Int. J.

Devl Neuroscience 37 (2014) 94–99

Contents lists available at ScienceDirect

International Journal of Developmental Neuroscience

journal homepage: www.elsevier.com/locate/ijdevneu

Directing mouse embryonic neurosphere differentiation toward an

enriched neuronal population
Ema F. Torrado a,1 , Cátia Gomes a,1 , Gisela Santos a , Adelaide Fernandes a,b , Dora Brites a,b,∗ ,
Ana S. Falcão a,b,∗
a
Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Avenida Professor Gama Pinto, 1649-003 Lisbon, Portugal
b
Department of Biochemistry and Human Biology, Faculdade de Farmácia, Universidade de Lisboa, Avenida Professor Gama Pinto, 1649-003 Lisbon,
Portugal

a r t i c l e i n f o a b s t r a c t

Article history: Neural stem cells (NSC) are self-renewing multipotent cells that have emerged as a powerful tool to
Received 22 April 2014 repair the injured brain. These cells can be cultured as neurospheres, which are floating aggregates of
Received in revised form 30 June 2014 neural stem/progenitor cells (NSPCs). Despite their high clonal expansion capacity, it has been suggested
Accepted 1 July 2014
that in neurospheres, only a small percentage of cells are capable of proliferation and that this system
is not efficient in terms of neurogenic competence. Thus, our aim was to develop a neurosphere culture
Keywords:
method with a highly proliferative stem/progenitor cell population and particularly with a prominent
Mouse neural stem cells
neurogenic potential, surpassing some of the claimed weaknesses of the neurosphere assay. In our model,
Neuronal differentiation
Neural progenitor cells
mouse neurospheres were harvested from neural tissue at E15 and after only 4 days in vitro (DIV), we
Mouse cortex E15 neurospheres have achieved highly proliferative primary neurospheres (81% Sox2 and 76% Ki67 positive cells) and a
rather low number of cells expressing glial and neuronal markers (∼10%). After inducing differentiation,
we have attained an enriched neuronal population (45% ␤-III-tubulin positive cells at 15 DIV). Using a
simple methodology, we have developed a NSPC model that can provide a valuable source of neuronal
precursors, thus offering a potential starting point for cell replacement therapies following CNS injury.
© 2014 ISDN. Published by Elsevier Ltd. All rights reserved.

1. Introduction However, these models have proven to not accurately recapitu-

In the last 20 years, neural stem cell (NSC) research has shown
to be a promising tool for studying and elucidating the mole-
cular and cellular properties of brain development, plasticity and
regeneration, as well as an experimental basis for new biome-
dical applications. In fact, NSC can offer a renewable source for
cell replacement therapy in neurodegenerative diseases (Suter and
Krause, 2008) and may be used in assays aimed to test promising
therapeutic compounds for pharmaceutical and neurotoxic prop-
erties (Fritsche et al., 2011). In the past, the in vitro systems that
were commonly used to study neurodevelopmental processes were
restricted to transformed cell lines from humans (e.g. SH-SY5Y) or
rodents (PC12), as well as primary cultures obtained from rodent
central nervous system (CNS) or fetal tissue derived from abortions.
∗ Corresponding authors at: Research Institute for Medicines (iMed.ULisboa),
Faculdade de Farmácia, Universidade de Lisboa, Avenida Professor Gama Pinto,
1649-003 Lisbon, Portugal. Tel.: +351 2179464900; fax: +351 217946491.
E-mail addresses: dbrites@ff.ul.pt (D. Brites), asfalcao@ff.ul.pt (A.S. Falcão).
1
These authors contributed equally to this paper.
late in vivo development processes of the nervous system, such as
proliferation, migration, differentiation and synaptogenesis (Breier
et al., 2010). NSC are multipotent cells that possess the ability to
self-renew and the potential to generate the three major cell types
of the CNS, i.e., neurons, astrocytes and oligodendrocytes (Conti
and Cattaneo, 2010). Since the first studies from Temple (1989),
NSC have been isolated from several brain regions and there are
many assays for their isolation and expansion (Conti and Cattaneo,
2010). Among these, the isolation of NSC through the neurosphere
formation assay is one of the most widely used for NSC prolif-
eration. Neurospheres were first described in 1992, using both
adult and the embryonic CNS (Reynolds and Weiss, 1992; Reynolds
et al., 1992), and are defined as three dimensional structures that
grow as floating aggregates and are composed by a heteroge-
neous mixture of multipotent NSC, proliferating neural progenitor
cells (NPC) and postmitotic neurons and glia (Jensen and Parmar,
2006). The neurosphere system has been used extensively since it
is thought to provide a three-dimensional environment that can
mimic the neurogenic niche (Giachino et al., 2009). In fact, the neu-
rosphere system is considered as a valuable in vitro model to study
neurodevelopmental processes, as it is maintained under defined

