Embryonic Stem-Derived Versus Somatic Neural Stem Cells: A Comparative Analysis of Their Developmental Potential and Molecular Phenotype

Article (PDF Available)inStem Cells 24(4):825-34 · May 2006with28 Reads
DOI: 10.1634/stemcells.2005-0313 · Source: PubMed
Reliable procedures to induce neural commitment of totipotent undifferentiated embryonic stem (ES) cells have provided new tools for investigating the molecular mechanisms underlying cell fate choices. We extensively characterized the developmental potential of ES-induced neural cells obtained using an adaptation of the multistep induction protocol. We provided evidence that ES-derived neural proliferating cells are endowed with stem cell properties such as extensive self-renewal capacity and single-cell multipotency. In differentiating conditions, cells matured exclusively into neurons, astrocytes, and oligodendrocytes. All these features have been previously described in only somatic neural stem cells (NSCs). Therefore, we consider it more appropriate to rename our cells ES-derived NSCs. These similarities between the two NSC populations induced us to carefully compare their proliferation ability and differentiation potential. Although they were very similar in overall behavior, we scored specific differences. For instance, ES-derived NSCs proliferated at higher rate and consistently generated a higher number of neurons compared with somatic NSCs. To further investigate their relationships, we carried out a molecular analysis comparing their transcriptional profiles during proliferation. We observed a large fraction of shared expressed transcripts, including genes previously described to be critical in defining somatic NSC traits. Among the genes differently expressed, candidate genes possibly responsible for divergences between the two cell types were selected and further investigated. In particular, we showed that an enhanced MAPK (mitogen-activated protein kinase) signaling is acting in ES-induced NSCs, probably triggered by insulin-like growth factor-II. This may contribute to the high proliferation rate exhibited by these cells in culture.
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Available from: Sergio Ferrari, Sep 26, 2014
Embryonic Stem–Derived Versus Somatic Neural Stem Cells: A
Comparative Analysis of Their Developmental Potential and
Molecular Phenotype
Stem Cell Research Department, San Raffaele Scientific Institute, Milan, Italy;
Department of Biomedical
Sciences, University of Modena and Reggio Emilia, Modena, Italy;
National Institute for Cancer Research,
Genoa, Italy;
Department of Oncology, Biology and Genetics, Genova University Medical School, Genova, Italy
Key Words. Neural stem cell Embryonic stem cell Neural differentiation Self-renewal Multipotency
Transcriptional profile
Reliable procedures to induce neural commitment of totipotent
undifferentiated embryonic stem (ES) cells have provided new
tools for investigating the molecular mechanisms underlying
cell fate choices. We extensively characterized the developmen-
tal potential of ES-induced neural cells obtained using an ad-
aptation of the multistep induction protocol. We provided ev-
idence that ES-derived neural proliferating cells are endowed
with stem cell properties such as extensive self-renewal capac-
ity and single-cell multipotency. In differentiating conditions,
cells matured exclusively into neurons, astrocytes, and oligo-
dendrocytes. All these features have been previously described
in only somatic neural stem cells (NSCs). Therefore, we con-
sider it more appropriate to rename our cells ES-derived
NSCs. These similarities between the two NSC populations
induced us to carefully compare their proliferation ability and
differentiation potential. Although they were very similar in
overall behavior, we scored specific differences. For instance,
ES-derived NSCs proliferated at higher rate and consistently
generated a higher number of neurons compared with somatic
NSCs. To further investigate their relationships, we carried out
a molecular analysis comparing their transcriptional profiles
during proliferation. We observed a large fraction of shared
expressed transcripts, including genes previously described to
be critical in defining somatic NSC traits. Among the genes
differently expressed, candidate genes possibly responsible for
divergences between the two cell types were selected and fur-
ther investigated. In particular, we showed that an enhanced
MAPK (mitogen-activated protein kinase) signaling is acting in
ES-induced NSCs, probably triggered by insulin-like growth
factor–II. This may contribute to the high proliferation rate
exhibited by these cells in culture. S
TEM CELLS 2006;24:
825– 834
Stem cells are defined by three fundamental features: the ability
to self-renew, to give rise to a differentiated progeny, and to
maintain these features over a long period of time [1, 2, 3]. In
particular, embryonic stem (ES) cells are cell lines derived from
the inner cell mass of the developing blastocyst, which generates
all the different cell types of the future organism [4]. During
development, the cells of the inner cell mass generate the three
germinal layers of the embryos and, conceivably, will then give
rise to tissue-restricted stem cells that generate the organs and
tissues of the future organism. With this in mind, it may be
possible to explore in vitro the conditions by which ES totipo-
tent cells can be induced to give rise to a specific population of
tissue-restricted stem cells able to grow and differentiate. Over
the years, several in vitro systems have been described for the
derivation of neural progeny from ES cells. Notably, some of
these allow the generation of mature cells through an interme-
diate step represented by progenitor cells able to grow for some
time in vitro and generate differentiated progeny upon changes
in culture conditions [5–7]. These protocols have been widely
used to generate a variety of differentiated neural cell types such
as astrocytes, oligodendrocytes, and glutamatergic, GABAergic,
Correspondence: Vania Broccoli, Ph.D., Stem Cell Research Department, Dipartmento di Biotecnologie, San Raffaele Scientific
Institute, Via Olgettina 58, 20132 Milan, Italy. Telephone: 39 02 26434612; Fax: 39 02 26434621; e-mail: broccoli.vania@
hsr.it Received July 12, 2005; accepted for publication December 5, 2005; first published online in S
December 9, 2005. ©AlphaMed Press 1066-5099/2006/$20.00/0 doi: 10.1634/stemcells.2005-0313
STEM CELLS 2006;24:825– 834 www.StemCells.com
and dopaminergic neurons [8, 9]. However, the nature of the in
vitro– derived neural progenitors is still elusive, and a better
characterization of their self-renewal features and neural poten-
tial over time is desirable.
