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Reconstructing human early embryogenesis in vitro with pluripotent stem cells

Berna Sozen, Victoria Jorgensen, Meng Zhu, Tongtong Cui, Magdalena Zernicka-Goetz

Preprint posted on March 12, 2021 https://www.biorxiv.org/content/10.1101/2021.03.12.435175v1

Witnessing the creation of human life under the microscope. Reconstructing human early embryogenesis in vitro with pluripotent stem cells

Selected by Martin Estermann

Background:

Human life starts at fertilization with the union of both the sperm and the ovum to form the zygote. This unique cell, through cleavage, undergoes continuous divisions without any increase in size, resulting in a solid sphere known as morula. Further division and cell differentiation results in a hollowed structure called blastocyst. Two main structures are defined, the inner cell mass, which will form the proper embryo and the trophoblast, which gives rise to the placenta and other extra embryonic tissues. Later in development, the inner cell mass differentiates into the epiblast and hypoblast, which will contribute to embryonic cells and extra embryonic tissue, respectively.

In humans, this early developmental process occurs naturally within the body of the mother, making it hard to study. In addition, human embryonic samples are difficult to obtain and are regulated by ethical and legal restrictions. Due to these reasons, there is a lack of knowledge regarding this critical developmental step. The generation of in vitro models that recapitulate this early process is therefore crucial to gain insight into this process.

With this in mind, the authors of the preprint combined human human expanded pluripotent stem cells (hEPSCs) with a cell aggregation culture method to allow the formation of 3D structures similar to human blastocysts. This research was able to recapitulate key features equivalent to those occurring between days 3 and 9/10 of human development, providing a key resource to continue exploring this process in vitro.

 

Key findings

1) Generation of human iPSC derived blastocyst-like structures

Human pluripotent stem cells were reprogrammed to a molecular state similar to earlier blastomere (hEPSCs) (Fig. 1A). These cells were isolated and cultured in a multi-inverted-pyramid microwell to allow cell aggregation and self-organization (Fig. 1B). The combination of ROCK and ALK5 inhibitors, WNT antagonists, BMP and FGF2 during the first 48 hours of culture induced the formation of compact cellular aggregates (Fig. 1C). Around days 3 and 4 of culture, cystic structures were visible (Fig. 1C). By day 6, the morphology of these structures resembled blastocysts, showing a similar average cell size and number to human blastocysts (Fig. 1D).

Fig. 1. Human iPSC derived blastocyst-like structures. (A) Phase-contrast hEPSCs image in 2D culture. (B) Schematic protocol of blastocyst formation from hEPSCs. (C) Representative phase-contrast images of hEPSC aggregates in 3D culture during different days (D) of culture. (D) Phase-contrast image of human blastocyst-like structures at day six of culture (D6). (Preprint Fig. 1B-E).

2) Molecular characterization of the hEPSCs derived blastocyst

 To characterize the different cell lineages present in the blastocyst-like structures, the expression levels of core genes for trophectoderm, epiblast and hypoblast were evaluated by qRT-PCR (Fig. 2A). Genes involved in trophectoderm (TE) formation were strongly upregulated during blastocyst formation, compared with the 2D cultures of hEPSCs. In addition, hypoblast marker genes PDGFRA and GATA6 also showed a strong induction, except for SOX17. As expected, NANOG and POU5F1 pluripotent epiblast genes showed similar levels to the hEPSCs cells, whereas the marker KRT17 was upregulated.

To confirm the proper spatial expression of those markers, immunofluorescence was performed. GATA3 expression was evaluated on days 4 (Fig. 2B) and 5 (Fig. 2C) of incubation, being  restricted to the trophectoderm. Surprisingly, GATA3 expression on day 5 was cytosolic rather than nuclear (Fig. 2C). This, together with a poor expression of E-CADHERIN (Fig. 2C), suggests a compromised trophectoderm differentiation in later stages. In contrast, KRT18, another TE marker, displayed a specific expression in the trophectodermal cells (Fig. 2D). Hypoblast/primitive endoderm marker FOX2A and epiblast/pluripotent marker SOX2 were detected in the inner mass-like cells (Fig. 2E).

Fig. 2. Characterization of the hEPSCs derived blastocyst. (A) Heatmap of trophectoderm (TE), epiblast (Epi) and hypoblast (Hypo) marker genes mRNA expression in blastocysts cultured for 4, 5 and 6 days, compared with hEPSCs cells grown in 2D. Immunofluorescence staining of blastocyst-like structures at day 4 (B), day 5 (D and E) and day 6 (C). (Preprint Fig. 3A-E).

3)  hEPSCs derived blastocyst can organize into post-implantation structures

To address if the in vitro generated blastocysts have the capacity to develop beyond implantation, they were transferred to a 96-well ultra-low attachment U-shaped plate and cultured in vitro with modified IVC1 media (Fig 3A). The structures reorganized into disk-shaped structures with SOX2 positive inner compartment surrounded by GATA3 positive extra embryonic compartment (Fig. 3B). The structure generated was similar to human post implantation embryos.

Fig. 3. Blastocyst can organize into post-implantation structures. (A) Illustration of the in vitro post implantation differentiation protocol (top) and corresponding phase-contrast images of a representative structure at each step (bottom). (B) Immunofluorescence staining of a post implantation-like structure at endpoint. GATA3 (cyan) indicates extra-embryonic structures and SOX2 (yellow) the embryonic compartment. (Preprint Fig. 4A and B).

Why we choose this paper:

There is a lack of knowledge in human early embryogenesis. Mouse model organisms, despite giving a great approximation of this process do not recapitulate this process accurately. There is a current demand on in vitro methodologies to study this process in humans, and this research provides the first step into this unknown field. This research showed a great approximation of the early embryogenesis process equivalent to  days 3 to 10 of human development. This protocol is not perfect and as the authors noted, there is a lot of room for improvement, but it is the first step towards unraveling the mysteries of human early embryogenesis and to generate new and improved in vitro models. This research marks a before and after in the field of human early development and differentiation.

Questions to the authors:

  • Is there any limitation (ethical or technical) to continue the development of blastocyst-like structures beyond post-implantation stages?
  • Are the epiblast cells generated with this methodology capable of differentiating in vitro into ectoderm, endoderm and mesoderm?
  • Repeated implantation failure is one of the most common causes of pregnancy failure in in vitro embryonic transference, despite the good quality of the embryos. How feasible would it be to evaluate the implantation success in vitro (in endometrial cultured cells) or in vivo in humanized mice?

 

Posted on: 6th May 2021

doi: https://doi.org/10.1242/prelights.28869

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