http://dx.doi.org/10.1016/j.ijdevneu.2014.07.001
0736-5748/© 2014 ISDN. Published by Elsevier Ltd. All rights reserved.
 E.F. Torrado et al. / Int. J. Devl Neuroscience 37 (2014) 94–99
serum-free conditions where the environmental cues are limited
to those of the adjacent cells (Jensen and Parmar, 2006). Another
important advantage of the neurosphere system is that it has a great
passage potential as compared to adherent monocultures, enabling
large scale in vitro expansion of neural stem and progenitor cells for
potential drug screening and cell replacement therapy (Sun et al.,
2011). However, this culture method also has some disadvantages.
Thus, although it can mirror the cell heterogeneity of the in vivo NSC
niche, the three-dimensional structure of neurospheres shows no
spatial cellular organization in terms of cell types that are typical of
these niches. Another weakness of the neurosphere system is that
there are some studies claiming that only a small percentage of cells
within a neurosphere are capable of proliferation (Louis et al., 2008;
Moors et al., 2009; Pastrana et al., 2011). In addition, it is described
that this model is not particularly efficient in terms of neurogenic
competence (Breier et al., 2010; Conti et al., 2005).
Thus, our aim was to develop a mouse neurosphere culture
method that could obviate some of the flaws above indicated,
i.e., with a high proliferating stem/progenitor cell population and
an efficient neurogenic potential after inducing differentiation.
Starting from E15 mouse cortices, we have obtained highly prolif-
erative neurospheres after 4 days in vitro (DIV) that also expressed
a low amount of glial and neuronal markers. After inducing neural
differentiation with the culture medium that is used in primary
neuronal cultures (Falcão et al., 2007), this system revealed to
contain a high percentage of neuronal cells relatively to that of
astrocytes and oligodendrocytes, as compared to some of the
currently available protocols. Thus, this simple methodological
approach may be potentially useful when considering therapeu-
tic applications of neuronal replacement in neurodegenerative
diseases and CNS injury.
2. Materials and methods
2.1. Animal procedures
Animal care followed the recommendations of European Convention for the Pro-
tection of Vertebrate Animals Used for Experimental and Other Scientific Purposes
(Council Directive 86/609/EEC) and National Law 1005/92 (rules for protection of
experimental animals). All animal procedures were approved by the Institutional
Animal Care and Use Committee.
2.2. Neurospheres cultures and characterization
Neurospheres were isolated from CD1 mouse fetuses at embryonic day 15.
Briefly, pregnant mice were euthanized by asphyxiation with CO2 and the fetuses
were rapidly decapitated. After removal of meninges and white matter, the neocor-
tices were collected in Hanks’ balanced salt solution without Ca2+ and Mg2+ (HBSS,
Invitrogen, Carlsbad, USA) and transferred to trypsin–EDTA (5 g/L trypsin–2 g/L
EDTA, Sigma, St. Louis, USA) plus 1 U/mL DNAse I (Sigma) solution, for 30 min at 37 ◦ C.
Following trypsinization, cells were washed three times with HBSS, resuspended in
a final volume of RHB-A® stem cell culture medium (SCS-SF-NB-01, StemCells Inc.,
Newark, USA) and mechanically dissociated using a Pasteur pipette. Approximately
1 × 106 cells/mL were plated on tissue culture uncoated plates in the presence of
10 ng/mL murine epidermal growth factor (EGF, PeproTech, London, UK), 10 ng/mL
murine basic fibroblast growth factor (bFGF, PeproTech) and 1% antibiotic antimy-
cotic solution (AB/AM, Sigma), and maintained at 37 ◦ C in a humidified atmosphere
of 5% CO2 . Under these proliferating conditions, the cells grow as free-floating neuro-
spheres. Proliferating medium was changed after 2 DIV by mechanical dissociation
of neurospheres and replating cells on uncoated tissue culture plates at a density of
1 × 106 cells/mL. At 4 DIV, a set of neurospheres were trypsinized to obtain a suspen-
sion of dissociated cells. These cells were then plated in tissue culture plates (with
coverslips) pre-coated with poly-d-lysine (PDL, Sigma), and fixed after 4 h with 4%
paraformaldehyde (w/v) in PBS. This way, instead of having cells in the 3D structure
of neurospheres, we have isolated cells that can be characterized by immunocyto-
chemistry. Since neurospheres are a heterogeneous population of cells composed
by NSC, NPC and differentiated cells, we have used different specific antibodies
against: the Sox2 (Sex determining region of Y chromosome-related high motility
group box2) protein (1:500, ab5603, Merck Millipore, Darmstadt, Germany), a self-
renewal marker of neural stem/progenitor cells (NSPC); the glial fibrillary acidic
