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Bovine blastocyst like structures derived from stem cell cultures

Carlos A. Pinzón-Arteaga, Yinjuan Wang, Yulei Wei, Leijie Li, Ana Elisa Ribeiro Orsi, Giovanna Scatolin, Lizhong Liu, Masahiro Sakurai, Jianfeng Ye, Leqian Yu, Bo Li, Zongliang Jiang, Jun Wu

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

A new livestock embryo model from a self-renewing source

Selected by Carly Guiltinan

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.

Figure 1. Morphological comparison of bovine IVF blastocysts and blastoids. (Preprint figures 1C and 1E)

(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

Read preprint (1 votes)

Author's response

Jun Wu & Zongliang Jiang shared

1) How did the lineage composition of blastoids (Fig. 1F) compare to that of similarly-staged blastocysts?

Overall, lineage composition of bovine blastoids is comparable to bovine blastocysts produced via in vitro fertilization (IVF). It is important to note that the lineage composition is dependent on the culture condition. We noticed that low number of hypoblast cells in commercially available IVC media, which could be improved by the addition of FLI (FGF2, LIF and IGF1). Furthermore, the single cell RNA-seq analysis (Figure 2F) showed that the hypoblast cluster (#4) was composed mainly of in vivo derived bovine blastocysts (66%) while bovine blastocysts produced by IVF (15%) and bovine blastoids (14%) were comparable. Thus, it seems that the current in vitro conditions are not optimal for hypoblast cells.

 

2) 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’s unclear to us what’s causing the different observations. One possibility is that bovine TSCs used for blastoid formation were already adapted to N2B27 basal medium while IVF blastocyst trophoblast cells were not. Another possibility is that bovine TSCs are more developmentally advanced than IVF blastocyst trophectoderm cells.

 

3) 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 bEPSC-portion of the blastoid following transfer (i.e., the embryonic disc) in future studies?

In future studies, we plan to perform more in-depth analysis of transferred blastoids in terms of lineage differentiation and composition, and compare with control blastocysts, which include immunofluorescence studies and scRNA-seq analysis, among others.

 

4) I am sure a question that many ask – where do you see this technology going in the future?

Continuing to accelerate the genetic improvement of livestock populations and improve their production efficiency are critical in our ability to meet the protein demands of a growing global population. Over the past half century, assisted reproductive technologies (ARTs) such as in vitro fertilization and somatic cell nuclear transfer have been used to improve reproductive efficiency of agricultural economic species; however, limitations remain, and better ARTs are warranted. Additionally, early embryonic mortality is a major cause of infertility in cattle, yet the molecular causes remain a mystery. In the future, we believe the bovine blastoid technology can not only be used for the development of novel in vitro breeding program, but also great resources to study molecule mechanisms underlying early embryonic development.

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