Somatic neural stem cells (NSCs) can be retrieved from the
embryonic neural tissue or from the neurogenetic regions of the
adult brain (subependymal layer and dentate gyrus) and can
self-renew in vitro for a long period of time and differentiate
into neurons, astrocytes, and oligodendrocytes [10, 11]. Their
self-renewal features have been clearly characterized using clo-
nogenic assays to evaluate single-cell potentialities [12, 13]. ES
cell– derived neural progenitors, on the other hand, have not
been studied as well, and their clonogenic potentials are still
unknown. Moreover, a thorough comparison between ES cell–
derived neural progenitors and somatic NSCs in terms of stable
growth, differentiation potential, and overall gene expression is
still missing. To fill this gap, we studied a population of ES-
derived cells that showed all the cardinal properties of stem cells
and thus defined ES-derived NSCs. Moreover, we extensively
compared them with NSCs isolated from embryonic neural
tissue, in terms of proliferation ability, neural differentiation
potential, and overall gene expression.
Materials and methods are available as supplemental online
Derivation of Homogenous and Stably Proliferating
Neural Progenitors from ES Cells
To differentiate ES cells toward a neural phenotype, we em-
ployed a protocol based on a three-step procedure established by
Okabe et al. [1] and Lee et al. [5], with a number of modifica-
tions. Briefly, whole undifferentiated ES cell colonies were
isolated from the fibroblast feeder layers by dispase treatment
and re-plated in bacteriological plates containing ES cell me-
dium with 20% serum replacement to obtain embryoid bodies
(EBs). After 4 days, EBs were transferred in matrigel-coated
plates and cultured in a serum-free medium enriched in insulin
(0.025 mg/ml) and transferrin (100 ug/ml) and completed with
fibroblast growth factor (FGF-2) (10 ng/ml) and epidermal
growth factor (EGF) (20 ng/ml) (see Materials and Methods).
This medium is normally used for selection and maintenance of
NSCs isolated from embryonic and adult brains [12, 14]. Fur-
thermore, matrigel-coated dishes allowed a better cell attach-
ment and spreading than did gelatin, used in previous studies.
Within 5 days, a homogenous population of cells expressing
the neural precursor markers Nestin and Vimentin had grown
[15, 16] (Fig. 1C–1E). The overall procedure required 8 days,
thus shortening the period of time needed to obtain neural
commitment as compared with the protocol of Okabe et al. After
the first selection phase, we were able to maintain stable cell
growth of undifferentiated neural progenitors in NSC culture
medium for several months.
To monitor neural induction in our cells, we tested a number
of neural-specific genes by reverse transcription–polymerase
chain reactions (RT-PCRs). Interestingly, the expression of mo-
lecular markers typical of totipotent ES cells such as Oct4,
Cripto, and Nanog was not detected in the neural progenitor
cultures (Fig. 1G; data not shown), whereas genes specific to
somatic NSCs, such as Sox2, and forebrain-specific genes such
as Emx2, Pax6, Lhx2, and Foxg1 were activated and detectable
at similar levels (Fig. 1G) [17]. Cultures of ES cell– derived
neural progenitors were stable for months without undergoing
either senescence or growth factor–independent proliferation.
They showed a stable doubling time (approximately 24 hours)
and a normal caryotype (2n 40) as tested up to 17 passages,
spanning 1 month of in vitro culture (Fig. 1B, 1F).
ES cell– derived neural progenitors were differentiated by
sequential removal of FGF-2 and EGF and finally switched to a
hippocampal culture medium (see Materials and Methods) [18].
