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Neurosphere and neurosphere-forming cells: morphological and ultrastructural characterization
Brain Research 993 (2003) 18 – 29 www.elsevier.com/locate/brainres
Research report
Neurosphere and neurosphere-forming cells: morphological and ultrastructural characterization Alessandra Bez a, Elena Corsini a, Daniela Curti b, Marco Biggiogera c, Augusto Colombo d, Roberto Francesco Nicosia e,f, Stefano Filippo Pagano a, Eugenio Agostino Parati a,* a
Laboratory of Neurobiology, Department of Neurobiology and Neurorestorative Therapies, National Neurological Institute ‘‘C. Besta’’, Via Celoria 11, 20133 Milan, Italy b Department of Physiological Science and Cellular and Molecular Pharmacology, University of Pavia, Pavia, Italy c Department of Animal Biology, University of Pavia, Pavia, Italy d Institute for Obstetric and Gynaecology ‘‘L. Mangiagalli’’, ICP, Milan, Italy e Veterans Affairs Puget Sound Health Care System, Seattle, WA, USA f Department of Pathology, University of Washington, Seattle, WA, USA Accepted 14 August 2003
Abstract Despite recent advances in our understanding of neural stem cell (NSC) biology, the free-floating structures generated by these cells in vitro, the ‘‘neurospheres’’, have not been fully characterized. To fill this gap, we examined neurospheres and neurosphere-derived NSCs by confocal microscopy, electron microscopy (EM) and cytofluorimetry. Here, we show that neurospheres and neurosphereforming cells are morphologically and functionally heterogeneous. Confocal microscopy reveals differences in cell size, viability, cytoplasmic content and in the presence and distribution of active mitochondria. By electron microscopy, neurospheres appear as complex structures in which biological events such as mitosis, apoptosis and even phagocytosis are influenced by NSCs localization within the architecture of the neurosphere. NSCs derived from neurospheres are not synchronized and are represented in all phases of the cell cycle. Cytofluorimetric studies demonstrate NSCs’ heterogeneity in cell size by forward scatter (FSC) analysis, and in cytoplasmic granularity by side scatter (SSC) profiling. These findings may contribute to our understanding of the morphogenesis of the neurospheres, particularly as this process relates to the high environmental adaptability of the NSCs and the reported existence of different subpopulations of neural stem cells. D 2003 Elsevier B.V. All rights reserved. Keywords: Neurosphere; Neurosphere-forming cells; Characterization
1. Introduction Since they were first described in the mouse brain, neural stem cells (NSCs) have been the subject of intensive investigation because of their potential therapeutic use in neurodegenerative disorders. Mouse NSCs were initially thought to derive from the striatum [27]; additional studies demonstrated that the site of origin of these NSCs was actually located in the adjacent subventricular zone (SVZ). Different areas of embryonic and adult mouse and human
* Corresponding author. Tel.: +39-2-2394387; fax: +39-2-270638217. E-mail address: [email protected] (E.A. Parati). 0006-8993/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2003.08.061
brains were later found to contain neural stem cells [2,3,8,12,24,30,31]. Various methods have been developed to isolate NSCs and characterize their capacity to proliferate and differentiate [2,6,11,28,29,37,40 –43]. NSCs can be isolated by cell sorting based on expression of individual surface antigens such as CD24 [5,9,13,33,38] and CD 133 [40] or physical properties such as forward scattering (cell size) and side scattering (granularity) [23]. NSCs are highly plastic cells capable of fate conversion/transdifferentiation events [4,14,20,22,26,34,44], which are essential for their engraftment and migration. This plasticity allows NSCs to home to and differentiate into neural tissues of interest, with resulting amelioration of signs and symptoms of neurodegenerative or cerebro-
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vascular diseases [1,10,18,21,35,39]. Recently, NSCs have even been shown to fuse with embryonic stem cells (ES) [45]. Though much is known about NSCs’ biology and behaviour, investigative efforts have only recently focused on the characterization of NSCs morpho-functional features and metabolic properties, which are largely unknown, particularly in humans. For a long time, human NSCs have been considered a quiescent population of undifferentiated and homogeneous cells, which could be activated by environmental cues in vivo or epigenetic stimuli in vitro. Recent data, however, suggest that NSCs are heterogeneous and may express not only nestin, which is a marker of precursor neural cells [16], but also CD34, CD31 and Tie2 [15,25], which were previously described only in extraneural tissues. In addition, studies demonstrating that neurosphere forming cells are ultrastructurally heterogeneous [17] and express different neural lineage-specific markers indicate the existence within these in vitro formed structures of distinct cellular phenotypes, which implies variable developmental commitments of parental clone-forming cells [36]. A gap, however, exists in our understanding of the cytoarchitecture of human neurospheres and of the morpho-functional characteristics of human neurosphere-forming NSCs. To fill this gap, we have evaluated human embryonal brain-derived neurospheres and neurosphere-derived NSCs by electron microscopy (EM), confocal microscopy and flow cytometry. Our results indicate that human neurospheres are highly dynamic structures with distinct radial gradients of cell proliferation, survival, apoptosis and phagocytosis. NSCs apoptotic and necrotic events increase as neurospheres enlarge and develop insufficiently nourished inner cores. In addition, NSCs derived from these neurospheres are morphologically heterogeneous, exhibit different sizes and cytoplasmic granularity and coexist in different phases of the cell cycle.
