Transcript levels were estimated by northern analysis or qRT PCR in Rcho 1 trophoblast cells from stem and differentiated states. Each of the genes was expressed at higher levels in the differen tiated cell state. Most of the differentiation associated genes were detected in placental tissues and approximately half showed elevated expression in late gestation necessary versus midgestation trophoblast tissues. Several of the validated differentiation associated genes have been previously reported as upregulated during trophoblast giant cell develop ment, while others have not been associated with tro phoblast lineages. Functions of the differentiation associated genes have been con nected to the regulation of cell movement and invasion, interactions with maternal immune and vascular systems, and the endocrine phenotype of trophoblast giant cells.

A subset of differentiation associated mRNAs highly expressed in rat placental samples was localized to the placentation site via in situ hybridization. Differentiation associated transcripts were all found in trophoblast giant cells and in most instances other tro phoblast lineages. Ecm1 mRNA is expressed in tropho blast giant cells and some progenitor trophoblast cells on gestation d11. 5. Tfpi, Cited2, and Rsp1 transcripts were localized to trophoblast giant cells on gestation d11. 5, including those penetrating into the uterine spiral arterioles. On gestation d18. 5, Tfpi, Cited2, and Rsp1 were also identified in spongiotrophoblast. Cgm4 and Grn transcripts were expressed in trophoblast giant cells, spongiotrophoblast, and invasive trophoblast cells on gestation d18.

5. H19 mRNA was expressed in all tro phoblast lineages on gestation d11. 5 and d18. 5. Fn mRNA was expressed in all trophoblast lineages on d18. 5. PI3K signaling and trophoblast differentiation The PI3K signaling pathway has been implicated in the regulation of trophoblast differentiation and was further investigated in this report. Initially we examined the effect of disruption of PI3K during trophoblast dif ferentiation on the distribution of actin filaments and DNA content. Actin filaments were not signifi cantly affected by the PI3K inhibitor treatment regimen used. However, inhibition of PI3K did affect ploidy. Disruption of PI3K resulted in a significant fraction of cells with increased DNA con tent, and thus the generation of giant cells with elevated ploidy levels.

The findings suggest that PI3K restricts the formation of trophoblast giant cells with high ploidy levels. Higher concentrations of PI3K inhibitors interfere with actin filament distribu tions and cell survival. Phenotypes of differentiating trophoblast cells treated with the PI3K inhibitor or vehicle were also assessed Dacomitinib by DNA microarray analysis. Some genes iden tified were negatively regulated and others positively regulated by PI3K signaling.