300 DEVELOPMENTAL STAGE OF BOVINE BLASTOCYSTS AND THE ADDITION OF LEUKEMIA INHIBITORY FACTOR (LIF) AND BASIC FIBROBLAST GROWTH FACTOR (bFGF) DURING CULTURE IN THE ESTABLISHMENT OF EMBRYONIC STEM CELLS-LIKE COLONIES

Abstract

The pluripotent stem cells have the capacity to generate cells of 3 germ layers (ectoderm, mesoderm, and endoderm). The greatest example of pluripotent stem cells are the cells of the inner cell mass (ICM) of the blastocyst, called embryonic stem cells (ESC). Several studies have been conducted to determine the best culture conditions of bovine blastocysts for the establishment of ESCbov. It is known that basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) suppress differentiation in ESC from mice and, but although LIF is important for the growth of ESC in humans, bFGF is not important in preventing differentiation (Prelle et al. 1994 J. Reprod. Fertil.). Because there are no reports in the literature concerning the use of LIF and bFGF in the bovine, the objectives of this study were to use bovine blastocysts in different developmental stages to establish colonies of ESCbov, comparing the action of bFGF and LIF in maintaining the pluripotency of ESCbov during culture. For this, we used bovine blastocysts (IVP) cultured in basal medium with addition of 2.5% FCS; the ICM of early blastocysts, expanded blastocysts, and hatched blastocysts were mechanically removed using 2 insulin needles. The ICM cells were plated in 6 wells containing medium specific for ESC, subdivided into 3 groups: Group 1 (medium + LIF), Group 2 (medium + bFGF), and Group 3 (medium + bFGF + LIF) over a monolayer of mitotically inactivated bovine fibroblasts (20 × 10⁴ cells/1.9 cm² per well). To confirm the pluripotency of the cells in culture, the colonies were marked with Oct-4, Nanog, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 antibodies. The cells were also immunostained for cytokeratin, α fetoprotein, and vimentin to detect spontaneous differentiation. Adhesion and growth of ICM to the monolayer was observed only when blastocysts (n = 160) were used. This adherence began about 2 days after plating. None of the ICM obtained from early blastocysts (n = 300) resulted in colony formation, and the ICM of hatched blastocysts obtained (n = 45) showed a tendency to detach from the monolayer and form cystic embryoid bodies. The use of LIF was fundamental in the establishment of colonies; however, bFGF was not a significant factor in the cultivation of ESC. The colonies presented imunofluorescence for Oct-4, Nanog, SSEA-3, and TRA-1-81, but were negative for SSEA-1, SSEA-4, and TRA-1-60. The cells in primary culture were negative for α-fetoprotein and vimentin, but some colonies showed positive staining for cytokeratin, demonstrating the contamination of the culture with trophoblastic cells. However, after the first passage, cells presented spontaneous differentiation for mesodermal and endodermal tissues. The addition of LIF and bFGF did not reduce spontaneous differentiation. The expanded blastocysts are the most adequate embryos for derivation and establishment of ESC-like colonies in the bovine. The addition of LIF is important for maintaining cells in culture; however, neither LIF nor bFGF prevented spontaneous differentiation after the first passage.