Establishment of bovine embryonic stem cells after knockdown of CDX2

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.

Changes in chromosome territory position within the nucleus reflect alternations in gene expression related to embryonic lineage specification

“…A drastic reduction in transcript content was noted from zygote to 8–16 cell stage. Relatively high expression in zygotes corresponds to data presented by Wu et al [ 51 ] who detected high levels of CDX2 transcripts in MII oocytes, which declined at the 8 cell stage, and similarly to our data rose in the subsequent developmental stages. Thus, after the drastic degradation of maternal mRNA, CDX2 gene needs to become accessible to the transcriptional machinery.…”

Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts

“…Most, if not all, of the reported bESC lines do not pass standard pluripotency tests, i.e., in vitro embryoid body formation, in vivo teratoma assay, and/or chimera formation. Moreover, they show poor derivation efficiencies, limited proliferation capacities, and loss of pluripotency markers after extensive passages (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16).…”

The past, present and future of bovine pluripotent stem cells: a brief overview

“…Also, equally problematic was that the cells differentiated within a few replication cycles. Although a line of putative bovine ESCs that were sub-cultured more than 30 times was reported after knocking out the CDX2 gene, no data on chimeras were included [17] . To date, only one report documented possible germline chimeras from bovine ESCs [18] .…”