Neural progenitors exposed to differentiating conditions com-
pleted their maturation within 9 days.
-III-Tubulin and micro-
tubule associated protein 2 (MAP-2)–positive neurons, glial
fibrillary acidic protein (GFAP)–positive astrocytes, and galac-
tocerebrosidase (GALC)–positive oligodendrocytes were
present in a ratio of 54% 4%, 34% 5%, and 12% 2%
(200 total cells counted per field, n 3), respectively (Fig. 2C).
Most of the neurons presented a bipolar shape, whereas a minor
fraction showed a multipolar morphology (Fig. 2D, 2E). Differ-
entiated cultures contained different types of neurons, including
glutamatergic (Fig. 2J), GABAergic (Fig. 2H), dopaminergic
(Fig. 2K), and cholinergic neurons (Fig. 2L) in a ratio of 53%
6%, 37 5%, 6% 4%, and 4% 2% (200 cells, n 3),
Figure 1. Establishment of homogenous cultures of ES cell– derived
NPs. (A): Outline of the overall protocol based on three major steps: I,
embryoid body formation; II, plating in matrigel-coated dishes and
cultured with NSC medium; and III, disaggregation after amplification.
(B): Determination of the cell growth in a window of 1 month. (C):
Phase-bright microphotograph showing the general morphology of the
ES-derived NSCs in their undifferentiated state. Immunofluorescence
for nestin (D) and vimentin (E) on proliferating ES-derived cells. (F):
Normal karyotype (2n 40) isolated from P17 cell cultures. (G):
Reverse transcription–polymerase chain reaction analysis of a series of
molecular markers of naı¨ve ES cells (Oct4) and forebrain neural stem
cells (Sox2, Emx2, Pax6, Lhx2, and Foxg1). Abbreviations: dNP, dif-
ferentiated embryonic stem cell– derived neural progenitors; EGF, epi-
dermal growth factor; ES, embryonic stem; FGF, fibroblast growth
factor; KSR, knockout serum replacement; LIF, leukemia inhibitory
factor; NP, neural progenitor; NSC, neural stem cell.
826 Characterization of ES-Derived Neural Stem Cells
respectively (Fig. 2F). Interestingly, we noted the widespread
formation of many synaptic contacts among neurons as assessed
by synapsin-I immunofluorescence (Fig. 2I). This molecule
reveals the organization of neural synapses in mature differen-
tiated neurons. Furthermore, we asked whether ES cell– derived
neural progenitor differentiation could be manipulated adding
specific growth factors or cytokines both in early cultures.
Remarkably, retinoic acid (RA) supplementation (5
M) dras-
tically strengthened the overall neuronal differentiation, reduc-
ing astrocyte formation, whereas a combination of RA (0.2
and Sonic Hedgehog (Shh-N, 300 nM) led to a detectable
increase in the amount of cholinergic neurons (from 5% 2%
to 13% 2%) (120 cells, n 3) (supplemental online Fig. 1).
Similar results were obtained differentiating ES cell– derived
cultures from late passages (P16). Thus, long-term cultures of
the ES cell– derived neural progenitors did not lose their ability
to respond to external cues, modifying their differentiation
potential accordingly. Notably, isolation and differentiation of
ES-derived NSCs were accomplished using four different ES
cell lines (TVB2, R1, YC5, and D3), indicating a widespread
applicability of this protocol to ES cells [19].
To evaluate the in vivo differentiation potential of ES cell–
derived precursors, we took advantage of the YC5 ES cell line
that constitutively expresses enhanced green fluorescent protein
(eGFP) [20]. Neural precursors derived from this line showed
stable growth and multilineage differentiation in vitro. We trans-
planted the cells into the lateral ventricles of embryonic day 14.5
(E14.5) mouse embryos, and results were analyzed between P1
and P5. A diffuse integration of eGFP cells was normally
observed in the neural parenchyma of different regions, depend-
ing on the area of graft integration (supplemental online Fig. 2).
Generally, the lateral cortex, the olfactory bulbs, and the ventral
forebrain showed many integrated eGFP cells. In particular, in
the lateral cortex, many GFP cells were detected in all the layers
and spread along the dorsal-ventral axis (supplemental online
Fig. 2). eGFP integrated cells expressed markers of differenti-
ated neurons and astrocytes as assessed by
-III-tubulin or
NeuN and GFAP immunofluorescence, respectively (supple-
mental online Fig. 2). In all the experiments analyzed (n 3),
neurons, astrocytes, and oligodendrocytes were scored, but the
ratio among the different cell types was highly variable, depend-
ing on the injection site.
