141 A NOVEL METHOD FOR PURIFICATION OF INNER CELL MASS AND TROPHECTODERM CELLS FROM BOVINE BLASTOCYSTS USING MAGNETIC ACTIVATED CELL SORTING

Abstract

The first distinct lineage differentiation in the mammalian embryo occurs at the blastocyst stage when blastomeres are segregated into inner cell mass (ICM) or trophectoderm (TE). Obtaining purified TE or ICM can be useful for understanding regulation of early development and differentiation. Although several methods have been reported to separate TE and ICM (e.g. immunosurgery, mechanical dissection using a micromanipulator, or manual selection following trypsinization), limitations exist with these methods. Here, we describe a simple and effective method to sort cells of the blastocyst using magnetic activated cell sorting (MACS) following disaggregation of the blastocyst into single cells using trypsin. Bovine blastocysts were produced in vitro and the zona pellucida removed with a short exposure to acidic Tyrode’s solution. Zona-free blastocysts were incubated with concanavalin A conjugated to fluorescein isothiocyanate (FITC) to label the outer layer of the blastocyst. The blastocysts were then exposed to Hoechst 33342 to label nuclei of all blastomeres. The blastocysts were treated with 0.05% (wt/vol) trypsin, and then disaggregated into single blastomeres by repeating pipetting using a finely drawn, flame-polished mouth micropipette. Single blastomeres were incubated with magnetic microbeads conjugated to anti-FITC and subjected to MACS separation. A fraction of sorted cells was observed under a fluorescence microscope. The remainder were subjected to mRNA extraction, and NANOG (ICM marker) and CDX2 (TE marker) mRNA were quantified by quantitative PCR. After disaggregation of the blastocyst, 2 types of single blastomeres were observed: cells that were positive for both FITC and Hoechst 33342 (TE cells) and cells that were negative for FITC but positive for Hoechst 33342 (ICM cells). Before MACS, about two-thirds of the disaggregated blastomeres labelled with Hoechst 33342 were also labelled with FITC, while one-third were FITC negative. After MACS, the percent of dual-labelled cells in the FITC positive fraction was 91.2%, whereas the incidence of dual-labelled cells in the FITC negative fraction was only 7.8 ± 3.0%. A total of 11.5 μg of RNA per blastocyst was recovered from cells isolated by MACS. This represents 80% of the RNA present in intact blastocysts and suggests a high rate of recovery of blastomeres during the purification process. Furthermore, relative expression level of NANOG was lower in the FITC-positive fraction than in the FITC-negative fraction (0.30 ± 0.05 v. 3.1 ± 0.6, respectively, relative to gene expression level in whole blastocysts). Conversely, the relative expression level of CDX2 was higher in the FITC-positive fraction than in the FITC-negative fraction (3.2 ± 0.09 v. 0.30 ± 0.9, respectively). Results indicate that highly purified TE cells or ICM cells can be collected using MACS. This simple method can be used to study differentiation of the mammalian embryo as well as to prepare embryonic cells of specific lineages for cell therapy.