protein (GFAP, 1:100, MAB360, Merck Millipore), an astrocytic marker; the neu-
ronal protein HuC/D (1:750, A-21271, Molecular Probes, Carlsbad, USA), an early
postmitotic neuronal precursor marker; the ␤-III-tubulin protein (1:500, MAB1637,
95
Merck Millipore), a component of microtubules regarded as a neuron-specific
marker; the chondroitin sulfate proteoglycan NG2 (1:200, AB5320, Merck Millipore),
that stains for oligodendrocyte precursor cells; and the myelin basic protein (MBP,
1:200, MAB387, Merck Millipore), which is a marker for mature oligodendrocytes.
We have also performed a proliferation assay, by using a specific antibody against
the nuclear protein Ki67 (1:25, sc-7846, Santa Cruz Biotechnology, Dallas, USA). For
each coverslip, nuclei were stained with Hoechst dye 33258 (Sigma). All images
were acquired using a fluorescence microscope (AxioScope A1, Zeiss, Oberkochen,
Germany) with an integrated camera (AxioCamHRm, Zeiss). Immune-positive cells
of each type and total cell number (at least 1000 cells per slide) were counted and
the values were presented as percentage of positive cells for each staining ±SEM.
2.3. Differentiation of neurospheres
After 4 DIV under proliferating conditions, another set of neurospheres was
induced to differentiate by culturing dissociated neurospheres in Neurobasal
medium (Invitrogen) supplemented with 0.5 mM l-glutamine, 2% B-27 Supplement
(Invitrogen), 30 mM glucose, 50 ng/mL bovine serum albumin, 1% fetal bovine serum
and 1% AB/AM in tissue culture plates (with coverslips) pre-coated with PDL, at
1 × 105 cells/cm2 (adapted from Smith et al., 2003). Every 3 days, half of old medium
was removed by aspiration and replaced by the same volume of fresh medium. Cells
were fixed at 3, 9 and 15 DIV under differentiating conditions, and processed for
characterization by immunocytochemistry, using the same neural markers previ-
ously indicated for neurospheres.
2.4. Statistical analysis
Results of at least three different experiments, performed in duplicate, were
expressed as mean ± SEM. Data was statistically evaluated using the two-tailed t
test on the basis of equal or unequal variance, as appropriate. We considered P < 0.05
to be statistically significant.
3. Results and discussion
The RHB-A® medium used for neurospheres proliferation and
expansion allowed a fast formation of the floating aggregates as
soon as 2 DIV after plating. In fact, this medium was previously
used for a more efficient production of NPC from embryonic stem
cells in monolayer cultures (Abranches et al., 2009). In addition, the
presence of the growth factors EGF and bFGF also promoted sus-
tained self-renewing divisions of NSC (Conti et al., 2005). After 4
DIV, neurospheres were characterized in order to validate the het-
erogeneous composition of these structures. Since there are some
studies claiming that only a small percentage of cells within the
neurosphere are capable of proliferation (Louis et al., 2008; Moors
et al., 2009; Pastrana et al., 2011), we have first determined the pro-
portion of stem/progenitor cells of these tridimensional structures
by determining the number of Sox2 positive cells. Sox2 plays a cru-
cial role in embryonic development and in the developing brain its
expression is first detected uniformly in the neural plate, in which
most of the cells are multipotent, controlling the expression of se-
veral developmentally important genes (Wegner and Stolt, 2005).
In mouse embryo brains, it is mainly expressed in the expanding
cell population of the ventricular zone, in which NPC are generated
to subsequently establish the functional neocortex (Bani-Yaghoub
et al., 2006). Furthermore, it is described that this transcription fac-
tor is essential for the maintenance of NSC, since inhibition of Sox2
signaling promotes an early exit from the cell cycle, while consti-
tutive expression inhibits neuronal differentiation (Graham et al.,
2003). Generally, the majority of studies use Nestin as a NSC marker.
However, this protein is also detected in immature neurons. In
fact, it was referred that when green fluorescent protein-labeled
Nestin positive cells are sorted and tested for neurosphere forma-
tion, less than 1% of the cells were able to form neurospheres, thus
suggesting that this marker is not NSC exclusive (Mignone et al.,
2004). Our results (Fig. 1) demonstrate that about 81% of the cells
that constitute the neurospheres were Sox2-positive and the Ki67
staining confirmed that most of these cells were in a proliferat-
ing state (76 ± 2.7%). Compared with the 31% of Sox2-positive cells
obtained by (Park et al., 2007) in embryonic mice primary neuro-
spheres, our procedure present a major advantage in allowing a
96 E.F. Torrado et al. / Int. J. Devl Neuroscience 37 (2014) 94–99

