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Multilineage Differentiation Potential of CNS Cell Progenitors in a Recent ssing oce & pr B o io i t e B f c Santacroce et al., J Bioproces Biotech 2014, 4:7 h o n l i a q n u DOI: 10.4172/2155-9821.1000186 r e u s o J Journal of Bioprocessing & Biotechniques ISSN: 2155-9821 Research Article Open Access Multilineage Differentiation Potential of CNS Cell Progenitors in a Recent Developed Gilthead Seabream (Sparus aurata L.) Nervous Model Maria Pia Santacroce1*, Antonella Tinelli2, Anna Selene Pastore1, Michele Colamonaco3 and Giuseppe Crescenzo3 1Unit of Aquaculture and Zooculture, Department of Veterinary Medicine, University of Bari “Aldo Moro”, Italy 2Unit of Pathology, Deptartment of Veterinary Medicine, University of Bari “Aldo Moro”, Italy 3Unit of Pharmacology and Toxicology, Department of Veterinary Medicine, University of Bari “Aldo Moro”, Italy Abstract Neural Progenitor Cells (NPCs) have gathered more and more attention in the field of Neural Stem Cells (NSCs). However, the multilineage differentiating behavior of these cells and their contribution to tissue regeneration, almost in lower vertebrate taxa, remain unknown. Since the early 1970s, many comparative studies have been performed using immunocytochemical screening on the brains of several vertebrate taxa, including teleosts, in order to identify these cells, even if the data are sometimes contrasting. This study aims: (1) to investigate in vitro the potential proliferative role of NPCs and Radial Glia Progenitors (RGP) in seabream neurogenesis; (2) to reveal the strict ability of fish NSCs to undertake the multilineage development and differentiation in neurons, astrocytes and oligodendrocytes. By the use of double Immunofluorescence (IF) analysis and phase contrast microscopy, we identified the multilineage differentiation and the exact cell morphology. We demonstrated that NSC can self-renew and differentiate into different types of neurons or glial cells during extended culturing. Mature neurons expressed specific neuronal markers; they could differentiate during long term culturing, generating an extensive neurite growth. Glia was found highly mitotic and could developed mature astrocytes and oligodendrocytes. Glial cells were assessed by Glial Fibrillary Acidic Protein (GFAP) reactivity; neurons and myelinating oligodendrocytes were immunostained with cell-specific markers. This work provide that the multilineage differentiation potential of seabream neural cell progenitors might be a useful tool for neurodegenerative diseases, being a promising approach for repairing the CNS injuries, also in other animals, as a new coming strategy for function recovery of damaged nerves. Keywords: Teleost cell culture; In vitro fish neurogenesis; Sparus proliferation of endogenous NPCs, their lateral migration and neuronal aurata; CNS cell progenitors differentiation at the injury site [11]. Introduction Studies on Neural Stem Cells (NSCs) defined these cells as self-renewing CNS cells that can differentiate into any of the three The generation of new neurons in the adult Central Nervous major neural cell lineages, specifically neurons, astrocytes and System (CNS), so called adult neurogenesis, is a fundamental feature oligodendrocytes [16,17]. of the vertebrate brain during the post-embryonic neural growth [1]. It is well known that in mammals and birds, constitutive turnover of Even though the maintenance of vertebrate NSCs in the neural neurons occurs not only during embryogenesis but also during adult epithelium, as their controlled differentiation, is regulated by a conserved stages [2-5]. Adult neurogenesis has been observed in rodents and genetic network, many pro-differentiation factors have been explored songbirds, where newborn neurons are added throughout life to highly for being involved in promoting the neural induction, as Fibroblast restricted spatial regions in the telencephalon [5-9]. In vivo in mammals, Growth Factor (FGF), Retinoic Acid (RA) and Epidermal Growth endogenous Neural Progenitor Cells (NPCs), almost generated along Factor (EGF) [18,19]. The mitogenic action of these growth factors the Sub Ventricular Zone (SVZ) in the adult brain, have been proposed can induce the in vitro proliferation and differentiation of NPCs that as a potential source of newborn neurons for the neural tissue repair began to divide within the first week of culture in vitro. The first signal after various brain insults, such as ischemic stroke or traumatic brain produced in the cluster of differentiating progenitors is the expression injury [10,11]. of nestin, an intermediate filament present in neuroepithelial stem cells, as revealed in cultures of albino mouse embryos [18]. Dissimilarly to birds and mammals, low non-mammalian vertebrates, as fish, amphibians