Cell cycle regulators contribute to extravillous trophoblast cell differentiation and function.

STUDY QUESTION: What is the contribution of cyclin-dependent kinase inhibitor 1C (CDKN1C) to the regulation of extravillous trophoblast (EVT) cell differentiation and function? SUMMARY ANSWER: Cell cycle regulators CDKN1C, cyclin-dependent kinase 7 (CDK7), and cyclin E1 (CCNE1) contribute to the development of a healthy placenta by promoting trophoblast stem (TS) cell differentiation to the invasive EVT cell lineage. WHAT IS KNOWN ALREADY: Early in gestation, cytotrophoblast cells expand and then differentiate into terminal cell lineages, including EVT cells, which invade into uterine tissue and facilitate redirection of maternal resources to the fetus. Cyclin-dependent kinase inhibitor 1C (CDKN1C) is a cell cycle regulator implicated in placental development. STUDY DESIGN, SIZE, DURATION: In this study, we used control versus gene-silenced human trophoblast stem cells to assess mechanisms regulating human TS cell differentiation to the EVT cell lineage. An 8-day EVT cell differentiation protocol was utilized. RNA sequencing data include n = 3 for control EVT cells and n = 3 for gene-silenced cells. Assessment of cell counts, DNA content, qPCR, and migration assays were performed with between n = 3 and n = 6. PARTICIPANTS/MATERIALS, SETTING, METHODS: In situ hybridization was used to evaluate CDKN1C, CDK7, and CCNE1 transcript expression in human placental tissue sections. A human trophoblast stem cell model was utilized to assess the contributions of CDKN1C, CDK7, and CCNE1 to EVT cell development. Gene perturbation was achieved through lentiviral delivery of short-hairpin RNAs to XY (CT29) and XX (CT27) human TS cell lines. Characterization of cell models and effects of gene silencing were measured by assessing cell morphology and number, reverse transcriptase-quantitative polymerase chain reaction, western blotting, flow cytometry, and co-immunoprecipitation. MAIN RESULTS AND THE ROLE OF CHANCE: CDKN1C was expressed in EVT cells of the placenta and in EVT cells derived from TS cells. Disruption of CDKN1C in TS cells affected cell proliferation, morphology, DNA content, migration, and transcript profiles. CDKN1C is a known modulator of CDK-CCN complexes. CDK7 and CCNE1 were prominently expressed in EVT cells of the placenta and in EVT cells derived from TS cells. CDKN1C was also shown to physically interact with CDK7 and CCNE1. Loss of CDK7 or CCNE1 impaired EVT cell differentiation. LARGE SCALE DATA: RNA-seq datasets are available at the Gene Expression Omnibus database under accession number GSE316875. LIMITATIONS, REASONS FOR CAUTION: In vitro experiments were used to evaluate contribution of cell cycle regulators to EVT cell development. Therefore, these methods do not completely recapitulate the in vivo cell environment. WIDER IMPLICATIONS OF THE FINDINGS: Collectively, these findings demonstrate the importance CDKN1C in regulating EVT cell differentiation and link its actions, at least in part, to CDK7 and CCNE1. FUNDING: Supported by an NIH F31predoctoral fellowship to RLS (HD104495), an NIH K99/R00 grants to KMV (HD107262) and AMI (HD115834), P20GM113117 to DKJ, NIH grants (HD020676, HD105734, HD112559), The Sosland Foundation, and the Donald C. Johnson Research Endowment Fund. DISCLOSURES: No disclosures to report.