ES Cell–Derived Neural Progenitors Are Clonogenic
and Multipotent
Clonal analysis is a stringent paradigm for testing multipotency
at the single-cell level. Although it has been extensively used in
the NSC field, it has rarely been applied to ES cell– derived
neural cultures. Individual ES-derived cells from different pas-
sages (P3, P7, and P11) were transferred into four-well chamber
slides (1 cell/1 well) pre-coated with matrigel. In the presence of
FGF-II/EGF, approximately 5% of these single cells prolifer-
ated, giving rise to a clone in approximately 3 weeks (Fig.
3A–3D). Upon differentiation, cells were tested for
lin, GFAP, and GalC immunofluorescence (Fig. 3E–3H). In all
cases (n 12), immunostainings revealed neural tri-lineage
commitment, confirming the multipotent nature of the clone
founder cells. The percentages of mature cell progenies (neu-
rons, astrocytes, and oligodendrocytes) over the whole differ-
entiated population derived from either a single clone or the
sister uncloned culture were very similar (Fig. 3I). This indi-
cated that each individual clonal culture maintained its differ-
entiation potentialities with no alteration. Finally, we tested the
growth rate of the three cultures derived by single-cell expan-
sion (no. 35 from P3, no. 102 from P7, and no. 155 from passage
P11) in comparison with their sister bulk cultures. The four
cultures exhibited a similar growth rate when measured for a
period of 4 weeks (Fig. 3J). These results suggest that the ES
cell– derived progenitors exhibit self-renewal features as tested
with stringent criteria.
Taken together, these data demonstrate that ES cell– derived
neural progenitors are self-renewing, multipotent, and clono-
genic and maintain these potentialities for a long period of time
in vitro. In conclusion, neural precursors derived from ES cells
are endowed with all the cardinal features of stem cell lines and
thus represent bona fide NSCs. We thus renamed them ES-
derived NSCs. This finding cleared the way for a comparison of
the cellular and molecular phenotypes of these two NSC lines in
similar experimental conditions.
Figure 2. Differentiation protocol and phenotypes of embryonic stem
(ES)-derived neural cells. (A, B): Low magnification of differentiated
cell cultures stained with
-III-tubulin (neurons) and GFAP (astrocytes).
(C): Quantitative analysis of neurons, glia, and oligodendroglia in the
differentiated cells. (D, E): Morphological characterization of the ES
cell– derived neurons stained with NF160 and MAP2. (F): Quantitative
analysis of the different types of neurons according to their neurotrans-
mitter content. (G, G): Mature oligodendrocyte highlighted in the ES
cell– derived cultures by GalC and O4 antibody stainings, respectively.
(H, J, K, L): Immunocytochemistry for neuron subtype–specific mark-
ers in the differentiated cell cultures using antibodies against GABA
(H), glutamate (J),TH(K), and ChAT (L). (I): Double staining for
synaptophysin (green) and
-III-tubulin (red) to assess the formation of
synaptic buttons in neurites of ES cell– derived neurons. Abbreviations:
-III-tubulin; ChAT, choline acetyltransferase; GABA,
nobutyric acid; GalC, galactocerebroside; GFAP, glial fibrillary acidic
protein; MAP2, microtubule associated protein 2; TH, tyrosine hydrox-
827Colombo, Giannelli, Galli et al.
ES-Derived NSCs and Somatic NSCs Compared
with In Vitro Phenotypes
We isolated somatic NSCs from forebrain regions of E14.5
mouse embryos to compare in vitro growth and differentiation
potential of somatic and ES-derived NSCs. Therefore, we main-
tained both cell lines in the same culture medium (see Materials
and Methods) and compared cells at the same passages (between
P8 and P14).
