Communication • DOI: 10.2478/s13380-011-0007-4 • Translational Neuroscience • 2(1) • 2011 • 43-48 Translational Neuroscience HUMAN NEUROSPHERES: FROM Emanuela Monni1,2, Terenzio Congiu3, STAINED SECTIONS TO THREE- Denise Massa1, Roxana Nat4,5, DIMENSIONAL ASSEMBLY Andrea Diana1,* Abstract 1Department of Cytomorphology, Human neurospheres are free-floating spherical clusters generated from a single neural stem cell and Laboratory of Neurogenesis and Neuropoiesis, comprising cells at different stages of maturation in the neuronal and glial lineages. Although recent University of Cagliari, Monserrato, findings have disproved the original idea of clonally derived neurospheres according to the paradigm of one 09042, Italy stem cell – one neurosphere, they still represent a valid model for growing neural stem cell cultures in vitro. While the immunocytochemical approach to the identification of stem cells, progenitor cells, and mature 2Laboratory of Neural Stem Cell Biology and cells has been extensively used, scant data are available about the ultrastructural arrangement of different Therapy, University Hospital, cell types within the neurosphere. This paper provides, by means of scanning electron microscopy, some new Lund, SE-221 84 Sweden insights into the three-dimensional assembly of human neurospheres, trying to correlate some parameters 3Department of Human Morphology, such as cell density, shape and growing strategies with the immunolocalization of some antigens such as nestin, GFAP, α-internexin and βIII-tubulin. The major findings from this study are: a) regardless of the stage University of Insubria, Varese, 21100, Italy of in vitro maturation, the growth of the spheres is the result of mitotic divisions producing the aspect of 4Department of Cellular and Molecular an irregular budding mechanism in the outermost layer look like; b) analysis of the volumetric composition Medicine, University of Medicine and of the inner core has revealed the presence of two alternative shape pattern (pyramidal vs rounded cells) Pharmacy Carol Davila, Bucharest, Romania possibly related to both the ongoing maturation stages and GFAP and internexin expression. 5Institute for Neuroscience, Innsbruck Medical University, Keywords Innsbruck, 6020, Austria • Human neurospheres • Neural stem cells • Scanning electron microscopy • Immunocytochemistry Received 9 March 2011 accepted 11 March 2011 © Versita Sp. z o.o. 1. Introduction several caveats have been raised regarding the in vitro stages, to assess the effectiveness of application of clonal features to those dynamic this method in providing further information, in In the last decade, a new boost to the structures [13]. On the other hand, neurosphere association with the immunolabeling of neural understanding of neurodegenerative culture systems represent intriguing models stem cells and their progeny. In this sense, our mechanisms has come from the discovery of by reason of their intrinsic multipotency, aim was to fill the correlation gap between three-dimensional floating cultures, called which is easily triggered by withdrawing basic the rigorous definition based on gene and/or neurospheres that growth in vitro in a “morula”- mitogens (EGF and FGF-2) and ultimately protein expression (the latter also addressed by like fashion [1,2]. Originally interpreted as neural leads to the maturation of all the neuronal this paper) and the dramatic complexity of shape stem cell (NSC) reservoirs, those embryoid and glial lineages [14] capable to develop in and geometry in contacting cells ultimately bodies have been found by biochemical and vitro, such as monolayered cells. However, their committed to building a three-dimensional molecular evidence to harbour a heterogeneous morphological and biochemical characterization model. cell population comprising neural progenitors at single cell level has almost exclusively relied and precursors [3-7]. An interesting feature on the use of immunocytochemical markers is that neurospheres isolated from different applied to sectioned neurospheres [15] or 2. Experimental procedures regions of the developing or adult brain neurosphere-dissociated cells [16]. Indeed very display unique characteristics with regard to few studies have investigated the closer spatial A collection of human embryos ranging from growth, differentiation, and gene expression relationships between cells based on their 5 to 12 gestational weeks (gw) was obtained [8-10]. Therefore, despite not being selectively volumetric assembly by means of ultrastructural from abortions performed at the Obstetric/ committed [11], they maintain distinct regional approaches [17-19] in particular by using Gynecology Department, “Carol Davila” and temporal identities. transmission electron