2. Material and methods 2.1. Establishment of human embryonic stem cell lines The use of human central nervous system (CNS) tissues was authorized by the Ethics Committee of ‘‘C. Besta’’ Neurological Institute and ‘‘L. Mangiagalli’’ Obstetric – Gynecological Clinic. Human NSCs were derived from the brains of 12-week-old human embryos obtained following the ethical guidelines of the European Network for Transplantation (NECTAR), as previously reported [25,42]. Briefly, the tissue was mechanically minced and the resulting cell suspension was plated in the presence of 20 ng/ml EGF and 10 ng/ml bFGF (both human recombinant) in an NS-A basal serum-free medium (Euroclone, Irvine, UK), optimised for neural stem cell growth and referred to as control medium. These experimental condi-
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tions promote the formation of spherical clusters called ‘‘neurospheres’’ from floating cultures of single cells. The resulting cell strains can be amplified in vitro and cryopreserved. They maintain multipotentiality even after several passages in vitro, as demonstrated by clonal analysis, and can differentiate into neurons, astrocytes and oligodendrocytes [24,42]. 2.2. Laser scanning confocal microscopy (LSCM) Neurospheres were dissociated to cell suspension and centrifuged at 1000 rpm for 10 min (Hermle Z300K). The pellet was resuspended in a buffer of the following composition (in mM): 142 NaCl, 2 KCl, 1.2 K2HPO4, 1 MgSO4, 10 HEPES, 1% glutamine, 1.3 CaCl2, 10 glucose, pH 7.4. Aliquots of cell suspensions were labeled with intravital fluorescent probes for 20 min at 37 jC. Mitochondrial transmembrane potential (/) was measured with 5,5V,6,6V tetracloro-1,1V,3,3V-tetraetilbenzimidazol carbocianine (JC-1, 1.5 AM) at ex 488 nm with an Argon laser and at em 527 nm (green fluorescence, monomeric form) and 590 nm (red fluorescence, J-aggregates). A ‘‘shift’’ from red to green fluorescence is observed upon depolarisation of mitochondria [7]. Cytosolic and mitochondrial calcium were investigated with Rhod2 (0.5 AM), at ex 543 and em 580 and Indo1 (5 AM) at ex 350 and em 400/500 with an UV laser. In some experiments, NSCs were stained also with the nuclear dye Hoechst 33342. The neurospheres, either intact or dissociated into individual cells, were double stained with the SYTO 59 vital nuclear dye (0.5 AM) and propidium iodide (0.15 AM) to estimate cell viability and position in the neurosphere. After labeling, the cells were washed with fresh buffer, left for at least 30 min in the dark, and then incubated for 5 min on polylysin-coated coverslips (19 mm diameter), washed again with fresh medium and perfused (0.5 ml/min) in a 300-Al chamber whose bottom is the coverslip. Image galleries were acquired at 0.8 – 1 Am interval on the z axis. Experiments were performed with a DMS IRBE SP2 (Leica) confocal microscope, equipped with an inverted microscope and a 63 oil objective (NA 1.3). Data analysis was performed with Leica software. 2.3. Electron microscopy For ultrastructural analysis, the neurospheres were fixed by adding glutaraldehyde directly to the growth medium to a final concentration of 2%, and left in fixative for 2 h. Neurospheres were then centrifuged, rinsed in Sorensen buffer (pH 7.2) and postfixed in 1% OsO4 for 2 h at room temperature. The pellet was then incorporated into a 2% agarose gel, dehydrated in ethanol and embedded in LR White resin. Semithin sections were routinely stained with toluidine blue. Ultrathin sections were stained with uranyl acetate and lead citrate. For immunolabeling, 100 AM
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bromouridine (BrU) was added to the growth medium prior to fixation and left for 10 min. The cells were then fixed with 4% paraformaldehyde for 2 h and processed as above. Thin sections were immunolabeled by the colloidal gold method for BrU for evaluation of RNPs. All the specimens were examined with a Zeiss EM 900 electron microscope operating at 80 kV. 2.4. Cytofluorimetric analysis Neurospheres from six different cell strains were analysed by flow cytometry. The neurospheres were collected at 24 h, 5 days and 10 days after plating in fresh medium and dissociated into single cells. The percentage of cells in G0/G1, S and G2/M phases, and the number of cells present in each phase were evaluated. Cell cycle was evaluated by using a staining kit for DNA analysis in flow cytometry (DAKO). Approximately 500,000 cells
were incubated for 1 h at 4 jC with propidium iodide (50 Ag/ml), resuspended in a lysis-containing buffer containing a lysis agent, a Rnase and a chromatin stabilizer, according to manufacturer’s instruction. Then cells were resuspended again, filtered through a 50-Am pore filter and measured on a DAKO Galaxy (DAKO) using FloMax software. Cytofluorimetric analysis was then performed to establish NSCs size. Microspheres of pre-defined sizes (NileredR Fluorescent beads, BD Biosciences; 1.7 – 2.2 Am; 2.5– 4.5 Am; 10– 14 Am; 15 – 19 Am) were resuspended in PBS and used as standard to establish NSCs size by cytofluorimetric analysis. Both cells and beads were analysed with the same setting of instrumental physical parameters (forward scatter [FSC], representing cell and bead size, and side scatter [SSC], representing cellular granularity). Cell size was calculated on a curve employing bead size on x axis and FSC values on the y axis.
Fig. 1. Phase contrast images of neurospheres (A – C) and confocal image of neurosphere-derived NSCs (D). Neurospheres were cultured under serum-free conditions in presence of EGF and bFGF. (A) Neurosphere with irregular rim and cilia-like cytoplasmic processes protruding from its surface (a). (B) Neurosphere with regular rim and well defined spherical shape. (C) Neurosphere adherent to an uncoated well exhibits pseudopod-like cytoplasmic processes even though it is cultured in serum-free medium supplemented with GFs (not differentiating culture conditions); this neurosphere detached and became floating again a few days later. (D) Neurosphere-derived cells stained intravitally with JC-1, a cationic dye that shifts from red to green fluorescence as a result of mitochondrial membrane depolarisation. Cytoplasmic staining obtained with this dye demonstrates morphological heterogeneity of NSCs. In particular, two kinds of cells (b and c) can be identified based on size, amount of cytoplasm and mitochondrial distribution. Other cellular phenotypes are more heterogeneous and less frequently observed. Bars: 50 Am (A – C) or 12 Am (D).
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To quantify the number of apoptotic and necrotic cells, we used human Annexin V-FITC Kit (Bender MedSystem). Cells at two different DIV, 12 and 19, and dimensions, between about 150 and 220 Am, respectively, were treated according to the manufacturer’s instructions. Neurospheres were dissociated to single cells, resuspended in the binding buffer of the kit and adjusted to a cell density of 2– 5 105. Annexin V-FITC (5 Al) was added to 195 Al of the cell suspension. Cells were then mixed and incubated for 10 min at room temperature. Finally, cells were washed, resuspended in 190 Al of binding buffer, mixed with 10 Al of propidium iodide (final concentration 1 Ag/ml) and analysed by FACS. 2.5. Immunocytochemistry For the analysis of the distribution of cells in mitosis, the neurospheres were plated on Matrigel and kept in growth medium containing BrdU (20 AM, Sigma) for 24 h. During this period, they spread so that it was possible identify peripheral and internal cells. The neurospheres were then fixed for 20 min in 4% paraformaldehyde in PBS (pH 7.4), washed and incubated with PBS 1 , 0.3% Triton containing 10% normal goat serum and a monoclonal anti-BrdU antibody (1:500, Chemicon) for 90 min at 37 jC. The neurospheres were processed using the standard avidin – biotin peroxidase procedure. Antigen unmasking was performed by treating the cultures with 2 M HCl for 1 h. After
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being rinsed in Tris buffer, the neurospheres were treated with biotinylated secondary antibody and incubated with the avidin– peroxidase complex ABC kit (Vector), according to the manufacturer’s instructions.