and reptiles, display a considerable amount of adult neurogenesis in more numerous brain regions, even *Corresponding author: Maria Pia Santacroce, Unit of Aquaculture and Zooculture, though such differences are poorly understood and investigated [11]. Department of Veterinary Medicine, University of Bari “Aldo Moro”, Italy, Tel: +39-080- The adult CNS of teleost fish exhibits a high capacity for neuronal 544-3919 Fax: +39-080-544-392; E-mail: [email protected] regeneration after injury [4]. Comparative studies in zebra fish and Received August 04, 2014; Accepted November 14, 2014; Published mammals show that the telencephalic Ventricular Zone (VZ) in the November 18, 2014 adult zebra fish brain generates NPCs that share characteristics with Citation: Santacroce MP, Tinelli A, Pastore AS, Colamonaco M, Crescenzo the NPCs in the mammalian SVZ: they migrate tangentially and G (2014) Multilineage Differentiation Potential of CNS Cell Progenitors in a differentiate into mature neurons into the Olfactory Bulb (OB) [5,11- Recent Developed Gilthead Seabream (Sparus aurata L.) Nervous Model. J Bioprocess Biotech 4: 186 doi: 10.4172/2155-9821.1000186 15]. Recently, Kishimoto et al. [11] developed an in vivo fish model of telencephalic injury to demonstrate the strong ability of adult Copyright: © 2014 Santacroce MP, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which zebra fish to undergo neuronal regeneration. Prodigious regenerative permits unrestricted use, distribution, and reproduction in any medium, provided processes were found in the injured zebra fish telencephalon, with the the original author and source are credited. J Bioproces Biotech ISSN:2155-9821 JBPBT, an open access journal Volume 4 • Issue 7 • 1000186 Citation: Santacroce MP, Tinelli A, Pastore AS, Colamonaco M, Crescenzo G (2014) Multilineage Differentiation Potential of CNS Cell Progenitors in a Recent Developed Gilthead Seabream (Sparus aurata L.) Nervous Model. J Bioprocess Biotech 4: 186 doi: 10.4172/2155- 9821.1000186 Page 2 of 12 In zebra fish, as in rodents, neuroblasts become quiescent after ml streptomycin, 200 µg/ml amphotericin B, 100 µg/ml gentamicin, 10 larval development; while in the adult brain, often after brain injury, mM HEPES, 25 mM NaHCO3 in Leibovitz’s L-15). The homogenate a Notch signaling pathway prompted by growth factors regulate the was then extracted by adding a cocktail of four enzymes including transition of NSCs cells to a proliferative state, into neuroblasts toward 0.05% Collagenase Type IV, 0.03% Hyaluronidase Type IV-S, 0.3% neurogenesis. However, the extrapolation on the NSCs self-renewing Dispase Type II, 0.03% DNase Type I for 20 min dissociations at 20°C capabilities arose mainly from in vivo studies of rodents and mammals, to the homogenate in DM. The enzyme digestion was blocked adding and we are still lacking evidences on the truly proliferative ability of Leibovitz’s L-15 medium supplemented with 10% Foetal Bovine Serum multipotent NSCs and whether these cells can undergo multilineage (FBS; Bio Whittaker, Lonza Walkersville Inc., Italy). The digestion differentiation to develop mature and self-renewing neurons or glial mixture was filtered through 104 µm and 60 µm stainless-steel filters, cells in brain. supernatant was discarded, and the pellet was resuspended in cold 1X Phosphate-Buffered Saline (PBS) 5% Fetal Calf Serum (FCS) and In mammals, NPCs have been isolated and characterized in vitro centrifuged twice at 150 g for 10 min at 4°C. The white pellet was from various regions of the fetal, adult and post-mortem brain tissues recovered, washed and dissociated by adding 0.45 µg ml-1 DNase I in of various species [20-25]. Accumulating evidence suggests that in cold PBS to dissociate cell clumps by Pasteur pipette, then centrifuged teleost fish brain, CNS has the capability to generate new neurons in the at 70 g for 5 min at 4°C. The final collected pellets were resuspended in adult CNS continuously and in large numbers, probably more than in 10% FBS/L-15 medium and cell number was counted. Viability of cells any other vertebrate taxon [4,26-34]. was estimated by trypan blue exclusion. Brain cell suspension yielded Nevertheless, none of these studies on fish has addressed in a 5.2×107 viable cells/g per tissue weight with a viability of 92.8 ± 0.06%. definite manner the involvement of radial glia (RDG) in the generation The suspension of purified neuroglia was adjusted to a density of of new neurons, by interplaying with the full development of the 1×106 cells/ml in basal culture medium (BM) consisting of Leibovitz’s fish nervous tissue in an in vitro microenvironment. Due to several L-15 with 2 mM L-glutamine, 10% FBS, 100 IU/ml penicillin, 100 µg/ discrepancies, arose the need for improving differentiation
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