Abstract
Bovine embryonic stem cells (bESCs) have not been successfully established yet. One reason could be that CDX2, as the trophectoderm regulator, expresses in bovine inner cell mass (ICM), which probably becomes a technical barrier for maintaining the pluripotency of bESCs in vitro. We hypothesized that CDX2 knockdown (CDX2-KD) could remove such negative effort, which will be helpful for capturing complete and permanent capacity of pluripotency. Expression and localization of pluripotent genes were not affected in CDX2-KD blastocysts. The CDX2-KD bESCs grew into monolayers on feeder layer. Pluripotent genes expressed at an improved levels and lasted longer time in CDX2-KD bESCs, along with down-regulation of DNA methylation on promoters of both OCT4 and SOX2. The cystic structure typical for trophoblast cells did not show during culturing CDX2-KD bESCs. CDX2-KD bESC-derived Embryoid bodies showed with compact morphology and with the improved levels of differentiations in three germ layers. CDX2-KD bESCs still carried the capacity of forming teratomas with three germ layers after long-term culture. In summary, CDX2 in bovine ICM was inducer of trophoblast lineage with negative effect on maintenance of pluripotency of bESCs. Precise regulation CDX2 expression to switch on/off will be studied next for application on establishment of bESCs.
Highlights
IntroductionBovine inner cell mass (ICM) cells that were isolated by immuno-surgery still showed trophoblast characteristics, such as cystic structure and cytoplasmic lipid inclusions during in vitro cultivation, suggested that the activation of CDX2 might induce trophoblast differentiation[19]
Space-temporal expressions for both mRNA and protein of CDX2 were first analyzed from oocytes to pre-implantation embryos, in order to design the strategy of CDX2 gene knockdown and to evaluate the knockdown effects on the cultured bovine ESCs (bESCs) afterward
For obtaining the expected knockdown effect, optimal candidate of Short hairpin RNAs (shRNA) structure for knockdown of CDX2 was first selected from a pool of our constructed DNA plasmids, including sequence fragments of shRNA-Control, shRNA488, shRNA621, shRNA672 and shRNA621 +672, which were tested in the over-expressed CDX2 bovine embryonic fibroblasts (O-C-bEFs)
Summary
Bovine ICM cells that were isolated by immuno-surgery still showed trophoblast characteristics, such as cystic structure and cytoplasmic lipid inclusions during in vitro cultivation, suggested that the activation of CDX2 might induce trophoblast differentiation[19]. This finding suggested that CDX2 could be negative regulator for pluripotency of bESCs. depletion of CDX2 in bovine embryos could recover pluripotent gene expressions from the repression state, benefit to establish bESCs. In this study, bovine CDX2-KD embryos were generated after somatic nuclear transfer mediated CDX2 knockdown. CDX2-KD bESCs showed the higher-level expression of pluripotent genes and the robust ability of in vitro and in vivo differentiations
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