3. Results A first analysis was performed by confocal and electron microscopy on whole neurospheres. This was followed by confocal microscopic and cytofluorometric evaluation of viable cell suspensions obtained after mechanical dissociation of the neurospheres. The cells used in all the experiments were fresh, never frozen and thawed, and the number of neurosphere dissociations (passages) varied from 7 to 16 (100 –180 DIV). We avoided passages that were too low or too high to elude the possibility of non-pure NSCs culture or an excessively long cell manipulation, respectively. Similar results were obtained using cell lines at different passages. 3.1. Neurospheres A cross comparison of neurospheres derived from the same brain, which were the starting materials of our experiments, revealed significant heterogeneity (Fig. 1A – C). Some neurospheres had a well-defined spherical shape (Fig. 1B), whereas others appeared as irregular cell clusters with uneven external rims (Fig. 1A). Ciliated-like cells (Fig.
Fig. 2. Confocal micrograph of neurosphere double stained with the SYTO 59 vital nuclear dye (blue) and propidium iodide (red) to estimate cell viability. SYTO 59 is a lipophilic dye capable of labeling nuclei of viable cells. Propidium iodide penetrates the cell membrane and labels nuclei only if cells are injured and no longer viable. Blue cells are therefore healthy whereas red cells are suffering or dead. Viable cells are mostly located at the periphery of the neurosphere. Bar: 50 Am.
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Fig. 3. Chromosome spread of representative neurosphere shows a mitotic figure (B, inset of A, arrow) in an external layer of the neurosphere. Bars: 50 Am (A), 8 Am (B).
1C) seemed to be on the surface of some neurospheres. Spheres of different sizes could be generated from cells plated at the same time and under identical culture conditions. Larger neurospheres had generally a dark core while smaller ones appeared more translucent. Neurospheres appeared typically as free-floating structures but sometimes, in spite of growth culture condition that favoured proliferation rather than differentiation (serum-free medium enriched with EGF and bFGF), they adhered to the uncoated wells (Fig. 1C) to become floating again a few days later.
Morphological heterogeneity was a peculiarity of neurosphere-forming cells, too (Fig. 1D). On this basis, we considered cells and spheres of six cell lines used for our experiments as a representative pool of NSCs lines in general. The presence of viable cells was particularly clear on the surface of the neurospheres. This phenomenon was consistently observed in all the spheres analysed, with some rare exceptions, which did not, however, influence the solidity of the finding. For instance, cells permeable to propidium iodide were hardly ever seen on the external
Fig. 4. Fluorescent (A) and phase contrast (B) images of paraformaldehyde-fixed neurospheres labeled with propidium iodide to highlight nuclear details, and light micrographic image of BrdU-labeled neurosphere spread over Matrigel substrate (C). The box in A and B marks a telophase occurring on the surface of the neurosphere. BrdU labeling demonstrates DNA synthesis in the majority of cells occupying the outer layers of the neurosphere. Bars: 50 Am.
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Fig. 5. Toluidine blue-stained semithin sections (A, B) and electron micrographs (C, D) of neurosphere. Apoptotic phenomena (highlighted in boxes in A and B) are rarely seen at the periphery of the neurosphere (A) and are identified mainly in its inner layers (B). Ultrastructural studies demonstrate phagocytosed apoptotic bodies (arrows) mostly in the internal layers of the neurosphere (C) and only rarely in peripheral cells (D). Note degradation of apoptotic body in C. Bars: 50 Am (A, B), 1 Am (C, D).
layers of the neurospheres, whereas cells positive for SYTO 59, a lipophilic dye capable of labeling nuclei of viable cells, were demonstrated on the neurosphere surface (Fig.
2). In addition, mitotic figures were found peripherally and were never noticed in the inner part of the spheres (Figs. 3 and 4). We verified these data by immunocytochemistry:
Fig. 6. Electron micrographs of neurosphere cells. Adjacent cells of the neurosphere are joined by a gap-junction (A, inset). Lysosomes (arrow) are demonstrated in the neurosphere cell cytoplasm (B). Bars: 5 Am.
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Fig. 7. Electron micrographs of neurosphere cells immunostained by the colloidal gold method for BrU, a precursor of RNA. RNA synthesis is more prominent in a representative cell at the periphery of the neurosphere (A) than in a cell from the inner layers (B). Colloidal gold particles are present on both perichromatin fibrils and nucleolus, evidence of high trascriptional activity. Bars: 1.5 Am (A), 5 Am (B).
When a neurosphere was plated on a matrix, it spread and flattened losing its spherical shape and becoming a monolayer of cells except for its center that, even if flat, was always composed of several cell layers. Only peripheral
cells, which as a consequence of the contact with an adhesive matrix spread forming pseudopodia-like cytoplasmic processes, stained for BrdU, that is, were actively synthesizing DNA after 24 h of incubation with BrdU
Fig. 8. Confocal images of NSCs stained with the intravital dye JC-1 to highlight morphologic features. NSCs, under appropriate culture conditions, may differentiate into oligodendrocytic (A), astrocytic (B) and neuronal (C) phenotypes.
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Fig. 9. Confocal micrograph of neurosphere-derived NSCs after mechanical dissociation and double staining with the fluorescent dyes JC-1 (green/red, cytoplasm/mitochondria) and Hoechst 33342 (blue, nuclei). Representative NSCs, including a three-cell cluster, exhibit similar characteristics: large size, cytoplasmic and mitochondria content, and nuclear/cytoplasmic ratio. These cells correspond to the cellular phenotype shown in Fig. 1D/c. Bar: 15 Am.
(Fig. 4). We have also documented, for the first time in the literature of human neurospheres, the presence of phagocytic events and, in particular, phagocytosis of apoptotic bodies; apoptosis and phagocytosis of apoptotic bodies never occurred at the periphery of the neurospheres but were typically seen in their inner regions, between the external 2nd to 3rd layers and their core (Fig. 5). The presence of lysosomes further supported the evidence of phagocytosis (Fig. 6). EM revealed that necrotic cells, like apoptotic cells, never localized in the external layers. The percentage of viable, apoptotic and necrotic cells in a neurosphere was quantified by cytofluorimetric analysis using a human Annexin V-FITC Kit. Neurospheres maintained in culture for 12 and 19 DIV, which corresponded to neurospheres of about 150 and 220 Am in diameter, contained 11.2% and 21% of apoptotic cells and 9 and 19.2% of necrotic cells, respectively. BrU incorporation experiments, which were performed to evaluate RNA synthesis, demonstrated a high trascriptional activity in peripheral cells and a definitely reduced one in the inner ones (Fig. 7). Neurosphere-forming cells were connected by weak junctional complexes (Fig. 6). Desmosoms were never observed and there was no evidence of fusion of plasmalemmas.
NSCs appeared as spherical and floating cells after dissociation but, under appropriate culture conditions, they differentiated into oligodendrocytic, astrocytic or neuronal
3.2. Cells dissociated from the neurospheres The second part of our study was carried out on NSCs obtained by mechanical dissociation of neurospheres.
