RESUMO
TEAD4 is a member of transcriptional enhancer factor (TEF) family of transcription factors and plays a pivotal role in regulating embryonic development and muscle regeneration. Known previously, dysfunction of TEAD4 in mouse myoblasts impairs myotube development. However, the effects of TEAD4 on multipotency of muscle-derived stem cells (MDSCs) have not been clearly understood. Recently, bovine MDSCs (bMDSCs) were successfully isolated from adult bovine muscle. Our derived bMDSCs could differentiate into mesodermal cells, including myotubes, adipocytes, and osteoid cells. Our results also revealed that bMDSCs had the capacity to develop into ectodermal and endodermal lineages including neuron-like cells and insulin-secreting cells. After TEAD4 knock-down (TEAD4-KD), bMDSCs still kept the original capacity to differentiate into neuron-like cells and insulin-secreting cells, as shown by acquisition of both neuronal and pancreatic markers normally expressed in differentiated cells. However, up-regulation of CAV3 and ßMHC failed during myogenesis of bMDSCs with TEAD4-KD, although TEAD4-KD in bMDSCs did not affect osteoid cells and myotube formation. More interestingly, adipogenic differentiation of TEAD4-KD bMDSCs was significantly suppressed. During adipogenic differentiation, TEAD4-KD systematically impaired upregulation of TEAD1, TEAD2, and TEAD3, as well as the activation of C/EBP2, ADD1, and PPARγ as the key transcription factors for adipogenic differentiation. Finally, TEAD4-KD led to the failure of adipogenesis from bMDSCs. Together, our results support that TEAD4 is essential during adipogenic differentiation of bMDSCs. It has little effect on myogenesis of bMDSCs, and does not affect ostegenesis, neurogenesis, or pancreatic differentiation of bMDSCs. Our findings will be helpful for future study on the roles of the TEAD family during differentiation of MDSCs, and for controlling MDSC differentiation for stem cell applications.
RESUMO
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.