Importance of WNT-dependent signaling for derivation and maintenance of primed pluripotent bovine embryonic stem cells

Abstract

The WNT signaling system plays an important but paradoxical role in the regulation of pluripotency. In the cow, IWR-1, which inhibits canonical WNT activation and has WNT-independent actions, promotes the derivation of primed pluripotent embryonic stem cells from the blastocyst. Here, we describe a series of experiments to determine whether derivation of embryonic stem cells could be generated by replacing IWR-1 with other inhibitors of WNT signaling. Results confirm the importance of inhibition of canonical WNT signaling for the establishment of pluripotent embryonic stem cells in cattle and indicate that the actions of IWR-1 can be mimicked by the WNT secretion inhibitor IWP2 but not by the tankyrase inhibitor XAV939 or WNT inhibitory protein dickkopf 1. The role of Janus kinase-mediated signaling pathways for the maintenance of pluripotency of embryonic stem cells was also evaluated. Maintenance of pluripotency of embryonic stem cells lines was blocked by a broad inhibitor of Janus kinase, even though the cells did not express phosphorylated signal transducer and activator of transcription 3 (pSTAT3). Further studies with blastocysts indicated that IWR-1 blocks the activation of pSTAT3. A likely explanation is that IWR-1 blocks differentiation of embryonic stem cells into a pSTAT3+ lineage. In conclusion, results presented here indicate the importance of inhibition of WNT signaling for the derivation of pluripotent bovine embryonic stem cells, the role of Janus kinase signaling for maintenance of pluripotency, and the participation of IWR-1 in the inhibition of activation of STAT3.

Regulation of NANOG and SOX2 expression by activin A and a canonical WNT agonist in bovine embryonic stem cells and blastocysts

“…Treatment with CHIR99201 also compromised blastocyst development. These latter results are consistent with other findings that inhibition of WNT signaling promotes development of ESC in the cow ( Bogliotti et al, 2018 ; Xiao et al, 2021 ; Zhao et al, 2021 ) while activation of WNT signaling inhibits ability of embryos to become blastocysts ( Denicol et al, 2013 ; Tríbulo et al, 2017 ). Thus, overactivation of WNT signaling can be inimical to development and maintenance of pluripotency in the cow.…”

“…Furthermore, the WNT agonist also reduced number of SOX2 + cells and number and percent of blastomeres positive for NANOG in the blastocyst. The present study is thus in agreement with previous reports (Bogliotti et al, 2018;Choi et al, 2019;Gao et al, 2019;Vilarino et al, 2020;Soto et al, 2021;Xiao et al, 2021;Zhao et al, 2021) that support the notion that inhibition of the canonical WNT signaling is essential for maintenance of the pluripotent state of ESC in several livestock species. A recent comprehensive analysis of signaling pathway modulation also was indicative that activation of canonical WNT signaling was associated with reduction in degree of stemness in both naïve and primed human ESC (Bayerl et al, 2021).…”

“…In other studies, loss of expression of SOX2 was associated with differentiation of bESC (Bogliotti et al, 2018;Soto et al, 2021;Xiao et al, 2021). It remains to be seen whether the downregulation of SOX2 by activin A affects pluripotency of bESC.…”

Atlas of receptor genes expressed by the bovine morula and corresponding ligand‐related genes expressed by uterine endometrium

“…Pharmacological activation of canonical WNT signaling reduces the percent of bovine embryos developing to the blastocyst stage (Aparicio et al, 2010;Denicol et al, 2013;Tríbulo et al, 2017). Moreover, maintenance of cells of the inner cell mass in a pluripotent state is facilitated by inhibition of WNT signaling (Xiao et al, 2021). Among the members of the WNT signaling system present in the bovine embryo and endometrium were the canonical WNT ligands NDP, expressed by the endometrium in conjunction with high expression of its receptor LGR4 in the embryo, and RSPO3, which was expressed by the embryo.…”

Cytokines That Serve as Embryokines in Cattle

“…It is not clear if LIF is needed to establish naïve-state pluripotency in bovine stem cells. To date, only bovine ESCs that resemble primed-state stem cells have been generated [102,103].…”