of similarly-staged blastocysts?
- For the 3D rotating culture period, there appeared to be a rapid growth period for the remaining blastoids from d14-18 of culture, which was not mirrored by IVF blastocysts (Extended Data Fig. 4H). Do you have any hypotheses to why this difference was observed?
- It was very exciting to see that blastoids can give rise to pregnancy recognition signaling following transfer, and it is great evidence for the functional capacity of the newly reported bTSCs. Could you share if/how you plan to characterize the continued development of the bEPSC-portion of the blastoid following transfer (i.e., the embryonic disc) in future studies?
- I am sure a question that many ask – where do you see this technology going in the future?
doi: https://doi.org/10.1242/prelights.33604
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