Characteristics of Whale Muller Glia in Primary and Immortalized Cultures

Abstract

[EN] Muller cells are the principal glial cells in the retina and they assume many of the functions carried out by astrocytes, oligodendrocytes and ependymal cells in other regions of the central nervous system. Muller cells express growth factors, neurotransmitter transporters and antioxidant agents that could fulfill important roles in preventing excitotoxic damage to retinal neurons. Vertebrate Muller cells are well-defined cells, characterized by a common set of features throughout the phylum. Nevertheless, several major differences have been observed among the Muller cells in distinct vertebrates, such as neurogenesis, the capacity to reprogram fish Muller glia to neurons. Here, the Muller glia of the largest adult mammal in the world, the whale, have been analyzed, and given the difficulties in obtaining cetacean cells for study, these whale glia were analyzed both in primary cultures and as immortalized whale Muller cells. After isolating the retina from the eye of a beached sei whale (Balaenoptera borealis), primary Muller cell cultures were established and once the cultures reached confluence, half of the cultures were immortalized with the simian virus 40 (SV40) large T-antigen commonly used to immortalize human cell lines. The primary cell cultures were grown until cells reached senescence. Expression of the principal molecular markers of Muller cells (GFAP, Vimentin and Glutamine synthetase) was studied in both primary and immortalized cells at each culture passage. Proliferation kinetics of the cells were analyzed by time-lapse microscopy: the time between divisions, the time that cells take to divide, and the proportion of dividing cells in the same field. The karyotypes of the primary and immortalized whale Muller cells were also characterized. Our results shown that W21M proliferate more rapidly and they have a stable karyotype. W21M cells display a heterogeneous cell morphology, less motility and a distinctive expression of some typical molecular markers of Muller cells, with an increase in dedifferentiation markers like alpha-SMA and beta-III tubulin, while they preserve their GS expression depending on the culture passage. Here we also discuss the possible influence of the animal's age and size on these cells, and on their senescence.