278 DIRECTED NEURONAL DIFFERENTIATION OF PLURIPOTENT CELL LINES DERIVED FROM PIG PARTHENOGENETIC EMBRYOS

Abstract

Embryonic stem cells (ESCs) can be differentiated into many cell types in vitro, thus providing a potentially unlimited supply of cells for cognitive in vitro studies and cell-based therapy. However, suitable animal models are crucial prerequisites for the development of safe preclinical protocols in biomedical research. Pig has been proposed as an alternative to the mouse model, due to the morphological and functional affinity of the porcine species with the human. We have previously obtained pluripotent cell lines from pig parthenogenetic blastocysts (Brevini et al. 2007 Theriogenology 67, 54–63). These cells expressed pluripotency-related molecules, such as Oct-4, Nanog, SSEA-4, etc., and were negative for markers specific for ectodermal, endodermal, and mesodermal differentiation (Brevini et al. 2007 Theriogenology 68, 206–213). In this study, we investigated their ability to differentiate into the neural lineage. Since no information was available in the literature, we tested and compared two differentiation protocols. Neurospheres were obtained by culturing, for 2 days, undifferentiated parthenogenetic pluripotent cells in hanging drops of DMEM:Ham F10 medium (1:1), supplemented with knockout (KO) serum replacement (10%), fetal calf serum (5%), retinoic acid (10 µm), and Sonic Hedgehog (100 ng mL^–1). In protocol A, neurospheres were disaggregated and plated in adherent cultures in neural progenitor basal medium supplemented with neural survival factor-1 (NSF-1, 2%) and brain-derived neurotrophic factor (BDNF, 20 ng mL^–1) for 3 weeks. In protocol B, cells were cultured in hanging drops for a further 7 days. Neurospheres were then plated in monolayer and cultured for 3 weeks in DMEM:Ham F12 medium (70:30), supplemented with epidermal growth factor (EGF; 200 ng mL^–1), basic fibroblast growth factor (bFGF; 200 ng mL^–1), B27 (2%), and N2 (1%). At the end of the culture period, cells were either harvested and screened by RT-PCR for neural differentiation-related markers or fixed and subjected to immunocytochemical fluorescent staining. Protocol A was not suitable for neural differentiation of pig pluripotent cell lines and did not seem to induce any differentiation. In contrast, protocol B could efficiently drive cells toward ectodermal differentiation and neural lineage. Neurospheres showed expression of neurofilament H after culture in hanging drops. At the end of the 21-day culture in monolayer, approximately 18% of the cells originally plated differentiated into more mature cell types of the neural lineage, as shown both by morphology and by staining with nestin and β-tubulin III antibodies. Our results indicate that pig pluripotent parthenogenetic cells can be induced to differentiate into both early and more mature neural subpopulations. These experiments, however, also indicate that the direct use of culture conditions developed for mouse and human cells is not always appropriate for pig cells, and the identification of specifically targeted protocols and media formulations for pig cells are required.

This work was supported by PRIN 2005 and FIRST 2006.