Fig. 1. Expression of undifferentiated and early post-mitotic neuronal cell markers in neurospheres derived from E15 mice cortices and in resulting differentiating cells
at 3, 9 and 15 days in vitro (DIV). (A) Representative results of one experiment are shown. Nuclei were stained with Hoechst dye (blue) and cells labeled for Sox2 (neural
stem/progenitor cells in green, upper panel) and HuC/D (early post-mitotic neurons in red, lower panel). Merged images of Sox2 and HuC/D are shown between the Sox2
and HuC/D columns and arrow heads represent cells that have positive double staining. Scale bar represents 20 ␮m. (B) Graph bars represent, for each developmental stage,
the percentage of cells (mean ± SEM) positive for Sox2 and HuC/D relatively to the total number of nuclei (at least 1000 cells per slide), from at least three independent
experiments, performed in duplicate. **P < 0.01 vs. the respective neural marker expression in neurospheres. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of the article.)

very high yield in NSPC content, corroborating once more the ben-
efits of our proliferation medium and validating this protocol as
a good source of NSPC for therapeutic applications, such as cellu-
lar transplantation. After inducing cell differentiation, there was a
marked decrease in Sox2 staining (P < 0.01) to 49% at 3 DIV, 19% at
9 DIV and 14% at 15 DIV, as expected. During the differentiation
process, since neuronal cells do not express this transcription fac-
tor, this labeling corresponds to persisting NSPC and proliferating
astrocytes that are referred to maintain Sox2 expression through-
out differentiation until they reach a quiescent state (Bani-Yaghoub
et al., 2006).
Neurospheres also expressed HuC/D, which is an early postmi-
totic neuronal indicator (Fujiyama et al., 2009) and thus, a marker
of neuronal precursors (Ghai et al., 2008). For a clearer perception
of the nature of these HuC/D positive cells, we have performed a
double staining with Sox2 (Fig. 1A) and observed that there was
a percentage of these HuC/D positive cells that also express the
NSC marker (i.e. 27 ± 4.7%, 26 ± 1.1%, 34 ± 3.4% and 42 ± 6.9% from
total HuC/D cells in neurospheres, and in cells at 3, 9 and 15 DIV of
differentiation, respectively). This means that some of the HuC/D
positive cells that have just exited the cell cycle also express mark-
ers of proliferation, and thus, we can identify this population as
early neuronal progenitor cells. After inducing differentiation we
have observed a rapid increase in neuronal precursors at 3 DIV
(31% of HuC/D positive cells, P < 0.01) until 9 DIV, where it reached
the maximum value of 42% (P < 0.01). After this time point, the
percentage of HuC/D positive cells started to decrease although
neuronal maturation was still occurring, which was observed by
the increase in the number of ␤-III-tubulin positive cells (Fig. 2).
Despite several differentiation protocols using embryonic mice
primary neurospheres, where most of the works did not succeed
in obtaining more than 30% of ␤-III-tubulin positive cells (Conti
and Cattaneo, 2010; Ahlenius et al., 2009; Cavallaro et al., 2008;
Smith et al., 2003; Ostenfeld et al., 2002), we have achieved 31%
of these cells at 9 DIV (P < 0.01) further increasing to 45% at 15
DIV (P < 0.01), meaning a continuous process of neuronal matu-
ration and a promising source of viable neurons (Fig. 2). Higher
values of ␤-III-tubulin positive cells have been recently achieved
by using a shaking purification method based on the differential
substrate attachment properties of neuronal and glial cells in a dif-
ferentiating neural stem cell progeny culture (Azari et al., 2014),
or by applying more complex and costly methodologies, such as a
purification step or cell sorting technology (Azari et al., 2011, 2012).
We consider that the high neurogenic potential of our neurosphere
model relies on the differentiation medium composition. This cul-
ture medium is the same we use to obtain pure primary cultures
of neurons (Falcão et al., 2007), and where the presence of Neu-
robasal medium associated with B-27 supplement were shown to
favor neuronal maturation and survival (Svendsen et al., 1995). In
addition, due to the short proliferation period of our neurospheres,
 E.F. Torrado et al. / Int. J. Devl Neuroscience 37 (2014) 94–99 97

