The Isolation of Binucleated Trophectoderm from Mid-Gestation Bovine Placentae Using Fluorescence Activated Cell Sorting.

Abstract

The trophectoderm of ruminant placentae is comprised of two main cell types; mononucleate cells (MNCs) and binucleate cells (BNCs). BNCs comprise approximately 20% of all trophectoderm cells in cattle, sheep, goats, and deer and are characterized as large, hyperploidic, binucleated cells that invade into the endometrium and fuse with maternal cells to form syncytial plaques. BNCs also produce several hormones, including placental lactogen and progesterone. Developing schemes for isolating BNCs from ruminant placentae will enhance our ability to understand BNC development and function. Therefore, the purpose of this study was to use fluorescence activated cell sorting (FACS) for isolating BNCs from mid-gestation bovine placentae. Pregnant bovine uteri (138.64 ± 6.41 days gestation) were collected at a nearby abattoir and transported to the laboratory on ice. Whole cotyledons (n=6/placenta) were dissected away from each placenta, diced into 5-6 mm cubes and incubated in DMEM containing 25 units/ml Dispase, 10% fetal bovine serum (FBS) and 10mM HEPES at 37°C for 1 h. The tissue homogenate was filtered using a 200μm mesh and centrifuged at 1,200 RPM for 10 minutes at room temperature. Cells were resuspended in Dulbecco's PBS containing 5% FBS and 10μM Vybrant DyeCycle Green at 37°C for 30 minutes. FACS was completed on the cells using a FACS-ARIA cell sorting system. Forward scatter (530nm±15nm) was monitored after excitation (488nM). In the first study (n=5 placentae), cells containing excitation intensities two- to four-times that of the peak value (i.e. diploid cells) were collected. Approximately 1 million cells were collected for each placental preparation. Cells were fixed and immuno-stained for placental lactogen (PL) using a polyclonal antibody directed against ovine PL (generously provided by Dr. Russ Anthony). Cells were examined with epifluorescence microscopy to determine the proportion of BNCs (detection of two nuclei within one plasma membrane) and PL-positive cells. Four individual slides were examined for each placental preparation, and four independent fields were counted on each slide (approximately 125-200 cells/ field). The percentage of BNCs were greater (P=0.0001) in the FASC-sorted preparation (73.8±3.3%) than placental cell preparations sampled before sorting (20.1 ±2.2%). Also, FACS-sorted preparations contained a greater proportion (P=.0002) PL-positive cells than the pre-sorted preparations (65 ±4.3% versus 15.9±1.7%). These cells also were viable after FACS. Membrane integrity is maintained in nearly all cells, and high quality RNA preparations were retrieved from sorted cells. In another study, MNCs and BNCs were sorted so that cells falling in the range of the diploid peak were collected as one fraction (putative MNC fraction) and those cells with 2 to 4 times greater fluorescence were collected as the BNC fraction (n=4 placentae). When compared with the pre-sorted preparations, which contained 12.76±1.17% BNCs, the FASC-sorted MNC population consisted of 5.79±0.77% BNCs whereas the BNC sorted preparations contained 83.07±1.05% BNCs (individual means differ; P<0.01). In conclusion, Dispase digestion and FACS is a useful approach for obtaining enriched populations of BNCs and MNCs from midgestation bovine cotyledons. It is anticipated that this technology will enable researchers to study various cell functions of BNCs and begin to understand what controls their formation. (poster)