Fig. 10. Flow cytometric analysis of representative NSCs suspension demonstrates heterogeneity of physical parameters as no subpopulation with defined FSC/SSC values can be identified.
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Fig. 11. Flow cytometric analysis of representative NSCs suspension labeled with propidium iodide and evaluated for cell cycle. (A) RN1, RN2 and RN3 gates identify cells in G0/G1, S, and G2/M phases, respectively. (B, C, D) Dot-plots showing cell size and organelle complexity of cells in G0/G1 phases (B), S phase (C) and G2/M phases (D) demonstrate that neurosphere-derived cells with different physical properties (SSC/FSC parameters) can be identified in all phases of the cell cycle.
phenotypes (Fig. 8). Cytofluorimetric analysis of NSCs revealed heterogeneity of NSCs size and granularity. Using NileredR Fluorescent beads of predefined sizes as a reference, the NSCs range in size by flow cytometry between 9.28 and 19.27 Am in diameter. After neurosphere dissociation, more than 80% of the recovered cells were viable. Some cells had many energized mitochondria, whereas others showed few mitochondria and/or low aerobic energy metabolism (Figs. 1D and 9). Analyses of calcium distribution allowed the observation of cells enriched in mitochondrial calcium depots. The mitochondrial calcium signal was higher in cells with very low cytoplasmic calcium (data not shown). The FSC/SSC profile of NSCs confirmed the heterogeneity of neurosphere-forming cells from the physical parameters point of view (Fig. 10). The neurosphere-derived cells were not synchronized, were represented in all the phases of the cell cycle and their dimensions varied with the cycle phase. The means of the values obtained from the six cell lines utilised were: 75.24% of cells in G0/G1 phase, 8.36% in S and 15.82% in G2/M. A typical cell cycle trend and a three dot-plots representing cell size and
organelle complexity/cellular granularity of each cell-cycle phase are shown in Fig. 11. Slight and nonsignificant fluctuations in the percentage of cells present in the
Fig. 12. Percentage of NSCs in different phases of the cell-cycle evaluated after being plated and maintained in the same medium for 1, 5 or 10 days. The absence of significant fluctuations over time suggests that the freshness of the medium and the depletion of GFs occurring after 10 days have no effects on the cell-cycle trend.
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different cell cycle phases could be observed from 1 to 10 DIV (Fig. 12).
4. Discussion Our results demonstrate that neurospheres derived from NSCs plated at the same time and under the same culture conditions differ in size and morphology. Neurospheres can grow considerably becoming darker as they enlarge. Their dark cores become the site of necrotic events probably because of reduced nourishment from the external medium. Smaller spheres, on the other hand, are translucent, have no dark cores and appear much healthier. Neurospheres may have regular or irregular shapes, and cells on their external layers may occasionally show cytoplasmic processes resembling cilia or pseudopodia. Neurospheres may temporarily adhere to the bottom of the culture dish, even under growing culture conditions, to spontaneously become floating again a few days later. Heterogenity characterizes not only the neurospheres but the neurosphere-forming cells as well. These cells have different sizes, granularity, metabolism, cytoplasmic content and are in different phases of the cell cycle. Moreover, they exhibit different features according to the layers of the spheres they occupy. The distribution of biological phenomena such as mitosis, transcription processes, apoptosis, phagocytosis and necrosis was distinctly influenced by the position cells took in the structure of the neurosphere. The cytoarchitecture of the neurosphere and the degree of cell viability and activity within it may depend on the access neurosphere-forming cells have to the culture medium and therefore on the availability of oxygen and nutrients and on the possibility to readily eliminate catabolites in the environment (culture medium). This interpretation would justify the higher cell activity (i.e., mitosis, protein syntesis) we observed at the periphery of the neurospheres where exchange of nutrients, oxygen and catabolites is facilitated. In contrast, apoptosis, phagocytosis, necrosis and low or absent mitotic and transcriptional activity are typical of the inner layers where nutritional exchanges are more difficult. This is confirmed by the higher percentage of apoptotic and necrotic cells found in neurospheres that are maintained in culture without being dissociated for 19 DIV and are larger than neurospheres dissociated after 12 DIV. The neurosphere properties described above can be interpreted as an adaptation of NSCs to in vitro culture conditions. The NSCs may organize in such clusters to survive the nonphysiological conditions of the in vitro environment. Cells probably optimise their interactions acquiring the most advantageous shape from a thermodynamycal point of view: the sphere. The neurosphere, in turn, develops a high degree of biological organization. The observed phagocytosis is probably the expression of a dynamically maintained equilibrium between the generation
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of new cells and the apoptosis or necrosis of older cells. A neurosphere may be considered a microsystem able to grow and survive until a threshold crucial point is reached when the self-restoration mechanisms fail; we can therefore consider the neurospheres an example of ‘‘environmental adaptability’’. This environmental adaptability may enable the NSCs to better express the plasticity they need for in vivo engraftment and fate conversion [4,14,20,22,26,34,44]. In light of the evidence regarding the neurosphere structure and its possible morphogenetic mechanisms, we can hypothesize that neurosphere-derived cell heterogeneity is due to their topographic distribution within the cytoarchitecture of the sphere. This morphologic heterogeneity may also be due, at least in part, to the presence of subpopulations of neurosphere-forming cells with distinct survival and proliferative behaviour. Previous studies have in fact demonstrated that NSCs are endowed with different degrees of stemness properties, as they are able to give origin to neurospheres with variable frequencies, and exhibit different EGF and bFGF responsiveness. We know, for example, that in the presence of EGF, cloned murine neurospheres are larger in diameter, whereas in the presence of bFGF, they are small and that exposure to both GFs produces both large and small spheres [46]; moreover, the EGF-responsive mouse population increases in size by asymmetric division of bFGF-responsive cells and by symmetric division of EGF responsive cells [19] EGF-, bFGF- and both GFs-responsive NSCs with different capacity to generate neurospheres have been also demonstrated in human fetal brain [34]. These cells can be also selectively isolated based on their size and PNA-binding activity or on the expression of cell surface markers such as CD133+ (CD45 , CD34 ) and CD24 (low levels) [31]. In summary, our study provides new insights into the behaviour of NSCs in vitro and the morphogenesis of neurospheres. This knowledge may help optimise methods for the isolation and in vitro expansion of NSCs toward the utilisation of these cells for in vivo studies and therapeutic applications.
Acknowledgements We thank Dr. M. Di Segni and Dr. G. Terzoli for kindly providing Fig. 3, Dr. P. Veglianese for Fig. 4A,B and Dr. S. Pozzi for Fig. 4C. The Laboratory of Neurobiology dedicates this work to its precious research scientist, Dr. Stefano Pagano, who passed away in his youth.
