Method of isolation and characterization of Girardia tigrina stem cells

Abstract

Abstract. Tissue regeneration is widely studied due to its importance for understanding the biology of stem cells, aiming at their application in medicine for therapeutic and various other purposes. The establishment of experimental models is necessary, as certain invertebrates and vertebrates have different regeneration abilities depending on their taxon position on the evolutionary scale. Planarians are an efficacious in vivo model for stem cell biology, but the correlation between planarian cellular and molecular neoblast pluripotency mechanisms and those of mammalian stem cells is unknown. The present study had the following objectives: i) Establish Girardia tigrina cell culture, ii) determine the time required for complete cell disintegration and iii) obtain neoblasts by cell subdivision. Twenty-four specimens were deprived of food for seven days. After this time, disintegration was performed by incubation protected at three temperatures for 48 h in an antibiotic, antimycotic and trypsin solution, after which the suspension was homogenized and centrifuged. Histopaque ® 1077 was used for cell separation and interphases were collected and monitored by optical and fluorescence microscopy. Optical microscopy analysis informed the nucleus-to-cytoplasm ratio, cell morphology and cell size. Under fluorescence microscopy, interphase 1 (I1) was subdivided into two groups and neoblasts were marked for characterization; one group was stained with 4',6-diamidino-2-phenylindole and the other was immunolabeled with octamer-binding transcription factor 4 (OCT4) and isolated and observed after 10 days of cultivation. Neoblasts predominated in I1 with a small amount of other cell types. In conclusion, sample disintegration with a trypsin and antibiotic solution was effective at 18˚C and Iscove's modified Dulbecco's medium supplemented with fetal bovine serum was adequate for the establishment of primary cell cultures after 48-h incubation and centrifugation. Antibody anti-OCT4 was used for the characterization of stem cells and was successfully labeled with concentrated neoblasts on interphase 1. IntroductionRegeneration is a complex event that occurs in several vertebrates and invertebrates (1). For regeneration to occur, one of the earliest signaling events following a lesion is the production of cells that are capable of rebuilding lost structures. The way that these events occur and the types of cells involved differ between animal groups (2).The choice of planarians as a regeneration study model is based on their plasticity, regeneration ability, rapid response and adult stem cell reservoir (3). Cell and molecular characterization of planarian stem cells has been achieved with various tools, such as inhibition of the function of genes involved in regeneration via RNA interference, western blotting, in situ hybridization and cell isolation by flow cytometry (4-8). These tools are extremely important alternatives for understanding the mechanisms associated with the maintenance and role of stem cells in vivo (9).Stem cell...

The complete mitochondrial genome of a planarian flatworm Girardia tigrina (Tricladida: Dugesiidae)

“…Girardia tigrina is a freshwater planarian species native to America, and has been introduced to other continents such as East Asia and Europe. Due to its large body size (approximately 10 mm in length), fast asexual reproductive rate and easy large-scale cultivation in the laboratory, G. tigrina has become an important model organism in many research fields such as development, stem cell, regeneration, neurogenesis and toxicology (Lopes et al 2015 ; Bach et al 2016 ). In this study, we reported the complete mitochondrial genome of G. tigrina and relevant phylogenetic analysis for the first time (GenBank accession No: MW972220).…”