microscopy. Therefore, University of Medicine and Pharmacy, Bucarest, Since then, the “neurosphere assay” has been this study set out to explore, by scanning Romania, according to the ethical legislation extensively explored as a bona fide method to electron microscopy (SEM), both intact human (nr. 134/25.01.2005 from the Bioethical define and measure NSCs behavior 12[ ] but neurospheres and their fragments at different Committee of the same university). * E-mail: [email protected] 43 Translational Neuroscience The time elapsed between the abortion, pixel chip. Image analysis was performed by 3. Results dissection and cell manipulation was means of the ImagePro Plus package (Media approximately 15 to 30 min [20]. Dissociated Cybernetics, Silver Springs, MD). Human neurospheres observed with an cells were cultured in selective medium inverted microscope showed after some mitotic containing DMEM-F12 (Gibco, Grand Island, 2.2 Scanning electron microscopy divisions a unique morphological feature NY), 1% B-27 with antioxidants (Gibco), preparation due to appearance of cilia-like protrusions epidermal growth factor (EGF) and fibroblast 2.2.1 Osmic maceration emerging from the outer cells, which tended growth factor-2 (FGF-2) (both at 20 ng/ml, R&D After extensive washing with PBS, to be reabsorbed with senescence (Figure 1A). Systems, Minneapolis, MN), heparin (5 μg/ml) “naïve” neurospheres were immediately Such physical extensions of the plasma and 1% gentamicin (Sigma-Aldrich, St. Louis, immersed in 0.25% glutaraldehyde and membrane were responsible for neurosphere MO). Passage of the cells was performed every 0.25% paraformaldehyde in 0.1 M Na- motility since they immediately disappeared in 14 days by selecting spheres to be sectioned cacodylate buffer (pH 7.2) for 20 min at room case of sudden adhesion to the bottom of the when they reached at least 400 µm in diameter temperature. After an initial reduction in size plastic surface (data not shown). In addition, with a tissue chopper, in accordance with of 1 mm3, the specimens were washed in when they underwent spontaneous fusion Svendsen et al. [21]. Those neurospheres that phosphate-buffered saline (PBS, pH 7.2) and (Figure 1B) these tiny irregular formations had not reached the critical mass for cutting then postfixed in a solution of 1% osmium retracted, and the neurospheres resumed to after 2 weeks were allowed to grow further tetroxide and 1.25% potassium ferrocyanide their original elliptical shape. in culture for no longer than 1 month. Free- for 3 h. Specimens were further cut into Experiments on sections taken at the floating neurospheres were fed every 4 days 0.2 mm-thick slices, followed by a second equatorial plane showed intense fluorescent by replacing half of the medium with fresh post-fixation in 1% osmium tetroxide and immunoreaction when samples were incubated medium until they reached the desired size. 1.25% potassium ferrocyanide for 2 h. The with specific anti-nestin antibodies (Figure 2A). slices were washed in PBS and immersed in Virtually all cells were nestin-positive regardless 2.1 Immunocytochemistry 0.1% osmium tetroxide in PBS for 48 h. They of tissue localization. The most represented of Free-floating, undifferentiated neurospheres were then dehydrated in graded ethanol and the other markers was α-internexin (Figure 2B), obtained from the complete medium subjected to critical point drying with CO2. while a consistent number of cells also were washed several times with PBS The slices mounted on aluminum stubs were expressed GFAP (Figure 2C). Interestingly, very and immediately fixed in 4% buffered coated with 10 nm of pure gold in a Emitech few cells were found to express the neuronal paraformaldehyde for 10 min at room K250 sputter-coater. specific marker βIII-tubulin which, however, was temperature. After three rinses in the same All the specimens were observed with a better visualized within the neuropil extending buffering solution, several neurospheres were Philips SEM-FEG XL-30 scanning electron from the cell bodies (data not shown). mounted in Tissue-Tek (Sakura Finetek Europe, microscope operated at 10kV. Netherlands), frozen and cut on a cryostat into 10 µm-thick sections. Before immunostaining, the neurosphere sections were pre-incubated with 10% normal serum (Sigma) and 0.2% TritonX-100. Primary antibodies were mouse anti-nestin (1:200, Millipore, cat no. AB5922), rabbit anti-GFAP (1:400 Dako), rabbit anti- α-internexin (1:100, Chemicon, cat. no. AB5354) mouse anti-βIII-tubulin (1:100, Sigma). Detection with secondary antibodies (Molecular Probes, Eugene, OR) was carried out using Alexa Fluor 488 F(ab’)2 fragment of goat anti-mouse IgG (1:1000) and Alexa Fluor 546 F(ab’)2 fragment of goat anti-rabbit IgG (1:1000). Observations were made