Fig. 2. Expression of neuronal and glial cell markers in neurospheres derived from E15 mice cortices and in resulting differentiating cells at 3, 9 and 15 days in vitro (DIV).
(A) Representative results of one experiment are shown. Nuclei were stained with Hoechst dye (blue) and cells labeled for ␤-III-tubulin (neurons in green, upper panel) and
glial fibrillary acidic protein (GFAP; astrocytes in red, lower panel). Scale bar represents 20 ␮m. (B) Graph bars represent, for each developmental stage, the percentage of
cells (mean ± SEM) positive for ␤-III-tubulin and GFAP relatively to the total number of nuclei (at least 1000 cells per slide), from at least three independent experiments,
performed in duplicate. *P < 0.05 and **P < 0.01 vs. the respective neural marker expression in neurospheres. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of the article.)

we have prevented a loss of their neurogenic potential by avoiding
long-term expansion (Wright et al., 2006).
Regarding astrocytes, most studies have demonstrated GFAP
expression on these floating cell aggregates from mouse origin,
in contrast with those from human origin which evidence weak
immunoreactivity to anti-GFAP antibodies (Sun et al., 2008). We
found that the percentage of GFAP positive cells in neurospheres
was very similar to the one obtained for neuronal precursors (∼10%,
Fig. 2), suggesting that these proliferating cells may have the same
neurogenic and astrogliogenic potential. After differentiation, this
glial phenotype reached a peak at 9 DIV (22%, P < 0.01) without any
further increase, while neuronal differentiation is still occurring.
To this it may have accounted the fact that our culture medium
was not supplemented with higher concentrations of fetal bovine
serum. Indeed, increased levels of this supplement in the differen-
tiation medium (at least ten times higher) were indicated to favor
the differentiation of stem cells into astrocytes (astrogliogenesis)
and consequently, an increase in GFAP positive cells (Brunet et al.,
2004; Abe et al., 2006).
Neurospheres have also the ability to differentiate into oligo-
dendrocytes, the myelin-forming cells of the CNS. NG2 positive cells
were shown to preferentially occupy the outer zone of the neu-
rosphere (Mokry et al., 2008) and in our model they represented
12% of all cells (Fig. 3). The presence of these oligodendrocytes
progenitors was not accompanied by the mature, MBP-positive,
myelin-generating, oligodendrocytes. This is not surprising since it
is well known that maturation of oligodendrocytes only takes place
at early postnatal life (Baumann and Pham-Dinh, 2001). In fact, it is
described that even if the neurosphere cultures were obtained from
the adult mouse hippocampus, which is considered a NSC niche
(Jhaveri et al., 2010), there is still no staining for this marker. NG2
and GFAP positive cells in percentages like our own were previously
documented in other neurosphere protocol (Fernandez et al., 2009).
Although the number of cells expressing MBP almost duplicated
from 3 to 9 DIV, we assume that our culture medium is not appro-
priate to further increment or at least to maintain constant the
number of MBP positive cells. The bovine serum albumin present in
our differentiating protocol, as well as the one in the B-27 supple-
ment, by containing some bone morphogenic protein-like activity
(Clark and Ding, 2007), may have contributed to inhibit further
oligodendrocyte precursor cell maturation (See et al., 2004). Nev-
ertheless, if desirable, both NG2 and MBP enriched cultures may be
obtained by magnetic cell sorting (Cizkova et al., 2009).
In conclusion, our data support that cell fate decisions of CNS
cells may be controlled efficiently by the presence of some extrin-
sic factors in proliferating/differentiating conditions (Johe et al.,
1996). Based on this statement, we have adapted some existing
protocols regarding neurospheres proliferation (Abranches et al.,
98 E.F. Torrado et al. / Int. J. Devl Neuroscience 37 (2014) 94–99

Fig. 3. Expression of progenitor and mature oligodendrocytes markers in neurospheres derived from E15 mice cortices and in resulting differentiating cells at 3, 9 and 15 days
in vitro (DIV). (A) Representative results of one experiment are shown. Nuclei were stained with Hoechst dye (blue) and cells labeled for NG2 (oligodendrocyte progenitor
cells, upper panel) and myelin basic protein (MBP; mature oligodendrocytes, lower panel). Scale bar represents 20 ␮m. (B) Graph bars represent, for each developmental
stage, the percentage of cells (mean ± SEM) positive for NG2 and MBP relatively to the total number of nuclei (at least 1000 cells per slide), from at least three independent
experiments, performed in duplicate. **P < 0.01 vs. the respective neural marker expression in neurospheres. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of the article.)