The two cell lines grew in different ways. Whereas somatic
NSCs proliferated better as clusters of cells floating freely in the
medium, generally called neurospheres, the ES-derived NSCs
grew optimally when adhering to a substrate (supplemental
online Fig. 3). In fact, ES-derived NSCs were unable to form
growing “spheres,” and their growth was impaired when unable
to attach to a substrate (data not shown). Under optimal condi-
tions for each of the cultures and using the same culture me-
dium, cells showed a different proliferation rate at similar pas-
sage numbers. In fact, ES-derived NSCs showed a two-time log
10 increase with respect to embryonic somatic NSCs after 6
passages, spanning less that 1 month of in vitro culture
(supplemental online Fig. 3). Then, we analyzed their differen-
tiation potential. Both cell lines were plated on laminin-coated
dishes and exposed to similar differentiation conditions (see
Materials and Methods). After 8 days, cells were processed for
immunofluorescence to detect neurons (
-III-tubulin), astroglia
(GFAP), and oligodendroglia (GalC). In all cases analyzed (n
18), the proportion of neuronal and glial cells was different
between the two cell lines. In fact, differentiated progenies
derived from either ES-derived or somatic NSCs contained 54%
4% and 40% 3% of neurons, 36% 2% and 52% 3%
of astrocytes, and 10% 3% and 8% 3% of oligodendro-
cytes, respectively (200 cells, n 5) (Fig. 4). Furthermore, we
assessed the differentiation potential of NSCs derived from
adult brains in similar conditions and we observed a further
general decrease in neuronal cell production (28% 4%) and a
corresponding enrichment in astrocytes (64% 5%) (200 cells,
n 5) (Fig. 4C, 4F, 4H). Overall, ES-derived NSCs displayed
a higher efficiency in neuronal differentiation compared with
somatic NSCs. We then assessed the neuronal subtype compo-
sition of the mature neurons in embryonic NSCs and ES-derived
NSCs. In both cases, glutamatergic neurons were the most
abundant phenotype (69% 6% and 53% 4%) (200 cells, n
5), whereas GABAergic neurons represented 29% 3% and
37% 3% of the entire differentiated cell population, respec-
tively (n 5) (supplemental online Fig. 4). However, only in
the mature progeny derived by ES-derived NSCs was it possible
to observe a portion of dopaminergic (6% 4%) and cholin-
ergic (4% 3%) neurons (200 cells, n 5) (Fig. 2I; supple-
mental online Fig. 4).
Figure 3. Clonogenic assays and analysis of bulk and cloned cell cultures. (A–D): Bright-field pictures of a growing clone. (E–H): Immunoreactivity
for markers of neuronal (
-III-tubulin [E, G]), astroglial (GFAP [F, G]), and oligodendroglial (GalC [H]) fate of a clone derived by single-cell
expansion. (I): Characterization of the mature phenotypes observed in both bulk and cloned cell cultures (nos. 35, 102, and 135). (J): Compared
analysis of the growth rate of the bulk and three cloned cell cultures over a period of 3 weeks. Abbreviations:
-III-tubulin; GalC,
galactocerebroside; GFAP, glial fibrillary acidic protein.
828 Characterization of ES-Derived Neural Stem Cells
ES-Derived NSCs and Somatic NSCs Compared
with Transcription Profiles
To test the molecular heterogeneity between ES-derived NSCs
and somatic NSCs and to score genes differently expressed in
the two cell populations, we performed a compared global
transcriptional profile by means of gene chip analysis compar-
ing cells between passages P9 and P10. Briefly, fluorescent-
labeled cRNAs derived from ESC-derived NSCs and E14.5
embryonic striatal NSCs (between P8 and P9) were hybridized
to the Affymetrix Mouse Expression 430 set (Santa Clara, CA,
http://www.affymetrix.com). This platform consists of two
GeneChip probe arrays (MOE430A and MOE430B) containing
more than 45,000 probe sets representing more than 39,000
transcripts and variants, including more than 34,000 well sub-
stantiated mouse genes. As shown in the scatter plot in Figure
5A, both NSC lines expressed a large number of common
sequences. In this category, 15,031 sequences were not changed
in the two cell populations. Although 3,158 and 2,482 showed
an increased expression in ES-derived NSCs and somatic NSCs,
respectively, as identified by Affymetrix MAS 5.0 absolute and
comparative analyses, the number of sequences, which can be
considered specifically expressed in either cell population, on
the basis of a ratio equal to or higher than threefold (i.e., a
signal-log ratio grater than or equal to 1.5 or less than or equal
to 1.5), was 276 in ES-derived NSCs (among them, 185
present only ES-derived NSCs) and 372 in somatic NSCs
(among them, 201 present only in somatic NSCs). Thus, less
than 2% of the studied transcripts were found significantly
changed, indicating a substantial transcriptional homogeneity of
the two cell populations.
Importantly, the Xist gene resulted as one of the most widely
differentially expressed (Fig. 5B). This gene is exclusively
expressed in the female lineage from the blastocyst stage on-
ward because it produces a main molecular switch that inacti-
vates one of the two X chromosomes. In fact, the ES cell lines
used for the transcriptional profile were all male (XY), and thus
the Xist gene was not expressed. Conversely, the NSCs were
Figure 4. Composition and morphology of differentiated cell cultures
derived from embryonic somatic NSCs (A, D), adult somatic NSCs (B,
E), and ES-derived NSCs (C, F, G). (A–C): Double stainings for
-III-tubulin and GFAP to visualize the ratio of neurons/astrocytes in
the overall differentiated cultures. (D–G): Neural morphology visual-
ized by
-III-tubulin immunostaining of neurons derived from either
somatic embryonic (D) and adult (E) NSCs or ES-derived NSCs (F, G)
with nuclei labeled with 4,6-diamidino-2-phenylindole. (H): Quantita-
tive analysis of the neural, glial, and oligodendroglial phenotypes ob-
tained by differentiation of somatic and ES-derived cell cultures. , p
.05 versus ES cell– derived neurons; ⴱⴱ, p .05 versus ES cell– derived
astrocytes. Abbreviations:
-III-tubulin; ES, embryonic stem;
GFAP, glial fibrillary acidic protein; NSC, neural stem cell.
