The potential of the Apc mutant ES cells to differentiate into ecto, meso and endodermal lineages was also evaluated and confirmed by the teratoma formation assay followed by immunohistochemistry examination, matching our past success obtained with ES clones obtained by two rounds of gene targeting by homologous recombination. As anticipated, no expression of neuroectodermal markers was observed in teratomas derived from ApcNN ES cells. ES cells is often cultured in serum free medium supplemented with LIF, GSK inhibitor and Mek inhibitor, the so called 2i medium. Using the serum zero cost culture supple mented using a single inhibitor, we found that ApcNN cells possess the highest colony forming capacity when cultured in LIF Mek inhibitor, suggesting that their constitutive Wnt signaling activity replaces the desire for further pathway activation by the GSK inhibitor.
Of note, culturing ApcNN ESCs in medium supplemented with CHIRON reduced the colony formation capacity of these cells suggesting that a very high dosage of Wnt signaling can compromise selleck chemicals the development of ApcNN cells. We also observed that ApcTT and ApcNT cells formed related selleckchem XL184 number of colonies in numerous culture disorders independently of CHIRON supplementation, possibly pointing on the Wnt independent effects of Apc mutations in these cells. Wnt signaling down regulates Tcf3 expression in mouse ESCs To elucidate the molecular mechanisms underlying the altered cell fate determination in Apc mutant ES cells, genome broad transcrip tional evaluation was performed on the newly derived cells. Unsupervised hierarchical clustering examination showed that international gene expression in ApcNN ESCs is currently influenced ahead of differentiation is induced, resolving ApcNN from WT expression profiles in different branches with the dendogram.
Amid the genes differentially expressed amongst ApcNN ES cells and their wild style counterparts, we discovered that, not like other pluripotency markers, Tcf3 was especially down regulated in ApcNN ES cells, an observation which was even further confirmed by qRT PCR and western blot analysis. Even further qRT PCR evaluation unveiled that the observed downregulation is distinct for Tcf3 but not for other members within the Tcf/Lef relatives. Whereas Tcf3 was down regulated in each ApcNN and ApcMin/Min ESCs, the latter encode for the most severely truncated Apc mutant allele and as a result to get a really higher level of Wnt signaling, other members of the Tcf/Lef family have been exclusively up regulated in ApcMin/Min ESCs. Accordingly, Wnt activation achieved in wild kind cells either by Wnt3a conditioned medium or by a GSK3 modest molecule inhibitor, confirmed that Tcf3 down regulation is really a unique response to canonical Wnt signaling in mouse ESCs.