276 CHARACTERIZATION OF BOVINE EPIBLAST OUTGROWTH COLONIES DERIVED FROM DAY 12 BLASTOCYSTS

Abstract

Isolation and culture of mouse embryonic stem (ES) cells has been performed for many years, and the improvements achieved throughout the last decade in the human field has evoked great hopes for future cell replacement therapies. However, despite certain similarities in the molecular regulation of pluripotency between man and mouse, there is a need for developing large animal models. The aim of our study was to isolate, culture, and characterize bovine ES-like cell colonies derived from the epiblast. Embryos were produced by in vitro maturation, fertilization, and culture. After 6 days of in vitro culture, blastocysts were transferred to synchronized heifers and allowed to develop for an additional 6 days in vivo. At Day 12 after insemination, embryos were collected by nonsurgical flushing. Embryonic discs were isolated from 15 blastocysts by microsurgery and cultured on mitomycin-inactivated mouse embryonic fibroblasts (SLN cells) in DMEM/F12 medium supplemented with 15% fetal calf serum (FCS), 5% knockout serum replacement (KSR), 106UmL^–1 leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), nonessential amino acids (NEAA), and nucleosides. After 4 (n = 6), 6 (n = 4), and 8 days (n = 5) of culture, the primary outgrowth colonies were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and exposed to antisera recognizing Oct-4 (pluripotency marker; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Vimentin (mesenchyme marker; Zymed Laboratories, South San Francisco, CA, USA), Cytokeratin-8 (trophectoderm marker; Becton, Dickinson and Co., Franklin Lakes, NJ, USA), and α-1-Fetoprotein (hypoblast marker; DakoCytomation, Glostrup, Denmark). The site of antigen-antibody reaction was revealed using the ABC-AEC-method and counterstained with hematoxylin. At Day 4, all colonies had developed a compact central core of cells with a low cytoplasm-to-nucleus ratio, surrounded by a monolayer of squamous cells. At Days 6 and 8, 3 out of 4 and 3 out of 5 colonies, respectively, still presented the compact core which occasionally was encapsulated by a squamous or cuboidal cell sheet. In the remaining colonies, a compact core was less defined. Oct-3/4 staining was observed in the nuclei of the compact core in 5 out of 6 colonies on Day 4, and in all colonies presenting a compact core on Days 6 and 8. However, whereas all nuclei in the core were stained on Days 4 and 6, only scattered nuclei were stained on Day 8. Vimentin staining was observed in the cytoplasm of cells in the compact core in 3 out of 6 Day 4 colonies, in all Day 6 colonies presenting a compact core, but not in any Day 8 colonies. In contrast, α-1-Fetoprotein staining intensity increased with culture period and was mostly observed in squamous monolayer portions. Cytokeratin-8 staining was weak and restricted to the cytoplasm of the cells encapsulating and surrounding the core in 2 Day 6 colonies and a single Day 8 colony. In conclusion, epiblasts isolated from Day 12 bovine blastocysts efficiently attach to feeder cells and develop outgrowth colonies with cores containing presumptive pluripotent cells (Oct-4). However, these cells to some degree lost Oct-4 expression toward Day 8 and were, in parallel, to some degree overgrown by cells of hypoblast (α-1-Fetoprotein) and trophectoderm (Cytokeratin-8) origin.