Figure 5. Compared global transcriptional profile of somatic and
ES-derived NSCs. (A): Scatter plot showing the distribution of hybrid-
ization signals of somatic (E14-NSCs) on the y-axis and ES-derived
(ES-NSCs) NSCs on the x-axis. Transcripts more abundant in E14-
NSCs are shown in red, transcripts more abundant in ES-NSCs are
shown in green, and unchanged transcripts are shown in yellow. (B):
Probe sets considered as positive controls for the microarray experi-
ment: Xist, H2-K, H2-D1, and H13. (C): Microarray data of selected
genes chosen for their proven or suggested role played in somatic NSCs.
(D–F): Signaling pathways whose main components show equal ex-
pression in both ES-derived and somatic NSCs. (E): Wnt-
schematic transduction pathway and Western blotting showing Wnt5A
-catenin protein levels in both NSC cell lines. (C, D): Signals are
shown as color-coded cells (black for “absent” or “marginal” transcripts
and blue to red for “present” transcripts: blue for signals 100; yellow
for signals between 101 and 300; light orange for signals between 301
and 600; dark orange for signals between 601 and 1,000; and red for
signals 1,001). The fold change is also shown as SLR. (F): Notch
transduction pathway and protein level analysis of Notch1 and Hes1.
(G): Hh schematic molecular pathway and analysis of Dhh and Smo
protein levels in both stem cell lines. Abbreviations: Bmpr1a, bone
morphogenetic protein receptor 1a; Bmpr2, bone morphogenetic protein
receptor 1; Ccnb1, cyclinb1; Ccnnd1, cyclind1; Ccnd2, cyclind2; Dhh,
desert hedgehog; Disp1, Dispatched1; Dll1, Delta1; Dnclc1, dynein
light chain 1; Dvl1, Dishevelled 1; E14, embryonic day 14; Egfr1,
epidermal growth factor 1; ES-NSC, embryonic stem–neural stem cell;
Fgfr1, fibroblast growth factor receptor 1; Fz2/9, Frizzled receptor 2, 9;
Fu, fused; Hdac, histone deacetylase; Hdgf, hepathoma-derived growth
factor; Hh, Hedgehog; Jag1, Jagged1; Nes, nestin; Nic, nicastrin, NISD,
notch intracellular domain; Nr3c1, nuclear receptor 3c1; Pdgfa, platelet-
derived growth factor A; PS1, presenilin1; Ptch1, Patched1; SLR, signal
log 2 ratio; Smo, Smoothened; Stmn1, Stathmin1; ThrA, thyroid recep-
tor A; Vegfb, vascular endothelial growth factor B; Vim, vimentin.
829Colombo, Giannelli, Galli et al.
derived by a pool of both male and female E14.5 embryos, and
thus Xist gene expression was clearly detectable in the screen-
ing. Another expected difference between the two cell lines was
represented by the low expression of some genes coding for
major histocompatibility complex (MHC) class I proteins in
ES-derived NSCs (H2-K, H2-D1, and H13) (Fig. 5C), as already
stated in previous reports describing the very low level of MHC
class I gene expression in mouse and human ES cells with
respect to many somatic cell lines [20, 21].
Among the 15,031 genes equally expressed in the two cell
lines, several genes encode for a variety of proteins already
known to play a critical role in somatic NSCs, such as the
cytoskeletal components nestin [15] and vimentin [16] and the
cytosolic proteins stathmin [22], Pten [23], and ShcA [24] (Fig.
5D). Much evidence has been reported recently on the key
functions, such as self-renewal and cell fate choices, played by
some transcription factors in controlling the cardinal properties
of somatic NSCs. Most of these transcription factors are ex-
pressed in both cell types at very similar levels, as detected for
Sox2 [25], Emx2 [14], and Olig1,2 [17]. Among many other
transcription factors equally expressed in both cell types,
FoxM1, Stat3, Myc, Jun, RelA, Zipro1, Lmo4, Nr3c1, and Miz1
showed a very high expression and may play essential roles that
are still unexplored [26]. Finally, chromatin re-modeling pro-
teins such as histone deacetylases Hdac-1, -2, and -3 [27] and
Hmga1, Hmgb1, and Hmgb3 [28] were highly expressed in both
cell lines, as expected with their function in modulating cell
cycle progression in a variety of cells (Fig. 5D).