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Research report
Neurosphere and neurosphere-forming cells: morphological and ultrastructural characterization Alessandra Bez a, Elena Corsini a, Daniela Curti b, Marco Biggiogera c, Augusto Colombo d, Roberto Francesco Nicosia e,f, Stefano Filippo Pagano a, Eugenio Agostino Parati a,* a
Laboratory of Neurobiology, Department of Neurobiology and Neurorestorative Therapies, National Neurological Institute ‘‘C. Besta’’, Via Celoria 11, 20133 Milan, Italy b Department of Physiological Science and Cellular and Molecular Pharmacology, University of Pavia, Pavia, Italy c Department of Animal Biology, University of Pavia, Pavia, Italy d Institute for Obstetric and Gynaecology ‘‘L. Mangiagalli’’, ICP, Milan, Italy e Veterans Affairs Puget Sound Health Care System, Seattle, WA, USA f Department of Pathology, University of Washington, Seattle, WA, USA Accepted 14 August 2003
Abstract Despite recent advances in our understanding of neural stem cell (NSC) biology, the free-floating structures generated by these cells in vitro, the ‘‘neurospheres’’, have not been fully characterized. To fill this gap, we examined neurospheres and neurosphere-derived NSCs by confocal microscopy, electron microscopy (EM) and cytofluorimetry. Here, we show that neurospheres and neurosphereforming cells are morphologically and functionally heterogeneous. Confocal microscopy reveals differences in cell size, viability, cytoplasmic content and in the presence and distribution of active mitochondria. By electron microscopy, neurospheres appear as complex structures in which biological events such as mitosis, apoptosis and even phagocytosis are influenced by NSCs localization within the architecture of the neurosphere. NSCs derived from neurospheres are not synchronized and are represented in all phases of the cell cycle. Cytofluorimetric studies demonstrate NSCs’ heterogeneity in cell size by forward scatter (FSC) analysis, and in cytoplasmic granularity by side scatter (SSC) profiling. These findings may contribute to our understanding of the morphogenesis of the neurospheres, particularly as this process relates to the high environmental adaptability of the NSCs and the reported existence of different subpopulations of neural stem cells. D 2003 Elsevier B.V. All rights reserved. Keywords: Neurosphere; Neurosphere-forming cells; Characterization
1. Introduction Since they were first described in the mouse brain, neural stem cells (NSCs) have been the subject of intensive investigation because of their potential therapeutic use in neurodegenerative disorders. Mouse NSCs were initially thought to derive from the striatum [27]; additional studies demonstrated that the site of origin of these NSCs was actually located in the adjacent subventricular zone (SVZ). Different areas of embryonic and adult mouse and human
* Corresponding author. Tel.: +39-2-2394387; fax: +39-2-270638217. E-mail address: [email protected] (E.A. Parati). 0006-8993/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2003.08.061
brains were later found to contain neural stem cells [2,3,8,12,24,30,31]. Various methods have been developed to isolate NSCs and characterize their capacity to proliferate and differentiate [2,6,11,28,29,37,40 –43]. NSCs can be isolated by cell sorting based on expression of individual surface antigens such as CD24 [5,9,13,33,38] and CD 133 [40] or physical properties such as forward scattering (cell size) and side scattering (granularity) [23]. NSCs are highly plastic cells capable of fate conversion/transdifferentiation events [4,14,20,22,26,34,44], which are essential for their engraftment and migration. This plasticity allows NSCs to home to and differentiate into neural tissues of interest, with resulting amelioration of signs and symptoms of neurodegenerative or cerebro-
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vascular diseases [1,10,18,21,35,39]. Recently, NSCs have even been shown to fuse with embryonic stem cells (ES) [45]. Though much is known about NSCs’ biology and behaviour, investigative efforts have only recently focused on the characterization of NSCs morpho-functional features and metabolic properties, which are largely unknown, particularly in humans. For a long time, human NSCs have been considered a quiescent population of undifferentiated and homogeneous cells, which could be activated by environmental cues in vivo or epigenetic stimuli in vitro. Recent data, however, suggest that NSCs are heterogeneous and may express not only nestin, which is a marker of precursor neural cells [16], but also CD34, CD31 and Tie2 [15,25], which were previously described only in extraneural tissues. In addition, studies demonstrating that neurosphere forming cells are ultrastructurally heterogeneous [17] and express different neural lineage-specific markers indicate the existence within these in vitro formed structures of distinct cellular phenotypes, which implies variable developmental commitments of parental clone-forming cells [36]. A gap, however, exists in our understanding of the cytoarchitecture of human neurospheres and of the morpho-functional characteristics of human neurosphere-forming NSCs. To fill this gap, we have evaluated human embryonal brain-derived neurospheres and neurosphere-derived NSCs by electron microscopy (EM), confocal microscopy and flow cytometry. Our results indicate that human neurospheres are highly dynamic structures with distinct radial gradients of cell proliferation, survival, apoptosis and phagocytosis. NSCs apoptotic and necrotic events increase as neurospheres enlarge and develop insufficiently nourished inner cores. In addition, NSCs derived from these neurospheres are morphologically heterogeneous, exhibit different sizes and cytoplasmic granularity and coexist in different phases of the cell cycle.
