Reaching the ‘14-day rule’ and Beyond

By Denny Yang Tze Te

Modern embryologists have been extensively using mouse embryos as a model to gain insights on human embryonic development. This model system has unique advantages such as short development timing, low phenotypic variability between inbred strains and around 99% of mouse genes have human homolog. However, embryologists believe it is rather inappropriate to extrapolate findings in one specie to another regardless of how similar they are. One mentionable striking difference is that mouse post-implantation embryo morphology is cup-shaped, whereas human is discoid shaped. (Taft, 2008) Ideally, to understand human embryonic development duly, it is inevitable to use human embryo as a model. 

In the late 1970s, bioethicists and scientists have reached a consensus on the limit of days which any biologists can work with human embryos after fertilization. This limit is known as the ’14-day rule’. (Shen, 2018) After this golden period, human embryo will cease to divide. More importantly, this is a time when the nervous system is first specified and represents the point of individuation. However, the ’14-day rule’ was also a hypothetical limit where no embryologists had ever been able to grow human embryos that long.  

It was not until 2016, a team led by developmental biologist Magdalena Zernicka-Goetz at University of Cambridge had finally reached the 14-day limit. Her rationale to grow human embryos in vitro for 14 days is to understand the molecular phenomenon of post-implantation blastocyst. Implantation of blastocyst occurs at around day 7 after fertilization and it was never well studied. It was thought post-implantation blastocysts development was dependent on the mother’s uterine lining, so it was never studied in vitro. It was also impossible to study post-implantation blastocysts in vivo as Magdalena mentioned ‘blastocyst invades into the body of the mother and so becomes hidden within the decidua’. The post-implantation blastocyst is too small to be observed with ultrasound and it was not possible to study them without harming the mother. (Shahbazi et al., 2016)

Magdalena’s team adapted the mouse culture system they established in 2015 to direct in vitro human embryo to undergo pre- to post-implantation transition without maternal tissues. The culture system was developed by their previous study on growing mouse embryos beyond post-implantation stage. (Bedzhov et al., 2015) They first plated the blastocysts donated from couples undergoing IVF treatment with high oxygen in vitro culture media 1 (IVC1). It was found that high oxygenic culturing condition preserves the epiblast pluripotency and permits further development of the pluripotent lineage in vitro. After 48 hours, IVC1 was replaced by another special IVC with KnockOut Serum Replacement. At the end of day 7 the blastocyst cavity collapsed and the embryo ‘implants’ itself to the dish. The attached embryo continued to grow up to day 14. The human embryo culture system successfully recapitulates human embryogenesis up to 14 days in vivo. (Shahbazi et al.. 2016) Due to ethical considerations, Magdalena’s team was obliged to stop the human embryo culture at day 14 before primitive streak formation and nervous system specification. (Pera, 2017)

Magdalena’s finding open doors for developmental biologist to study the molecular phenomenon during post-implantation blastocyst development. Magdalena believes understanding the molecular processes of blastocysts could potentially educate clinical embryologist regarding in vitro fertilization (IVF). (Shahbazi et al., 2016) It was estimated that 30% of the pregnancy loss happens during the implantation stage. (Bashiri, Halper and Orvieto, 2018) If clinical embryologists can predict which embryo will successfully develop before transfer via IVF, this could potentially reduce the rate of early post-implantation failure.

The ’14-day rule’ was the major limitation which stops Magdalena’s team to further study the human embryo development in vitro. In other words, the ’14-day rule’ does not allow scientists to use Magdalena’s model to study gastrulation in human embryos in vitro. Gastrulation is a critical stage in embryonic development which happens after 14 days in human. This stage of embryonic develop results in formation of 3 germ layers namely ectoderm, mesoderm, and endoderm. The specification of germ layers will later differentiate into various tissues and organ systems in human. 

In 2020, Martinez Arias’ team developed an in vitro human model showing gastrulation-like event without directly growing human embryos. The model is called ‘gastruloids’, a 3D structure made up of clusters of human embryonic stem cells. Human embryonic stem cells are derived from pre-implantation epiblast of human embryos. Gastruloid is generated by culturing 2D embryonic stem cells with WNT agonist and these cells would prime toward a primitive-streak-like state. WNT signaling is a key signaling pathway that triggers mesoderm differentiation during gastrulation. Over time, these cells become organized and go through gastrulation-like event, much as they do in the normal embryos. Martinez’s team believe this model does not break the ’14-day rule’ because gastruloids do not develop any hint of neural cells nor cells that are necessary for the embryo to interact with maternal decidua. (Moris et al., 2020)

The field of developmental biology is a very appealing field of research in modern era. Understanding these fundamental morphological and molecular processes of embryonic development have clinical significances, for instance, the improvement of IVF treatment. However, there are ethical constrains when working with human embryos. The developmental biologists mentioned in this article have all complied to the ’14-day rule’ whilst open doors for future researches to explore the mysteries in early embryonic development. 

References:

Bashiri, A., Halper, K. and Orvieto, R., 2018. Recurrent Implantation Failure-update overview on etiology, diagnosis, treatment and future directions. Reproductive Biology and Endocrinology, [online] 16(1), pp.2-18. Available at: <https://rbej.biomedcentral.com/articles/10.1186/s12958-018-0414-2&gt; [Accessed 28 February 2021].

Bedzhov, I., Bialecka, M., Zielinska, A., Kosalka, J., Antonica, F., Thompson, A., Franze, K. and Zernicka-Goetz, M., 2015. Development of the anterior-posterior axis is a self-organizing process in the absence of maternal cues in the mouse embryo. Cell Research, [online] 25(12), pp.1368-1371. Available at: <https://www.nature.com/articles/cr2015104&gt; [Accessed 28 February 2021].

Moris, N., Anlas, K., van den Brink, S., Alemany, A., Schröder, J., Ghimire, S., Balayo, T., van Oudenaarden, A. and Martinez Arias, A., 2020. An in vitro model of early anteroposterior organization during human development. Nature, [online] 582(7812), pp.410-415. Available at: <https://www.nature.com/articles/s41586-020-2383-9&gt; [Accessed 28 February 2021].

Pera, M., 2017. Human embryo research and the 14-day rule. Development, [online] 144(11), pp.1923-1925. Available at: <https://dev.biologists.org/content/144/11/1923&gt; [Accessed 28 February 2021].

Shahbazi, M., Jedrusik, A., Vuoristo, S., Recher, G., Hupalowska, A., Bolton, V., Fogarty, N., Campbell, A., Devito, L., Ilic, D., Khalaf, Y., Niakan, K., Fishel, S. and Zernicka-Goetz, M., 2016. Self-organization of the human embryo in the absence of maternal tissues. Nature Cell Biology, [online] 18(6), pp.700-708. Available at: <https://www.nature.com/articles/ncb3347&gt; [Accessed 28 February 2021].

Shen, H., 2018. The labs growing human embryos for longer than ever before. [online] Nature. Available at: <https://www.nature.com/articles/d41586-018-05586-z&gt; [Accessed 28 February 2021].

Taft, R., 2008. Virtues and limitations of the preimplantation mouse embryo as a model system. Theriogenology, [online] 69(1), pp.10-16. Available at: <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2239213/&gt; [Accessed 28 February 2021].