We scored a similar presence not only of single genes but
also of entire transduction pathways implicated in regulating
somatic NSC behavior. For instance, three transduction path-
ways highly related to proliferation and self-renewal activity of
somatic NSCs, such as the Wnt–
-catenin (
-cat), Notch, and
Hedgehog (Hh) pathways, were similarly expressed in ES-
derived NSCs [28]. In particular, many single components of the
-cat pathway were detected in both cell types, including the
Wnt receptors Frizzled-1, -2, and -9 (Fzd-1, -2, and -9), the
transduction components Dishevelled1 (Dvl1), Glicogen
sinthase kinase 3 (GSK3-
), APC, Axin, as well as many
protein kinases (Csnk1e, Csnk1a1, and Csnk2a2) and phospha-
tases (Ppp2cb, Ppp2r1a, and Ppp2r5c), which are known to
regulate the activity of the Wnt pathway. Finally,
-cat and its
associated transcription factors Tcf4 and (to a lesser extent)
Tcf3 were equally detected (Fig. 5E). Both cell lines expressed
Wnt family members and in particular Wnt5a and Wnt7b, sug-
gesting the existence of a functional Wnt autocrine loop, which
might contribute to NSC maintenance. In particular, we detected
the presence of Wnt5A and
-cat proteins, suggesting the func-
tional activity of this pathway in ES-derived NSCs (Fig. 5E).
Another main transduction pathway implicated in somatic NSC
proliferation activity is the Notch pathway [29]. Both Notch-1
and -2, their ligands Delta-1 to -3 (Dll-1 to -3), and Jagged1
(Jag1) are highly expressed (Fig. 5F). The downstream effectors
of this pathway, Hes1 and Hes5, are also detected in the mi-
croarray. Furthermore, some other essential components of the
pathway such as RBP-Jk, Presenilin-1 (PS1), Nicastrin (Nic),
and Lunatic and Radical Fringe were enriched in both popula-
tions. Supporting some functional involvement of this pathway,
we identified the presence of both Notch1 and Hes1 proteins
(Fig. 5F) by Western blotting. Finally, we considered the Hh
pathway, which has been recently found to be involved in
supporting somatic NSC proliferation both in vitro and in vivo
[30, 31]. Both the Hh receptor Patched1 (Ptch1) and its core-
ceptor Smoothened (Smo) as well as the downstream effectors
Gli2-Gli3 were found expressed in both cell lines (Fig. 5G).
Interestingly, we found only Desert Hedgehog (Dhh), out of the
whole Hh ligand family, to be expressed in both cell types.
Indeed, we confirmed these results by Western blot analysis,
which indicated the presence of both Dhh and Ptch1 proteins
(Fig. 5G). Taken together, these data show that somatic and
ES-derived NSCs share a great part of their molecular pheno-
type, including important molecular components and specific
signal transduction pathways, suggesting the similar nature and
potentials of the two cell lines.
Furthermore, this study identified 245 sequences specifi-
cally enriched in either of the two NSC lines (supplemental
online Table 1). We identified three groups of genes among
them that may unravel the molecular pathways at the basis of the
differences of the two cell lines. Some of the genes most
differently expressed coded for proteins of the extracellular
matrix (ECM) such as collagen proteins such as Col1a1, Col3a1,
and Col1a2 (Fig. 6A). Furthermore, other important compo-
nents of the ECM, such as fibronectin (Fn) and fibrillins
(Fbn-1 and -2), were upregulated in ES-derived NSCs [32].
To independently confirm the reproducibility of the array
data, RT-PCRs were performed. Semiquantitative amplifica-
tion reactions for Col1a1, Fn1, and Fbn1 confirmed gene
expression diversity in the two cell populations and showed
even greater differences as in the case of Fbn1 (6.5-fold
increase in ES-derived NSCs vs. a 3.5-fold increase in the
gene-chip analysis) (Fig. 6A). The enhanced expression of
extracellular components in ES-derived cells may explain
why these cells show a higher ability to attach and grow over
many types of surfaces in respect to somatic NSCs, which
typically cannot grow on a naı¨ve substrate.
Among the 75 sequences found enriched in the ES-derived
NSCs and corresponding unambiguously to known genes, eight
(Nkx2.2, Irx2, En1, Hoxa1, Hoxa2, Hoxa3, Hoxa4, and Hoxa5)
were represented by genes coding for transcription factors
mostly or exclusively active along the caudal neural tube. Dur-
ing embryonic development, a series of transcription factors
regionalize the neural tube along the anterior-posterior (A-P)
axis. Growing evidence supports the hypothesis that these mol-
ecules are producing a molecular identity that specifies the
different regions of the neural tube such as forebrain, midbrain,
and hindbrain. Many of these transcription factors were ex-
pressed in both cell lines; however, we found a general enrich-
ment in ES-derived NSCs of mRNAs of transcription factors
that act on the spinal cord. Nkx2.2, Irx2, and En1 are genes
expressed starting from the mid-hindbrain region all along the
ventral spinal cord, whereas the Hox genes are active in the most
caudal regions of the body. We confirmed these findings by
means of semiquantitative RT-PCRs (Fig. 6B) and widened this
data set by analysis other Hox genes not included in the mi-
croarray platform, such as Hoxa9, Hoxa10, and Hoxa13 (Fig.
