ITGA1 is upregulated in response to oxygen over time in a BMP4 model of trophoblast

Integrin alpha-1 (ITGA1) interacts with the extracellular matrix, particularly collagen type 1 and degraded laminin (Fazleabas et al. 1997). Extravillous trophoblasts (EVT) in the mature human placenta express ITGA1 as they invade from the anchoring villi deeply into the maternal endometrium and myometrium in weeks 8–13 of gestation (Zhou et al. 1993). Similarly, in the baboon, ITGA1 is expressed by invading cytotrophoblast in the peri-implantation period(Fazleabas et al. 1997). Genbacev et al isolated primary first trimester CTBs and cultured them in either 20% oxygen (atmospheric) or 2% oxygen. Only CTBs cultured at 20% oxygen expressed ITGA1 (Genbacev et al. 1996), indicating a role for oxygen in regulating ITGA1 protein expression. In preeclampsia, which is associated with both poor trophoblast invasion and oxidative stress, there is a failure of EVTs to express ITGA1 (Zhou et al. 1993).

Human embryonic stem cells (hESCs) are efficiently differentiated into cells of the human trophoblast (TB) lineage by treatment with BMP4, A83–01 (an Activin/NODAL signaling inhibitor), and PD173074 (an FGF2 signaling inhibitor), termed BAP treatment (Amita et al. 2013). The resulting TB population expresses genes indicative of cytotrophoblast (CTB), syncytiotrophoblast (STB), and extravillous trophoblast (EVT) and can invade through a Matrigel matrix. Gene and protein expression profiles of the hES-derived TB suggest that they most resemble TB found very early in human pregnancy (Amita et al. 2013). Here, we tested whether oxygen regulates expression of ITGA1 in TB derived from BAP-treated hESC as it does in EVT isolated from human placentae late in the first trimester.

We plated male (H1) and female (H9) hES cells on Matrigel-coated coverslips and fixed them in 4% paraformaldehyde following 2, 4, 5, 6, or 8 days of BAP treatment. They were immunostained for ITGA1, for KRT7 to mark CTB and EVT lineages, and for CGB to mark STB. Briefly, coverslips were incubated in primary antibody (anti-ITGA1 Santa Cruz #sc-81733; anti-KRT7 Santa Cruz #sc-17116; anti-CGB abcam #ab53087) at a dilution of 1:100 for ITGA1 and 1:500 for KRT7 and CGB in 5% BSA+2.5% donkey serum overnight in 4C, followed by incubation with secondary antibodies raised in donkey conjugated to 488nm, 568nm, and 647nm AlexaFluor dyes at a dilution of 1:300 for 1 h, and counterstained with DAPI. ITGA1+ areas largely overlapped with CGB+ syncytial areas. Staining intensity was analyzed within three randomly-selected positive regions from each coverslip, in each of 3 biological replicates by using CellProfiler software. ITGA1 expression was more highly upregulated in H1 and H9 cells over days 0–8 of BAP-differentiation when the cells were exposed to a 20% oxygen environment compared to a 5% oxygen environment (p<0.003 Figure 1B). The expression of ITGA1 by CGB+ cells in the hES-derived TB cultures is consistent with the idea that these cells resemble the invasive, primitive syncytium of the peri-implantation period in human pregnancy. Furthermore, they can recapitulate oxygen-regulated gene expression in human TB.

Figure 1.

ITGA1 expression in BAP-differentiated hESCs exposed to a high oxygen environment (20%, atmospheric) and a lower, physiological oxygen environment (5%). (A) Immunofluorescence images of H1 (top) and H9 (bottom) hESCs treated for 6 days with BAP. ITGA1 (green) is expressed in syncytialized cells also stained positive for CGB (red). (B) Expression of ITGA1 over time, quantified as total fluorescence intensity per image, averaged across three images per experiment, and three experiments per time point. Both H1 and H9 cells significantly upregulate ITGA1 over time in response to high oxygen (2-way ANOVA interaction of time and oxygen=***p<0.0001, **p<0.003).