Trophectoderm complementation assay for IPS cells: a proposal for assessing pluripotency in cattle

Abstract

After the emergence of induced cell reprogramming, performed through the addition of "Yamanaka" transcription factors (Oct4, Sox2, Klf4 and cMyc) to somatic cells, the amount of researches regarding the generation and maintenance of pluripotency has increased greatly. Still, further investigations on the pluripotent state of these cells are still demanding, since different protocols, characterizations and purposes have being used. One major uncertainty about the iPS is how preserved the pluripotent state actually is. Of the many tools used to investigate the pluripotent state of stem cells (embryonic or induced) one of the most promising is the production of embryonic chimeras. Within this context, from Tetraploid Complementation Assay (TCA) it is possible to generate a new whole individual whose genotype is mostly/exclusively derived from the pluripotent stem cells used in the chimeric embryo, at least in mice. However, in cattle the TCA does not have the same efficiency, so we developed a new strategy in order to achieve this goal. This new strategy was called as trophectoderm complementation assay (TrCA) and has a patent process pending. Thus, this proposal aims to generate bovine induced Pluripotent Stem Cells (biPSC) in their naive state of cell differentiation. From these biPSC we will produce embryonic chimeras with different methodologies (TCA and TrCA) in order to generate new individuals derived mostly/exclusively from the biPSC. Hence, the biPSC will be cultured in supplementation with differentiation inhibitors (2i, 2i+LIF and LIF) previously known to enable the maintenance of pluripotency. The biPSC will be assessed for the expression of pluripotency gene markers and used for the formation of embryonic chimeras (experiment I). After selecting the best culture system for biPS, chimeric embryos will be produced in vitro by the aggregation of biPSC with embryos (diploid or tetraploid) or with fragments of trophectoderm from in vitro produced embryos. In order to verify the embryonic competence, we will assess chimeric blastocyst on the expression of genes related to the embryo quality, on the organization of organelles and nucleus by ultrastructural analysis (descriptive) and also the immunolocalization for differentiation of embryonic tissues (experiment II). Additionally, chimeric embryos will be transferred to recipients and pregnancy will be diagnosed by ultrasound examination. Once established a pregnancy (up to the 60th day of gestation), we will collected the embryonic and extraembryonic tissues for their origin assessment (biPS or embryo, experiment III). Our research has might potentially provide important and novel knowledge about the maintenance status of pluripotent biPSC and also about the capability of generating new whole individuals through the TrCA in cattle. (AU)