P–195 The influence of hormones and initial cell number on the size of self-assembled embryo-like structures
Authors: Niethammer M , Knöspel F , Ban Z , Schneider M R 
Published: August 2021

Abstract

Study question
Do hormonal treatments and initial cell number influence the formation of embryo-like structures (ELS) during their development in regard to size?

Summary answer
The chosen initial cell number for ELS-assembly seems to influence the ELS size only until day 4, while hormones affect embryo size throughout their development.

What is known already
The initial cell number is an important parameter for the development of ELS, which might help to better understand how embryos regulate their size. Previous studies on differently sized natural murine embryos revealed that an initial difference in size at the early stage is compensated until E6.75. Normal-size embryos experience an increased mitotic activity before E6.75, whereas larger sized embryos show an increased apoptotic activity, indicating an important control point of cell turnover by adapting mitotic activity and cell survival. Embryo development is strongly dependent on appropriate β-estradiol and progesterone levels.

Study design, size, duration
The first set of experiments interrogated the influence of initial cell number (two conditions) on the size of formed ELS during the first 3 days (D1–3). The second set included two different hormonal treatments and the two conditions of initial cell number (the same as in the first experiments) generating four different groups. For each day one Aggrewell (generating 1200 ELS/well) per condition was harvested. Experiments were repeated at least three times.

Participants/materials, setting, methods
ELS are generated by self-assembly in microwell-chamber plates combining embryonic stem cells, trophoblast stem cells and extraembryonic endoderm stem cells. Cells were cultured with and without addition of β-estradiol and progesterone, starting with different initial cell numbers (106 vs. 42 cells/ELS). ELS were harvested, stained, and at least 40 randomly picked ELS per condition were measured and statistically analyzed with Two-way ANOVA and Tukey’s multiple comparison test. Results show the average area ± SD.

Main results and the role of chance
The results show a continuous increase in the size of ELS during the first three days of cultivation, with significant lower values (on D1-D3) when ELS were assembled from 42 initial cells (D1: 224.1±87.7 μm²; D3: 674.0± 84.4 μm²) compared to ELS formed with 106 initial cells (D1: 467.1±224.1 μm²; D3: 1275.0±348.0 μm²). Onward on the course of self-assembly, ELS with 42 initial cells were still smaller on D4 (1465.7±657.6 μm²) compared to ELS formed with 106 initial cells (2028.6±522.4 μm²). However, these differences could not be measured on D5 (106 initial cells: 1892.2±603.7 μm²; 42 initial cells: 1855±448.5 μm²), D6 (106 initial cells: 2143.3±622.1 μm²; 42 initial cells: 1788.4±585.5 μm²) and D7 (106 initial cells: 2146.7±628.1 μm²; 42 initial cells: 2319.5±778.8 μm²). Differences between the conditions with and without hormonal treatments (HT) could also be detected especially when ELS were generated with 42 cells: on D4 ELS with HT (1730.4±852.4 μm²) were significantly larger than without hormones (1201.2±462.9 μm²). In contrast, on D7 HT influenced the size of ELS distinctly depending on the initial cell number (42 cells: 1989.2±558.3 μm² with HT vs. 2649.7±999.4 μm² without HT; 106 cells: 2334.9±770.2μm² with HT vs. 1958.6±486.1 μm² without HT).

Limitations, reasons for caution
An even cell distribution is crucial for reproducible ELS-formation. Unfortunately, the used techniques for cell seeding led to an uneven distribution within the microwells. Moreover, different orientation of ELS during the size assessment might be an additional reason for the high variance of ELS size within one condition.
Wider implications of the findings: Even if the results seem to be in accordance with the observations made with natural embryos regarding compensation of size until E6.75, additional experiments need to be conducted. Further investigations should be carried out by testing different culture formats to obtain a more even cell distribution during the cultivation.

Trial registration number
Not applicable