2009) and differentiation (Smith et al., 2003) and developed a very
simple experimental procedure with highly proliferative primary
neurospheres that in differentiating conditions ensure an enhanced
neurogenic potential. Since it is described that the proliferation of
NSPC as floating aggregates is not particularly efficient in terms
of neurogenic competence, the method here described seems use-
ful for cell differentiation into an enriched neuronal population, as
well as for studying mechanisms that underlie neurogenesis and/or
gliogenesis. More importantly, it may also be adequate to human
embryonic tissue application, as human neurosphere cultures have
a greater capacity to divide in vitro and to generate neuronal cells
(Ostenfeld et al., 2002). In fact, one of the major obstacles to the
use of NSPC in neuronal replacement therapy is the limited abil-
ity of these cells to generate sufficient number of neurons either
in vitro or after transplantation in animal models of neurodegener-
ative disease and CNS damage (Smith et al., 2003). Therefore, our
model supports neurospheres as a suitable system for regenera-
tive efforts following injury or disease, opening new avenues for
transplantation experimental approaches of functional neuronal
cells.
Acknowledgments
This work was supported by the strategic project PEst-
OE/SAU/UI4013/2011-2014 and the PTDC/SAU-NEU/64385/2006
grants from Fundação para a Ciência e a Tecnologia (FCT), Lisbon,
Portugal (to DB). ASF holds a post-doctoral research assistant posi-
tion (C2007-FFUL/UBMBE/02/2011) granted by FCT. The funding
organization had no role in study design, data collection and ana-
lysis, decision to publish, or preparation of the manuscript.
References
Abe, T., Takahashi, S., Suzuki, N., 2006. Metabolic properties of astrocytes differen-
tiated from rat neurospheres. Brain Res. 1101, 5–11.
Abranches, E., Silva, M., Pradier, L., Schulz, H., Hummel, O., Henrique, D., Bekman,
E., 2009. Neural differentiation of embryonic stem cells in vitro: a road map to
neurogenesis in the embryo. PLoS ONE 4, e6286.
Ahlenius, H., Visan, V., Kokaia, M., Lindvall, O., Kokaia, Z., 2009. Neural stem
and progenitor cells retain their potential for proliferation and differentiation
into functional neurons despite lower number in aged brain. J. Neurosci. 29,
4408–4419.
Azari, H., Osborne, G.W., Yasuda, T., Golmohammadi, M.G., Rahman, M., Deleyrolle,
L.P., Esfandiari, E., Adams, D.J., Scheffler, B., Steindler, D.A., Reynolds, B.A., 2011.
Purification of immature neuronal cells from neural stem cell progeny. PLoS ONE
6, e20941.
Azari, H., Sharififar, S., Fortin, J.M., Reynolds, B.A., 2012. The neuroblast assay: an
assay for the generation and enrichment of neuronal progenitor cells from dif-
ferentiating neural stem cell progeny using flow cytometry. J. Vis. Exp. 62, 3712.
Azari, H., Sharififar, S., Darioosh, R.P., Fortin, J.F., Rahman, M., Reynolds, B.A., 2014.
Purifying immature neurons from differentiating neural stem cell progeny using
a simple shaking method. Stem Cell Res. Ther. 4, 178.
Bani-Yaghoub, M., Tremblay, R.G., Lei, J.X., Zhang, D., Zurakowski, B., Sandhu, J.K.,
Smith, B., Ribecco-Lutkiewicz, M., Kennedy, J., Walker, P.R., Sikorska, M., 2006.
Role of Sox2 in the development of the mouse neocortex. Dev. Biol. 295, 52–66.
 E.F. Torrado et al. / Int. J. Devl Neuroscience 37 (2014) 94–99
Baumann, N., Pham-Dinh, D., 2001. Biology of oligodendrocyte and myelin in the
mammalian central nervous system. Physiol. Rev. 81, 871–927.
Breier, J.M., Gassmann, K., Kayser, R., Stegeman, H., De, G.D., Fritsche, E., Shafer, T.J.,
