Neurosphere and neurosphere-forming cells: morphological and ultrastructural characterization

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Brain Research 993 (2003) 18–29

Research report

Neurosphere and neurosphere-forming cells:

morphological and ultrastructural characterization

Alessandra Beza, Elena Corsinia, Daniela Curtib, Marco Biggiogerac, Augusto Colombod,Roberto Francesco Nicosiae,f, Stefano Filippo Paganoa, Eugenio Agostino Paratia,*

aLaboratory of Neurobiology, Department of Neurobiology and Neurorestorative Therapies, National Neurological Institute ‘‘C. Besta’’,

Via Celoria 11, 20133 Milan, ItalybDepartment of Physiological Science and Cellular and Molecular Pharmacology, University of Pavia, Pavia, Italy

cDepartment of Animal Biology, University of Pavia, Pavia, Italyd Institute for Obstetric and Gynaecology ‘‘L. Mangiagalli’’, ICP, Milan, Italy

eVeterans Affairs Puget Sound Health Care System, Seattle, WA, USAfDepartment 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 neurosphere-

forming 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 brains were later found to contain neural stem cells

Since they were first described in the mouse brain,

neural stem cells (NSCs) have been the subject of inten-

sive 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

0006-8993/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2003.08.061

* Corresponding author. Tel.: +39-2-2394387; fax: +39-2-270638217.

E-mail address: [email protected] (E.A. Parati).

[2,3,8,12,24,30,31].

Various methods have been developed to isolate NSCs

and characterize their capacity to proliferate and differen-

tiate [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 conver-

sion/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-

A. Bez et al. / Brain Research 993 (2003) 18–29 19

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 fea-

tures and metabolic properties, which are largely un-

known, 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 previ-

ously 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 com-

mitments of parental clone-forming cells [36]. A gap,

however, exists in our understanding of the cytoarchitec-

ture 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 neuro-

spheres 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 nour-

ished 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 fol-

lowing 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 recombi-

nant) 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-

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 cryopre-

served. They maintain multipotentiality even after several

passages in vitro, as demonstrated by clonal analysis, and

can differentiate into neurons, astrocytes and oligodendro-

cytes [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 glu-

cose, 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 car-

bocianine (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 mito-

chondrial 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 neuro-

sphere. 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

A. Bez et al. / Brain Research 993 (2003) 18–2920

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 ana-

lysed 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

Fig. 1. Phase contrast images of neurospheres (A–C) and confocal image of neu

conditions in presence of EGF and bFGF. (A) Neurosphere with irregular rim

Neurosphere with regular rim and well defined spherical shape. (C) Neurosphere a

even though it is cultured in serum-free medium supplemented with GFs (not differ

again a few days later. (D) Neurosphere-derived cells stained intravitally with JC

mitochondrial membrane depolarisation. Cytoplasmic staining obtained with this

kinds of cells (b and c) can be identified based on size, amount of cytoplasm and m

and less frequently observed. Bars: 50 Am (A–C) or 12 Am (D).

were incubated for 1 h at 4 jC with propidium iodide

(50 Ag/ml), resuspended in a lysis-containing buffer con-

taining 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 resus-

pended 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.

rosphere-derived NSCs (D). Neurospheres were cultured under serum-free

and cilia-like cytoplasmic processes protruding from its surface (a). (B)

dherent to an uncoated well exhibits pseudopod-like cytoplasmic processes

entiating culture conditions); this neurosphere detached and became floating

-1, a cationic dye that shifts from red to green fluorescence as a result of

dye demonstrates morphological heterogeneity of NSCs. In particular, two

itochondrial distribution. Other cellular phenotypes are more heterogeneous


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, resus-

pended in 190 Al of binding buffer, mixed with 10 Al ofpropidium 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 contain-

ing 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 per-

formed by treating the cultures with 2 M HCl for 1 h. After

Fig. 2. Confocal micrograph of neurosphere double stained with the SYTO 59 vi

SYTO 59 is a lipophilic dye capable of labeling nuclei of viable cells. Propidium io

and no longer viable. Blue cells are therefore healthy whereas red cells are suffering

Bar: 50 Am.

