High throughput screening platform for embryonic development in vitro

João Francisco Ribeiro Pereira Simões

Key information

Authors:

João Francisco Ribeiro Pereira Simões (João Francisco Ribeiro Pereira Simões Crispim)

Supervisors:

Erik Vrij (Erik Vrij); Cláudia Alexandra Martins Lobato da Silva (Cláudia Lobato da Silva)

Published in

06/11/2013

Abstract

The goal of this project is to develop an in vitro model of early embryonic development of the mouse using stem cells and therefore mimic the developmental period before the implantation of the blastocyst in the uterine wall. Embryonic stem cells (ESC) and Trophoblast stem cells (TSC) are seeded inside agarose microwells that allow the cells to self-organize into structures mimicking the morula state of embryonic development. The research project described in this thesis focuses on improving the coating efficiency of the TSC on the aggregated ESC cells. The results of the coating process allowed concluding that inhibition of the ROCK pathway is crucial in order to have better coating efficiencies. The establishment of adherens junctions between the two lineages if fundamental for a proper coating; inhibition of the ROCK pathway increases the levels of E-cadherin and therefore promotes the establishment of these junctions between the two lineages. To further increase the coating efficiency, a high throughput screen was done using a library of FDA approved small molecules. Among the 1200 compounds screened 16 hits were found that could potentially increase the coating efficiency. The final step of the thesis was to perform a secondary screen to confirm the possible hits as positive hits. The compounds trichlorfon and indapamide were identified as positive hits. Trichlorfon, at a concentration of 20 μM, increased the coating efficiency by two standard deviations compared to the control. For 5 μM, indapamide gave a coating efficiency one standard deviation higher than the positive control. O objectivo deste projecto é desenvolver um modelo in vitro de desenvolvimento embrionário do rato usando células estaminais e, portanto, imitar o período de desenvolvimento antes implantação do embrião no útero. As células estaminais embrionárias e células estaminais trofoblásticas são pipetadas para micropoços de agarose que permite que as células se organizem em estruturas que imitam o estado da mórula. O projecto nesta tese tem como objectivo melhorar a eficiência do revestimento das TSC à volta dos agregados celulares de células estaminais embrionárias. Para aumentar ainda mais a eficiência de revestimento, foi realizado um “high throughput screen” utilizando a bibliotecta Prestwick. Entre os compostos 1200 analisados, foram encontrados 16 compostos que poderiam potencialmente aumentar a eficiência de revestimento. A etapa final da tese foi realizar um “screen” secundário para confirmar os possíveis “hits” como “hits” positivos. De entre os 16 compostos classificados como possiveis “hits”, 2 deles foram confirmados como “hits”positivos.