2010. Neural progenitor cells as models for high-throughput screens of devel-
opmental neurotoxicity: state of the science. Neurotoxicol. Teratol. 32, 4–15.
Brunet, J.F., Grollimund, L., Chatton, J.Y., Lengacher, S., Magistretti, P.J., Villemure,
J.G., Pellerin, L., 2004. Early acquisition of typical metabolic features upon dif-
ferentiation of mouse neural stem cells into astrocytes. Glia 46, 8–17.
Cavallaro, M., Mariani, J., Lancini, C., Latorre, E., Caccia, R., Gullo, F., Valotta, M., DeBi-
asi, S., Spinardi, L., Ronchi, A., Wanke, E., Brunelli, S., Favaro, R., Ottolenghi, S.,
Nicolis, S.K., 2008. Impaired generation of mature neurons by neural stem cells
from hypomorphic Sox2 mutants. Development 135, 541–545.
Cizkova, D., Cizek, M., Nagyova, M., Slovinska, L., Novotna, I., Jergova, S., Radonak,
J., Hlucilova, J., Vanicky, I., 2009. Enrichment of rat oligodendrocyte progenitor
cells by magnetic cell sorting. J. Neurosci. Methods 184, 88–94.
Clark, J.H., Ding, S., 2007. Chemically-defined culture of embryonic stem cells. In: Sul-
livan, C., Chad, A.C., Eggan, K. (Eds.), Human Embryonic Stem Cells: The Practical
Handbook, vol. 918. Wiley, West Sussex, pp. 81–90.
Conti, L., Cattaneo, E., 2010. Neural stem cell systems: physiological players or
in vitro entities? Nat. Rev. Neurosci. 11, 176–187.
Conti, L., Pollard, S.M., Gorba, T., Reitano, E., Toselli, M., Biella, G., Sun, Y., Sanzone,
S., Ying, Q.L., Cattaneo, E., Smith, A., 2005. Niche-independent symmetrical self-
renewal of a mammalian tissue stem cell. PLoS Biol. 3, e283.
Falcão, A.S., Silva, R.F.M., Pancadas, S., Fernandes, A., Brito, M.A., Brites, D., 2007.
Apoptosis and impairment of neurite network by short exposure of immature
rat cortical neurons to unconjugated bilirubin increase with cell differentiation
and are additionally enhanced by an inflammatory stimulus. J. Neurosci. Res. 85,
1229–1239.
Fernandez, M., Paradisi, M., Del, V.G., Giardino, L., Calza, L., 2009. Thyroid hormone
induces glial lineage of primary neurospheres derived from non-pathological
and pathological rat brain: implications for remyelination-enhancing therapies.
Int. J. Dev. Neurosci. 27, 769–778.
Fritsche, E., Gassmann, K., Schreiber, T., 2011. Neurospheres as a model for develop-
mental neurotoxicity testing. Methods Mol. Biol. 758, 99–114.
Fujiyama, T., Yamada, M., Terao, M., Terashima, T., Hioki, H., Inoue, Y.U., Inoue, T.,
Masuyama, N., Obata, K., Yanagawa, Y., Kawaguchi, Y., Nabeshima, Y., Hoshino,
M., 2009. Inhibitory and excitatory subtypes of cochlear nucleus neurons are
defined by distinct bHLH transcription factors, Ptf1a and Atoh1. Development
136, 2049–2058.
Ghai, K., Stanke, J.J., Fischer, A.J., 2008. Patterning of the circumferential marginal
zone of progenitors in the chicken retina. Brain Res. 1192, 76–89.
Giachino, C., Basak, O., Taylor, V., 2009. Isolation and manipulation of mammalian
neural stem cells in vitro. Methods Mol. Biol. 482, 143–158.
Graham, V., Khudyakov, J., Ellis, P., Pevny, L., 2003. SOX2 functions to maintain neural
progenitor identity. Neuron 39, 749–765.
Jensen, J.B., Parmar, M., 2006. Strengths and limitations of the neurosphere culture
system. Mol. Neurobiol. 34, 153–161.
Jhaveri, D.J., Mackay, E.W., Hamlin, A.S., Marathe, S.V., Nandam, L.S., Vaidya, V.A.,
Bartlett, P.F., 2010. Norepinephrine directly activates adult hippocampal pre-
cursors via beta3-adrenergic receptors. J. Neurosci. 30, 2795–2806.
Johe, K.K., Hazel, T.G., Muller, T., Dugich-Djordjevic, M.M., McKay, R.D., 1996. Single
factors direct the differentiation of stem cells from the fetal and adult central
