Inhibition of Phosphoinositide-3-Kinase Signaling Promotes the Stem Cell State of Trophoblast

Abstract

Trophoblast stem cells (TSCs) are a heterogeneous cell population despite the presence of fibroblast growth factor (FGF) and transforming growth factor β (TGFB) as key growth factors in standard culture conditions. To understand what other signaling cascades control the stem cell state of mouse TSCs, we performed a kinase inhibitor screen and identified several novel pathways that cause TSC differentiation. Surprisingly, inhibition of phosphoinositide-3-kinase (PI3K) signaling increased the mRNA and protein expression of stem cell markers instead, and resulted in a tighter epithelial colony morphology and fewer differentiated cells. PI3K inhibition could not substitute for FGF or TGFB and did not affect phosphorylation of extracellular signal-regulated kinase, and thus acts independently of these pathways. Upon removal of PI3K inhibition, TSC transcription factor levels reverted to normal TSC levels, indicating that murine TSCs can reversibly switch between these two states. In summary, PI3K inhibition reduces the heterogeneity and seemingly heightens the stem cell state of TSCs as indicated by the simultaneous upregulation of multiple key marker genes and cell morphology. STEM CELLS 2019;37:1307-1318 SIGNIFICANCE STATEMENTTrophoblast stem cells (TSCs) are crucial to the development of a healthy placenta. However, not much is known about the signaling pathways that sustain their self-renewal and drive differentiation. This study uses a kinase inhibitor screen to identify novel pathways that regulate TSCs. Specifically, the authors identified that inhibition of phosphoinositide-3-kinase (PI3K) signaling increases the expression of stem cell markers in these cells. This is in contrast to other cell types, where PI3K inhibition leads to differentiation and apoptosis. The data suggest that just like embryonic stem cells, TSCs exhibit a dynamic stem cell state.

Fetal and trophoblast PI3K p110α have distinct roles in regulating resource supply to the growing fetus in mice

“…Moreover, p110α in both the trophoblast and epiblast-derived lineages contributed to the formation of the capillary network and the thinness of the diffusion barrier in the placental exchange region. These findings are consistent with previous work demonstrating that the PI3K pathway regulates trophoblast differentiation (Kent et al, 2010; Lee et al, 2019) and defects in p110α signaling impair developmental angiogenesis and placental exchange region morphogenesis (Graupera et al, 2008; Lelievre et al, 2005; Sferruzzi-Perri et al, 2016). However, we did not find expression of any markers of known differentiated trophoblast subtypes (eg Syna, Synb, Gcm1, Ctsq ), endothelial cells (eg Pecam1 ) or angiogenesis (eg Ang1/2, Tie2, Vegf ) in placentas that lacked p110α (in the Hom-P compared to Het-U placenta by RNA-seq) thatwould explain the morphological phenotype observed.…”

TET1 and 5-Hydroxymethylation Preserve the Stem Cell State of Mouse Trophoblast

“…While globally, 5hmC enrichment levels at CGIs were not strongly correlated with transcriptional activity of associated genes, there were a few notable exceptions ( Figures 2 E and S3 A). Cdx2 , a core TSC transcription factor known to be downregulated early in TSC differentiation ( Tanaka et al., 1998 , Latos et al., 2015a , Lee et al., 2019 ), lost 5hmC from a CGI associated with a putative 3′-enhancer in differentiated TSCs ( Watts et al., 2011 ). Interestingly, depletion of 5hmC was also observed at Lefty2 , a transcription factor that is similarly downregulated upon TSC differentiation ( Latos et al., 2015a ) and indeed is one of the most strongly downregulated genes in Tet1 KO TSCs ( Chrysanthou et al., 2018 ) ( Figure 2 E).…”

Transcriptomic analysis reveals differential gene expression, alternative splicing, and novel exons during mouse trophoblast stem cell differentiation

“…S 3 ; manuscript for the complete screen in preparation). A similar kinase inhibitor screen recently identified that several Aurora kinase inhibitors (including CCT137690, MLN8237, AT9283, and TC-A-2317) reduce CDX2 (a stem cell marker) expression in TSCs, thereby inducing their differentiation [ 43 ]. Thus, we conclude that Aurora A is required for maintaining the stemness of TSCs and inhibition of its activity results in their differentiation.…”

“…S 3 ). A recent kinase inhibitor screen for inducers of TSC differentiation also identified Aurora kinase inhibitors including CCT137690 [ 43 ]. CDK1 activity is required for activation of Aurora A during G2/M transition [ 45 ].…”