2. Material and methods 2.1. Establishment of human embryonic stem cell lines The use of human central nervous system (CNS) tissues was authorized by the Ethics Committee of ‘‘C. Besta’’ Neurological Institute and ‘‘L. Mangiagalli’’ Obstetric – Gynecological Clinic. Human NSCs were derived from the brains of 12-week-old human embryos obtained following the ethical guidelines of the European Network for Transplantation (NECTAR), as previously reported [25,42]. Briefly, the tissue was mechanically minced and the resulting cell suspension was plated in the presence of 20 ng/ml EGF and 10 ng/ml bFGF (both human recombinant) in an NS-A basal serum-free medium (Euroclone, Irvine, UK), optimised for neural stem cell growth and referred to as control medium. These experimental condi-
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tions promote the formation of spherical clusters called ‘‘neurospheres’’ from floating cultures of single cells. The resulting cell strains can be amplified in vitro and cryopreserved. They maintain multipotentiality even after several passages in vitro, as demonstrated by clonal analysis, and can differentiate into neurons, astrocytes and oligodendrocytes [24,42]. 2.2. Laser scanning confocal microscopy (LSCM) Neurospheres were dissociated to cell suspension and centrifuged at 1000 rpm for 10 min (Hermle Z300K). The pellet was resuspended in a buffer of the following composition (in mM): 142 NaCl, 2 KCl, 1.2 K2HPO4, 1 MgSO4, 10 HEPES, 1% glutamine, 1.3 CaCl2, 10 glucose, pH 7.4. Aliquots of cell suspensions were labeled with intravital fluorescent probes for 20 min at 37 jC. Mitochondrial transmembrane potential (/) was measured with 5,5V,6,6V tetracloro-1,1V,3,3V-tetraetilbenzimidazol carbocianine (JC-1, 1.5 AM) at ex 488 nm with an Argon laser and at em 527 nm (green fluorescence, monomeric form) and 590 nm (red fluorescence, J-aggregates). A ‘‘shift’’ from red to green fluorescence is observed upon depolarisation of mitochondria [7]. Cytosolic and mitochondrial calcium were investigated with Rhod2 (0.5 AM), at ex 543 and em 580 and Indo1 (5 AM) at ex 350 and em 400/500 with an UV laser. In some experiments, NSCs were stained also with the nuclear dye Hoechst 33342. The neurospheres, either intact or dissociated into individual cells, were double stained with the SYTO 59 vital nuclear dye (0.5 AM) and propidium iodide (0.15 AM) to estimate cell viability and position in the neurosphere. After labeling, the cells were washed with fresh buffer, left for at least 30 min in the dark, and then incubated for 5 min on polylysin-coated coverslips (19 mm diameter), washed again with fresh medium and perfused (0.5 ml/min) in a 300-Al chamber whose bottom is the coverslip. Image galleries were acquired at 0.8 – 1 Am interval on the z axis. Experiments were performed with a DMS IRBE SP2 (Leica) confocal microscope, equipped with an inverted microscope and a 63 oil objective (NA 1.3). Data analysis was performed with Leica software. 2.3. Electron microscopy For ultrastructural analysis, the neurospheres were fixed by adding glutaraldehyde directly to the growth medium to a final concentration of 2%, and left in fixative for 2 h. Neurospheres were then centrifuged, rinsed in Sorensen buffer (pH 7.2) and postfixed in 1% OsO4 for 2 h at room temperature. The pellet was then incorporated into a 2% agarose gel, dehydrated in ethanol and embedded in LR White resin. Semithin sections were routinely stained with toluidine blue. Ultrathin sections were stained with uranyl acetate and lead citrate. For immunolabeling, 100 AM
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bromouridine (BrU) was added to the growth medium prior to fixation and left for 10 min. The cells were then fixed with 4% paraformaldehyde for 2 h and processed as above. Thin sections were immunolabeled by the colloidal gold method for BrU for evaluation of RNPs. All the specimens were examined with a Zeiss EM 900 electron microscope operating at 80 kV. 2.4. Cytofluorimetric analysis Neurospheres from six different cell strains were analysed by flow cytometry. The neurospheres were collected at 24 h, 5 days and 10 days after plating in fresh medium and dissociated into single cells. The percentage of cells in G0/G1, S and G2/M phases, and the number of cells present in each phase were evaluated. Cell cycle was evaluated by using a staining kit for DNA analysis in flow cytometry (DAKO). Approximately 500,000 cells
were incubated for 1 h at 4 jC with propidium iodide (50 Ag/ml), resuspended in a lysis-containing buffer containing a lysis agent, a Rnase and a chromatin stabilizer, according to manufacturer’s instruction. Then cells were resuspended again, filtered through a 50-Am pore filter and measured on a DAKO Galaxy (DAKO) using FloMax software. Cytofluorimetric analysis was then performed to establish NSCs size. Microspheres of pre-defined sizes (NileredR Fluorescent beads, BD Biosciences; 1.7 – 2.2 Am; 2.5– 4.5 Am; 10– 14 Am; 15 – 19 Am) were resuspended in PBS and used as standard to establish NSCs size by cytofluorimetric analysis. Both cells and beads were analysed with the same setting of instrumental physical parameters (forward scatter [FSC], representing cell and bead size, and side scatter [SSC], representing cellular granularity). Cell size was calculated on a curve employing bead size on x axis and FSC values on the y axis.
Fig. 1. Phase contrast images of neurospheres (A – C) and confocal image of neurosphere-derived NSCs (D). Neurospheres were cultured under serum-free conditions in presence of EGF and bFGF. (A) Neurosphere with irregular rim and cilia-like cytoplasmic processes protruding from its surface (a). (B) Neurosphere with regular rim and well defined spherical shape. (C) Neurosphere adherent to an uncoated well exhibits pseudopod-like cytoplasmic processes even though it is cultured in serum-free medium supplemented with GFs (not differentiating culture conditions); this neurosphere detached and became floating again a few days later. (D) Neurosphere-derived cells stained intravitally with JC-1, a cationic dye that shifts from red to green fluorescence as a result of mitochondrial membrane depolarisation. Cytoplasmic staining obtained with this dye demonstrates morphological heterogeneity of NSCs. In particular, two kinds of cells (b and c) can be identified based on size, amount of cytoplasm and mitochondrial distribution. Other cellular phenotypes are more heterogeneous and less frequently observed. Bars: 50 Am (A – C) or 12 Am (D).
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To quantify the number of apoptotic and necrotic cells, we used human Annexin V-FITC Kit (Bender MedSystem). Cells at two different DIV, 12 and 19, and dimensions, between about 150 and 220 Am, respectively, were treated according to the manufacturer’s instructions. Neurospheres were dissociated to single cells, resuspended in the binding buffer of the kit and adjusted to a cell density of 2– 5 105. Annexin V-FITC (5 Al) was added to 195 Al of the cell suspension. Cells were then mixed and incubated for 10 min at room temperature. Finally, cells were washed, resuspended in 190 Al of binding buffer, mixed with 10 Al of propidium iodide (final concentration 1 Ag/ml) and analysed by FACS. 2.5. Immunocytochemistry For the analysis of the distribution of cells in mitosis, the neurospheres were plated on Matrigel and kept in growth medium containing BrdU (20 AM, Sigma) for 24 h. During this period, they spread so that it was possible identify peripheral and internal cells. The neurospheres were then fixed for 20 min in 4% paraformaldehyde in PBS (pH 7.4), washed and incubated with PBS 1 , 0.3% Triton containing 10% normal goat serum and a monoclonal anti-BrdU antibody (1:500, Chemicon) for 90 min at 37 jC. The neurospheres were processed using the standard avidin – biotin peroxidase procedure. Antigen unmasking was performed by treating the cultures with 2 M HCl for 1 h. After
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being rinsed in Tris buffer, the neurospheres were treated with biotinylated secondary antibody and incubated with the avidin– peroxidase complex ABC kit (Vector), according to the manufacturer’s instructions.
