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Published on: July 30, 2016
In vitro differentiation of macaque extravillous trophoblasts in a low oxygen environment
Logan T Keding1, Avery R Heselton2, Emily Ren2
1Wisconsin National Primate Research Center, University of Wisconsin-Madison, 1223 Capitol Ct, Madison, WI, 53715, USA; Department of Obstetrics and Gynecology, University of Wisconsin-Madison, 202 South Park St, Madison, WI, 53715, USA.
Introduction:
Early primate placental development occurs within a low oxygen (O2) environment, whereas in vitro differentiation of trophoblasts is performed at supraphysiologic O2 levels. Macaque trophoblast stem cells (TSCs) are capable of differentiation to extravillous trophoblasts (EVTs) in vitro, yet the morphological heterogeneity observed across cells lines necessitates evaluation of optimal culture conditions. Our objectives were to determine the impact of oxygen on the in vitro differentiation of macaque TSCs and to refine the molecular characterization of TSC-differentiated EVTs.
Methods:
Macaque TSCs were differentiated to EVTs in either 20% or 5% O2. Gene and protein expression profiles were compared between TSCs and EVTs and between differentiation conditions. Immunohistochemical analysis was performed on early gestation macaque placental tissues to assess in vivo expression of Ki-67, NCAM1 and monkey chorionic gonadotropin (mCG).
Results:
EVTs differentiated in 20% O2 had significantly higher expression of CGA, CGB and NOTCH2 and decreased HIF1A expression compared to those cultured in 5% O2. Regardless of oxygen condition, nearly all EVTs expressed NCAM1 and Mamu-AG, the macaque-specific homolog of human EVT marker HLA-G. In vivo placental expression of NCAM1 was restricted to EVTs within the trophoblastic shell and endovasculature, revealing a macaque EVT marker within the placenta.
Discussion:
Reduced oxygen minimally impacted macaque EVT differentiation in vitro. Elevated expression of the endovascular EVT marker NOTCH2 potentially suggests that 20% O2 supported differentiation of more mature EVTs. Altogether, a standard 20% O2 environment supports macaque EVT differentiation in vitro and the results further validate the identity of macaque TSC-differentiated EVTs.
