90 Establishment and characterization of Day 30 equine chorionic girdle and allantochorion cell lines

Abstract

Establishing cell lines is a good model for experimental applications to study molecular mechanisms and cell-specific gene expression. Equids have a diffuse epitheliochorial placenta, where the invasive trophoblast is represented by the chorionic girdle (CG) and the noninvasive trophoblast by the allantochorion (AC). Embryonic CG cells are unique to horses and have a crucial role in equine chorionic gonadotropin (eCG) production and maintenance of pregnancy during the first trimester. This study had three objectives: (1) establishing a stable cell line from Day 30 CG cells and AC using lentivirus encoding hTERT; (2) characterisation of Day 30 CG cells and AC cell morphology and expression of eCG α (eCGA) and β (eCGB) subunits, major histocompatibility complex class II (MHCII), and Kisspeptin receptor (KISS1R) in CG and AC cells; (3) investigating eCG protein production in vitro from Day 30 CG and AC cells. Three mares (n = 3) were used to collect Day 30 conceptuses by non-surgical uterine lavage on Day 30 of pregnancy. All 3 conceptuses were dissected for CG and AC cells then cultured in vitro to confluency in cell culture plates. Second-generation lentiviral particles were generated using a three-vector system including transfer vector pLV-hTERT-IRES-hygro, and human telomerase reverse transcriptase (hTERT) lentivirus was utilised to establish stable hygromycin-resistant equine embryonic cell lines. Reverse-transcription PCR (RT-PCR) was used to study gene expression in cells and radioimmunoassay was used to investigate protein presence in the media. We established a hygromycin-resistant Day 30 CG and AC cell lines that express eCGA, eCGB, and hTERT and confirmed using RT-PCR yielding the predicted bands. The cell lines were maintained for 16 passages (7 ± 2 days/passage), 10 of which were cultured after the lentiviral infection steps. Also, we characterised CG cells as fast-growing, large, binucleated, and epithelioid, and AC cells as rapid-growing showing smaller, squamous, mononucleate, epithelioid, and elongated fibroblastic cells. The RT-PCR results showed eCGA and eCGB subunits are expressed by both Day 30 CG and AC cells, but MHCII and KISS1R genes were not expressed in either of cells. Moreover, radioimmunoassay results showed that Day 30 CG cells did produce eCG protein (35.42 ng mL⁻¹) in vitro earlier than what previous literature has shown. However, Day 30 AC cells did not produce eCG protein (0.042 ng mL⁻¹) in vitro, and both CG and AC cell lines stopped secreting eCG in the media after the lentiviral infection. To conclude, establishing stable and hygromycin-resistant cell lines from Day 30 equine CG and AC cells using lentivirus encoding pLV-hTERT-IRES-hygro is attainable. Also, equine chorionic gonadotropin eCG protein is produced in vitro as early as Day 30 from CG cells.