nervous system. Genes Dev. 10, 3129–3140.
99
Louis, S.A., Rietze, R.L., Deleyrolle, L., Wagey, R.E., Thomas, T.E., Eaves, A.C., Reynolds,
B.A., 2008. Enumeration of neural stem and progenitor cells in the neural colony-
forming cell assay. Stem Cells 26, 988–996.
Mignone, J.L., Kukekov, V., Chiang, A.S., Steindler, D., Enikolopov, G., 2004. Neural
stem and progenitor cells in nestin-GFP transgenic mice. J. Comp. Neurol. 469,
311–324.
Mokry, J., Karbanova, J., Filip, S., Cizkova, D., Pazour, J., English, D., 2008. Phenotypic
and morphological characterization of in vitro oligodendrogliogenesis. Stem
Cells Dev. 17, 333–341.
Moors, M., Rockel, T.D., Abel, J., Cline, J.E., Gassmann, K., Schreiber, T., Schuwald, J.,
Weinmann, N., Fritsche, E., 2009. Human neurospheres as three-dimensional
cellular systems for developmental neurotoxicity testing. Environ. Health Per-
spect. 117, 1131–1138.
Ostenfeld, T., Joly, E., Tai, Y.T., Peters, A., Caldwell, M., Jauniaux, E., Svendsen, C.N.,
2002. Regional specification of rodent and human neurospheres. Brain Res. Dev.
Brain Res. 134, 43–55.
Park, J.H., Ahn, J.I., Kim, S.Y., Park, K.S., Lee, Y.D., Yamaguchi, M., Chung, H.J., 2007.
Genetic modification does not affect the stemness of neural stem cells in nestin
promoter-GFP transgenic mice. Neurosci. Lett. 421, 185–190.
Pastrana, E., Vargas, V.S., Doetsch, F., 2011. Eyes wide open: a critical review of
sphere-formation as an assay for stem cells. Cell Stem Cell 8, 486–498.
Reynolds, B.A., Tetzlaff, W., Weiss, S., 1992. A multipotent EGF-responsive striatal
embryonic progenitor cell produces neurons and astrocytes. J. Neurosci. 12,
4565–4574.
Reynolds, B.A., Weiss, S., 1992. Generation of neurons and astrocytes from isolated
cells of the adult mammalian central nervous system. Science 255, 1707–1710.
See, J., Zhang, X., Eraydin, N., Mun, S.B., Mamontov, P., Golden, J.A., Grinspan, J.B.,
2004. Oligodendrocyte maturation is inhibited by bone morphogenetic protein.
Mol. Cell. Neurosci. 26, 481–492.
Smith, R., Bagga, V., Fricker-Gates, R.A., 2003. Embryonic neural progenitor cells: the
effects of species, region, and culture conditions on long-term proliferation and
neuronal differentiation. J. Hematother. Stem Cell Res. 12, 713–725.
Sun, T., Wang, X.J., Xie, S.S., Zhang, D.L., Wang, X.P., Li, B.Q., Ma, W., Xin, H., 2011.
A comparison of proliferative capacity and passaging potential between neu-
ral stem and progenitor cells in adherent and neurosphere cultures. Int. J. Dev.
Neurosci. 29, 723–731.
Sun, Y., Pollard, S., Conti, L., Toselli, M., Biella, G., Parkin, G., Willatt, L., Falk, A.,
Cattaneo, E., Smith, A., 2008. Long-term tripotent differentiation capacity of
human neural stem (NS) cells in adherent culture. Mol. Cell. Neurosci. 38,
245–258.
Suter, D.M., Krause, K.H., 2008. Neural commitment of embryonic stem cells:
molecules, pathways and potential for cell therapy. J. Pathol. 215, 355–368.
Svendsen, C.N., Fawcett, J.W., Bentlage, C., Dunnett, S.B., 1995. Increased survival of
rat EGF-generated CNS precursor cells using B27 supplemented medium. Exp.
Brain Res. 102, 407–414.
Temple, S., 1989. Division and differentiation of isolated CNS blast cells in microcul-
ture. Nature 340, 471–473.
Wegner, M., Stolt, C.C., 2005. From stem cells to neurons and glia: a Soxist’s view of
neural development. Trends Neurosci. 28, 583–588.
Wright, L.S., Prowse, K.R., Wallace, K., Linskens, M.H., Svendsen, C.N., 2006. Human
progenitor cells isolated from the developing cortex undergo decreased neuro-
genesis and eventual senescence following expansion in vitro. Exp. Cell Res. 312,
2107–2120.