Effect of Small Molecule Supplements during In Vitro Culture of Mouse Zygotes and Parthenogenetic Embryos on Hypoblast Formation and Stem Cell Derivation

Abstract

Small molecule inhibitors are organic components that modulate signalling pathways and have the ability to change the differentiation state of cells. They have been used to increase the efficiency of induced pluripotent stem cell generation and to support stem cell derivation and culture. In this study, we aimed to evaluate the effects of small molecules on the development of mouse zygotes and parthenogenetic embryos. Three inhibitors (SC-1, PD0325901 and BIO) were added to the culture medium from the 2-cell stage onwards. We have observed that addition of an inhibitor of the fibroblast growth factor (FGF) pathway (SC-1 or PD0325901) compromises the segregation of hypoblast from the inner cell mass (ICM). Given no difference was observed in size of the ICM, but more epiblast cells were found in these embryos, we can conclude that this is caused by redirection of all ICM cells to the epiblast. We also determined the consequences of reduced hypoblast and increased epiblast formation on stem cell derivation efficiency. No significant difference was found between derivation rates from treated embryos as compared to controls. However, only under 2i + ROCKi conditions, stem cells could be derived with an efficiency of more than 90%. Addition of BIO, an activator of the WNT pathway, did not have any effects on hypoblast development or stem cell derivation. We have demonstrated that FGF signalling is crucial for hypoblast generation and small molecules can be efficiently used to inhibit this process both in zygotes and parthenogenetic embryos.