3. Results A first analysis was performed by confocal and electron microscopy on whole neurospheres. This was followed by confocal microscopic and cytofluorometric evaluation of viable cell suspensions obtained after mechanical dissociation of the neurospheres. The cells used in all the experiments were fresh, never frozen and thawed, and the number of neurosphere dissociations (passages) varied from 7 to 16 (100 –180 DIV). We avoided passages that were too low or too high to elude the possibility of non-pure NSCs culture or an excessively long cell manipulation, respectively. Similar results were obtained using cell lines at different passages. 3.1. Neurospheres A cross comparison of neurospheres derived from the same brain, which were the starting materials of our experiments, revealed significant heterogeneity (Fig. 1A – C). Some neurospheres had a well-defined spherical shape (Fig. 1B), whereas others appeared as irregular cell clusters with uneven external rims (Fig. 1A). Ciliated-like cells (Fig.
Fig. 2. Confocal micrograph of neurosphere double stained with the SYTO 59 vital nuclear dye (blue) and propidium iodide (red) to estimate cell viability. SYTO 59 is a lipophilic dye capable of labeling nuclei of viable cells. Propidium iodide penetrates the cell membrane and labels nuclei only if cells are injured and no longer viable. Blue cells are therefore healthy whereas red cells are suffering or dead. Viable cells are mostly located at the periphery of the neurosphere. Bar: 50 Am.
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Fig. 3. Chromosome spread of representative neurosphere shows a mitotic figure (B, inset of A, arrow) in an external layer of the neurosphere. Bars: 50 Am (A), 8 Am (B).
1C) seemed to be on the surface of some neurospheres. Spheres of different sizes could be generated from cells plated at the same time and under identical culture conditions. Larger neurospheres had generally a dark core while smaller ones appeared more translucent. Neurospheres appeared typically as free-floating structures but sometimes, in spite of growth culture condition that favoured proliferation rather than differentiation (serum-free medium enriched with EGF and bFGF), they adhered to the uncoated wells (Fig. 1C) to become floating again a few days later.
Morphological heterogeneity was a peculiarity of neurosphere-forming cells, too (Fig. 1D). On this basis, we considered cells and spheres of six cell lines used for our experiments as a representative pool of NSCs lines in general. The presence of viable cells was particularly clear on the surface of the neurospheres. This phenomenon was consistently observed in all the spheres analysed, with some rare exceptions, which did not, however, influence the solidity of the finding. For instance, cells permeable to propidium iodide were hardly ever seen on the external
Fig. 4. Fluorescent (A) and phase contrast (B) images of paraformaldehyde-fixed neurospheres labeled with propidium iodide to highlight nuclear details, and light micrographic image of BrdU-labeled neurosphere spread over Matrigel substrate (C). The box in A and B marks a telophase occurring on the surface of the neurosphere. BrdU labeling demonstrates DNA synthesis in the majority of cells occupying the outer layers of the neurosphere. Bars: 50 Am.
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Fig. 5. Toluidine blue-stained semithin sections (A, B) and electron micrographs (C, D) of neurosphere. Apoptotic phenomena (highlighted in boxes in A and B) are rarely seen at the periphery of the neurosphere (A) and are identified mainly in its inner layers (B). Ultrastructural studies demonstrate phagocytosed apoptotic bodies (arrows) mostly in the internal layers of the neurosphere (C) and only rarely in peripheral cells (D). Note degradation of apoptotic body in C. Bars: 50 Am (A, B), 1 Am (C, D).
layers of the neurospheres, whereas cells positive for SYTO 59, a lipophilic dye capable of labeling nuclei of viable cells, were demonstrated on the neurosphere surface (Fig.
2). In addition, mitotic figures were found peripherally and were never noticed in the inner part of the spheres (Figs. 3 and 4). We verified these data by immunocytochemistry:
Fig. 6. Electron micrographs of neurosphere cells. Adjacent cells of the neurosphere are joined by a gap-junction (A, inset). Lysosomes (arrow) are demonstrated in the neurosphere cell cytoplasm (B). Bars: 5 Am.
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Fig. 7. Electron micrographs of neurosphere cells immunostained by the colloidal gold method for BrU, a precursor of RNA. RNA synthesis is more prominent in a representative cell at the periphery of the neurosphere (A) than in a cell from the inner layers (B). Colloidal gold particles are present on both perichromatin fibrils and nucleolus, evidence of high trascriptional activity. Bars: 1.5 Am (A), 5 Am (B).
When a neurosphere was plated on a matrix, it spread and flattened losing its spherical shape and becoming a monolayer of cells except for its center that, even if flat, was always composed of several cell layers. Only peripheral
cells, which as a consequence of the contact with an adhesive matrix spread forming pseudopodia-like cytoplasmic processes, stained for BrdU, that is, were actively synthesizing DNA after 24 h of incubation with BrdU
Fig. 8. Confocal images of NSCs stained with the intravital dye JC-1 to highlight morphologic features. NSCs, under appropriate culture conditions, may differentiate into oligodendrocytic (A), astrocytic (B) and neuronal (C) phenotypes.
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Fig. 9. Confocal micrograph of neurosphere-derived NSCs after mechanical dissociation and double staining with the fluorescent dyes JC-1 (green/red, cytoplasm/mitochondria) and Hoechst 33342 (blue, nuclei). Representative NSCs, including a three-cell cluster, exhibit similar characteristics: large size, cytoplasmic and mitochondria content, and nuclear/cytoplasmic ratio. These cells correspond to the cellular phenotype shown in Fig. 1D/c. Bar: 15 Am.
(Fig. 4). We have also documented, for the first time in the literature of human neurospheres, the presence of phagocytic events and, in particular, phagocytosis of apoptotic bodies; apoptosis and phagocytosis of apoptotic bodies never occurred at the periphery of the neurospheres but were typically seen in their inner regions, between the external 2nd to 3rd layers and their core (Fig. 5). The presence of lysosomes further supported the evidence of phagocytosis (Fig. 6). EM revealed that necrotic cells, like apoptotic cells, never localized in the external layers. The percentage of viable, apoptotic and necrotic cells in a neurosphere was quantified by cytofluorimetric analysis using a human Annexin V-FITC Kit. Neurospheres maintained in culture for 12 and 19 DIV, which corresponded to neurospheres of about 150 and 220 Am in diameter, contained 11.2% and 21% of apoptotic cells and 9 and 19.2% of necrotic cells, respectively. BrU incorporation experiments, which were performed to evaluate RNA synthesis, demonstrated a high trascriptional activity in peripheral cells and a definitely reduced one in the inner ones (Fig. 7). Neurosphere-forming cells were connected by weak junctional complexes (Fig. 6). Desmosoms were never observed and there was no evidence of fusion of plasmalemmas.
NSCs appeared as spherical and floating cells after dissociation but, under appropriate culture conditions, they differentiated into oligodendrocytic, astrocytic or neuronal
3.2. Cells dissociated from the neurospheres The second part of our study was carried out on NSCs obtained by mechanical dissociation of neurospheres.