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 dissocia-

tion of the neurospheres. The cells used in all the experi-

ments 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 experi-

ments, 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.

tal nuclear dye (blue) and propidium iodide (red) to estimate cell viability.

dide penetrates the cell membrane and labels nuclei only if cells are injured

or dead. Viable cells are mostly located at the periphery of the neurosphere.

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 con-

ditions. 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 prolifer-

ation 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.

Fig. 4. Fluorescent (A) and phase contrast (B) images of paraformaldehyde-fixed n

light micrographic image of BrdU-labeled neurosphere spread over Matrigel subst

the neurosphere. BrdU labeling demonstrates DNA synthesis in the majority of c

Morphological heterogeneity was a peculiarity of neuro-

sphere-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

eurospheres labeled with propidium iodide to highlight nuclear details, and

rate (C). The box in A and B marks a telophase occurring on the surface of

ells occupying the outer layers of the neurosphere. Bars: 50 Am.

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.

Fig. 6. Electron micrographs of neurosphere cells. Adjacent cells of the neur

demonstrated in the neurosphere cell cytoplasm (B). Bars: 5 Am.

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:

osphere are joined by a gap-junction (A, inset). Lysosomes (arrow) are

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 mono-

layer of cells except for its center that, even if flat, was

always composed of several cell layers. Only peripheral

Fig. 8. Confocal images of NSCs stained with the intravital dye JC-1 to highlig

differentiate into oligodendrocytic (A), astrocytic (B) and neuronal (C) phenotype

cells, which as a consequence of the contact with an

adhesive matrix spread forming pseudopodia-like cytoplas-

mic processes, stained for BrdU, that is, were actively

synthesizing DNA after 24 h of incubation with BrdU

ht morphologic features. NSCs, under appropriate culture conditions, may

s.

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.

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 phago-

cytic 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 neuro-

sphere was quantified by cytofluorimetric analysis using a

human Annexin V-FITC Kit. Neurospheres maintained in

culture for 12 and 19 DIV, which corresponded to neuro-

spheres 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, demon-

strated a high trascriptional activity in peripheral cells and a

definitely reduced one in the inner ones (Fig. 7). Neuro-

sphere-forming cells were connected by weak junctional

complexes (Fig. 6). Desmosoms were never observed and

there was no evidence of fusion of plasmalemmas.

3.2. Cells dissociated from the neurospheres

The second part of our study was carried out on NSCs

obtained by mechanical dissociation of neurospheres.

NSCs appeared as spherical and floating cells after

dissociation but, under appropriate culture conditions, they

differentiated into oligodendrocytic, astrocytic or neuronal

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.

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 neuro-

sphere dissociation, more than 80% of the recovered cells

were viable. Some cells had many energized mitochon-

dria, 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 mitochon-

drial calcium signal was higher in cells with very low

cytoplasmic calcium (data not shown). The FSC/SSC

profile of NSCs confirmed the heterogeneity of neuro-

sphere-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


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 exter-

nal medium. Smaller spheres, on the other hand, are

translucent, have no dark cores and appear much health-

ier. 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 con-

tent and are in different phases of the cell cycle. More-

over, they exhibit different features according to the

layers of the spheres they occupy. The distribution of

biological phenomena such as mitosis, transcription pro-

cesses, 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 periph-

ery of the neurospheres where exchange of nutrients,

oxygen and catabolites is facilitated. In contrast, apopto-

sis, 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 thermody-

namycal 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

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 con-

sider 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 cytoarch-

itecture of the sphere. This morphologic heterogeneity may

also be due, at least in part, to the presence of subpopula-

tions of neurosphere-forming cells with distinct survival and

proliferative behaviour. Previous studies have in fact dem-

onstrated 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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Neurosphere and neurosphere-forming cells: morphological and ultrastructural characterization

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Transcript of Neurosphere and neurosphere-forming cells: morphological and ultrastructural characterization