Structure and ultrastructure of neurospheres spontaneously generated in culture from sheep ovarian cortical cells.

Abstract

Background: Neurospheres derived from adult stem cells of non-neural tissues represent a promising source of neural stem cells (NSCs) and neural progenitor cells (NPCs) for autologous cell therapy in neurological diseases. Analyzing the fine structure of neurospheres can provide pivotal information regarding the phenotype of spheroid-integrating cells, being useful for cell characterization during differentiation, and oncogenic transformation. Neurospheres can be generated by culturing ovarian cortical cells under particular conditions. In order to assess the reliability of use of these spheroids in regenerative medicine, developmental biology, and to state whether they share morphological features with central nervous system (CNS) derived neurospheres, they were analyzed by light and transmission electron microscopy. Sheep ovarian cortical cells were cultured in serum-free medium for 21 days to generate neurospheres. On days 10, 15 and 21 of culture, expression of pluripotency (Nanog, Oct4, Sox2), and NSC/NPC (nestin, Pax6, P75NTR) transcripts by qRT-PCR, immunolocalization of NSC/NPC antigens (nestin, Pax6, P75NTR), structure, and ultrastructure, were analyzed in spheroids. Results: Spheroids expressed transcripts and antigens of pluripotency, and NSC/NPC. Structural analyses revealed that cells were arranged in a peripheral sheet cover comprised by a few layers of spindle-shaped cells exhibiting frequent mitoses, and large round cells; and an inner core, with intermediate-sized cells showing frequent events of apoptosis and necrosis. Ultrastructural analyses showed that in the inner core, most cells were electron dense (dark), showing frequent apoptotic and degenerative events; the outer epithelium-like sheet, was formed by low electron dense cells (light flat cells; LFCs) showing signs of intense metabolic activity, abundant cytoplasmic organelles, phagosomes, lysosome-like structures, apical microvilli, and filament bundles of cytoskeleton elements, indicating that LFCs play a role in extracellular exchanges. Apical-basal polarization of LFCs evidenced by frequent intercellular contacts, like adherens junctions and cytoplasmic interdigitations, was lost in some cells whose contacts with neighboring cells were loose intedigitations and expanded cytoplasmic processes. This might anticipate disengaging/aggregating of light protruding cells (LPCs) from/to the neurosphere, as consistently observed in cultures by inverted microscopy. Conclusions: Neurospheres spontaneously generated from ovarian cortical cells in culture, share most structural and ultrastructural characteristics of neurospheres derived from the CNS.