317-POS

Abstract

Objectives Infection with human cytomegalovirus (HCMV) during pregnancy may cause spontaneous abortions, premature delivery, pre-eclampsia, or permanent neurological disabilities in infants infected in utero. Recent studies demonstrated that HCMV infection inhibits cytotrophoblast (CTB) differentiation into an invasive phenotype resulting in shallow placentation, however the mechanisms involved remain unclear. The lack of an in vitro model that recapitulates the early stages of trophoblast differentiation has hindered efforts to determine the effect of HCMV infection during CTB differentiation. We sought to develop an in vitro model of placental trophoblast differentiation that will allow detailed examination of factors that regulate trophoblast differentiation, as well as, the effect of HCMV infection on trophoblast differentiation and function. Methods Primary trophoblast progenitor cells (TBPCs) isolated from chorions of first-trimester human placentas (generously provided by Dr. M. McMaster) were allowed to attach to collagen beads and cultured in rotating wall vessel (RWV) bioreactors. Differentiation of TBPCs was assessed by immunofluorescence staining, Western blot and qRT-PCR analyses for loss of chorionic villi progenitor markers and gain of invasive cytotrophoblast markers. The invasive capacity of cells was monitored by fibrin clot assays. Results Undifferentiated TBPCs express Cytokeratin 7, HMGA2 and OCT4. TBPCs cultured in RWVs, without added growth factors, initially proliferated and formed aggregates. After 5 days in RWVs, TBPCs downregulated expression of OCT4 and upregulated expression of HLA-G, a marker of invasive cytotrophoblasts. Importantly, TBPCs cultured in RWVs displayed invasive ability in the fibrin clot assay. Multinucleated cells characteristic of syncytiotrophoblasts were not observed. Both undifferentiated and differentiated TBPCs were susceptible to infection with clinical and laboratory-adapted strains of HCMV. Conclusions We have developed an in vitro model of placental trophoblast differentiation that will allow detailed examination of factors that regulate trophoblast differentiation, as well as, pathological placental development that increases risk of pre-eclampsia. Disclosures K.F. Swan: None. M. Ferris: None. G. Pridjian: None. C.A. Morris: None. D.E. Sullivan: None.