Fig. 10. Flow cytometric analysis of representative NSCs suspension demonstrates heterogeneity of physical parameters as no subpopulation with defined FSC/SSC values can be identified.
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Fig. 11. Flow cytometric analysis of representative NSCs suspension labeled with propidium iodide and evaluated for cell cycle. (A) RN1, RN2 and RN3 gates identify cells in G0/G1, S, and G2/M phases, respectively. (B, C, D) Dot-plots showing cell size and organelle complexity of cells in G0/G1 phases (B), S phase (C) and G2/M phases (D) demonstrate that neurosphere-derived cells with different physical properties (SSC/FSC parameters) can be identified in all phases of the cell cycle.
phenotypes (Fig. 8). Cytofluorimetric analysis of NSCs revealed heterogeneity of NSCs size and granularity. Using NileredR Fluorescent beads of predefined sizes as a reference, the NSCs range in size by flow cytometry between 9.28 and 19.27 Am in diameter. After neurosphere dissociation, more than 80% of the recovered cells were viable. Some cells had many energized mitochondria, whereas others showed few mitochondria and/or low aerobic energy metabolism (Figs. 1D and 9). Analyses of calcium distribution allowed the observation of cells enriched in mitochondrial calcium depots. The mitochondrial calcium signal was higher in cells with very low cytoplasmic calcium (data not shown). The FSC/SSC profile of NSCs confirmed the heterogeneity of neurosphere-forming cells from the physical parameters point of view (Fig. 10). The neurosphere-derived cells were not synchronized, were represented in all the phases of the cell cycle and their dimensions varied with the cycle phase. The means of the values obtained from the six cell lines utilised were: 75.24% of cells in G0/G1 phase, 8.36% in S and 15.82% in G2/M. A typical cell cycle trend and a three dot-plots representing cell size and
organelle complexity/cellular granularity of each cell-cycle phase are shown in Fig. 11. Slight and nonsignificant fluctuations in the percentage of cells present in the
Fig. 12. Percentage of NSCs in different phases of the cell-cycle evaluated after being plated and maintained in the same medium for 1, 5 or 10 days. The absence of significant fluctuations over time suggests that the freshness of the medium and the depletion of GFs occurring after 10 days have no effects on the cell-cycle trend.
A. Bez et al. / Brain Research 993 (2003) 18–29
different cell cycle phases could be observed from 1 to 10 DIV (Fig. 12).
4. Discussion Our results demonstrate that neurospheres derived from NSCs plated at the same time and under the same culture conditions differ in size and morphology. Neurospheres can grow considerably becoming darker as they enlarge. Their dark cores become the site of necrotic events probably because of reduced nourishment from the external medium. Smaller spheres, on the other hand, are translucent, have no dark cores and appear much healthier. Neurospheres may have regular or irregular shapes, and cells on their external layers may occasionally show cytoplasmic processes resembling cilia or pseudopodia. Neurospheres may temporarily adhere to the bottom of the culture dish, even under growing culture conditions, to spontaneously become floating again a few days later. Heterogenity characterizes not only the neurospheres but the neurosphere-forming cells as well. These cells have different sizes, granularity, metabolism, cytoplasmic content and are in different phases of the cell cycle. Moreover, they exhibit different features according to the layers of the spheres they occupy. The distribution of biological phenomena such as mitosis, transcription processes, apoptosis, phagocytosis and necrosis was distinctly influenced by the position cells took in the structure of the neurosphere. The cytoarchitecture of the neurosphere and the degree of cell viability and activity within it may depend on the access neurosphere-forming cells have to the culture medium and therefore on the availability of oxygen and nutrients and on the possibility to readily eliminate catabolites in the environment (culture medium). This interpretation would justify the higher cell activity (i.e., mitosis, protein syntesis) we observed at the periphery of the neurospheres where exchange of nutrients, oxygen and catabolites is facilitated. In contrast, apoptosis, phagocytosis, necrosis and low or absent mitotic and transcriptional activity are typical of the inner layers where nutritional exchanges are more difficult. This is confirmed by the higher percentage of apoptotic and necrotic cells found in neurospheres that are maintained in culture without being dissociated for 19 DIV and are larger than neurospheres dissociated after 12 DIV. The neurosphere properties described above can be interpreted as an adaptation of NSCs to in vitro culture conditions. The NSCs may organize in such clusters to survive the nonphysiological conditions of the in vitro environment. Cells probably optimise their interactions acquiring the most advantageous shape from a thermodynamycal point of view: the sphere. The neurosphere, in turn, develops a high degree of biological organization. The observed phagocytosis is probably the expression of a dynamically maintained equilibrium between the generation
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of new cells and the apoptosis or necrosis of older cells. A neurosphere may be considered a microsystem able to grow and survive until a threshold crucial point is reached when the self-restoration mechanisms fail; we can therefore consider the neurospheres an example of ‘‘environmental adaptability’’. This environmental adaptability may enable the NSCs to better express the plasticity they need for in vivo engraftment and fate conversion [4,14,20,22,26,34,44]. In light of the evidence regarding the neurosphere structure and its possible morphogenetic mechanisms, we can hypothesize that neurosphere-derived cell heterogeneity is due to their topographic distribution within the cytoarchitecture of the sphere. This morphologic heterogeneity may also be due, at least in part, to the presence of subpopulations of neurosphere-forming cells with distinct survival and proliferative behaviour. Previous studies have in fact demonstrated that NSCs are endowed with different degrees of stemness properties, as they are able to give origin to neurospheres with variable frequencies, and exhibit different EGF and bFGF responsiveness. We know, for example, that in the presence of EGF, cloned murine neurospheres are larger in diameter, whereas in the presence of bFGF, they are small and that exposure to both GFs produces both large and small spheres [46]; moreover, the EGF-responsive mouse population increases in size by asymmetric division of bFGF-responsive cells and by symmetric division of EGF responsive cells [19] EGF-, bFGF- and both GFs-responsive NSCs with different capacity to generate neurospheres have been also demonstrated in human fetal brain [34]. These cells can be also selectively isolated based on their size and PNA-binding activity or on the expression of cell surface markers such as CD133+ (CD45 , CD34 ) and CD24 (low levels) [31]. In summary, our study provides new insights into the behaviour of NSCs in vitro and the morphogenesis of neurospheres. This knowledge may help optimise methods for the isolation and in vitro expansion of NSCs toward the utilisation of these cells for in vivo studies and therapeutic applications.
Acknowledgements We thank Dr. M. Di Segni and Dr. G. Terzoli for kindly providing Fig. 3, Dr. P. Veglianese for Fig. 4A,B and Dr. S. Pozzi for Fig. 4C. The Laboratory of Neurobiology dedicates this work to its precious research scientist, Dr. Stefano Pagano, who passed away in his youth.
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