Immortalization of porcine placental trophoblast cells through reconstitution of telomerase activity

Placental trophoblast cells (PTCs) play a critical role in histotrophic nutrient absorption, gaseous exchange, endocrine activities, and barrier function between the maternal and fetal systems. Establishment of immortalized porcine PTCs will help us to investigate the potential effects of different viruses on porcine trophoblast. In the present study, primary porcine PTCs were isolated from healthy gilts at Day 30 to Day 50 of gestation through collagenase digestion, percoll gradient centrifugation, and anti-CD9 immunomagnetic negative selection. To provide stable and long lifespan cells, primary PTCs were transfected with human telomerase reverse transcriptase (hTERT) gene. One porcine placental trophoblast cell line, named as hTERT-PTCs, was chosen for characterization. Human telomerase reverse transcriptase-PTCs achieved an extended replicative lifespan without exhibiting any neoplastic transformation signs in vivo or in vitro. The morphologic and key physiological characteristics of the immortalized PTCs were similar to primary PTCs. The immortalized PTCs retained original cell polarity and normal karyotype, expressed trophoblast-specific marker cytokeratin 7 and E-cadherin but did not express vimentin and major histocompatibility complex class I antigens as well as primary PTCs. Human telomerase reverse transcriptase-PTCs secreted low levels of chorionic gonadotrophin subunit and placental lactogen thk were coincident with primary PTCs. Taken together, our results demonstrated that the porcine PTCs could be immortalized through reconstitution of telomerase activity. The immortalized PTCs maintained its original characteristics and can be used as a model cells line to study the pathologic changes of porcine placental trophoblast in viruses infectious diseases. (C) 2016 Elsevier Inc. All rights reserved.