6B; data not shown). In all cases, these genes were expressed in
ES-derived NSCs but were represented at a low or undetectable
level in somatic NSCs. These data strongly suggested that
ES-derived NSCs displayed the positional molecular informa-
tion of the whole A-P axis of the developing neural tube.
830 Characterization of ES-Derived Neural Stem Cells
Conversely, somatic NSCs showed a general low expression or
absence of members of the Hox gene family and other transcrip-
tion factors of the Nkx and Irx classes, which are critical for the
establishment and shaping of the spinal cord.
Among the 75 known transcripts enriched in the ES-derived
NSCs, insulin-like growth factor (IGF)-II is coding for the
growth factor most expressed in a relative and absolute manner
in these cells. IGF-II belongs to the IGF family and shares many
pleiotropic activities in neural cells, such as supporting cell
proliferation, migration, and survival, with the other members of
the family, IGF-I and IGF-III [33, 34]. IGF receptors are ex-
pressed in the germinal zone of the developing neural tissue as
well as in the subventricular zone (SVZ) of adult mouse brain.
Targeted mutations of members of this family lead to deficits in
both body and brain development [35]. IGF signals are trans-
duced through the two IGF receptors (IGF-1R and -2R), which
are receptor tyrosine kinases closely related to the insulin re-
ceptor. Activation of these receptors results in tyrosine phos-
porylation of cytoplasmic insulin receptor substrates (IRSs) that
in turn can activate the Ras-MAP kinase or the PI3 kinase
(phosphatidylinositide-3 kinase)-Akt pathways [35]. Interest-
ingly, all these intracellular mediators of the IGF pathway were
highly expressed in ES-derived NSCs at an equivalent level to
those detected in somatic NSCs, as shown by gene-chip and
RT-PCR analyses (Fig. 6C). Also, the IGF binding proteins 2
and 4, which have been implicated in regulating the overall
actions of the IGF molecules [36], were expressed at compara-
ble levels (Fig. 6C). These data prompted us to determine
whether the IGF signaling was effectively upregulated in the
ES-derived NSCs and to correlate this molecular diversity with
a particular aspect of the in vitro behavior of these cells.
The Enhanced IGF Signaling Is Responsible
for the High Proliferation Rate of ES-Derived
NSCs In Vitro
To verify whether the IGF-II enhanced expression resulted in
the functional activation of the downstream targets, we evalu-
ated the relative amounts of either phosphorylated MAPK (mi-
togen-activated protein kinase) and phophorylated Akt, two key
molecules of the Ras-Raf-MAPK and PI3-Kinase (PI3K) path-
ways, respectively. Indeed, we found an increase in the phos-
phorylated forms of MAPK and Akt, in the ES-derived in
respect to the somatic NSCs, without any clear increase of the
total amount of these proteins (Fig. 7A). Thus, a sustained
increase in IGF signaling was exhibited by ES-derived NSCs
Figure 6. Classes of genes differentially expressed in ES-derived
NSCs (ES-NSCs) versus somatic NSCs (E14-NSCs). (A): Microarray
and semiquantitative reverse transcription-polymerase chain reaction
(RT-PCR) analysis of ES-derived NSC– enriched transcripts coding for
extracellular matrix proteins. (B): Gene expression analysis by Gene-
Chip and RT-PCR of transcripts coding for transcription factors con-
tributing to caudal neural tube identity. (C): Microarray and RT-PCR
study of genes coding for IGF signaling pathway. In tables, signals are
shown as color-coded cells (black for “absent” transcripts and blue to
red for “present” transcripts, as in Figure 5). The fold change is also
shown as SLR. Abbreviations: aNSC, adult neural stem cell; Col1a1,
collagen 1a1; Col3a1, collagen 3a1, Col1a2, collagen 1a2; Col6a3,
collagen 6a3; E14-NSC, embryonic day 14 –neural stem cell; ES-NSC,
embryonic stem–neural stem cell; Fn1, fibronectin 1; Fbn1, fibrillin 1;
Fbn2, fibrillin 2; Itga9, Integrin a9; SLR, signal log 2 ratio.
Figure 7. Active IGF signaling is enhanced in ES-derived NSCs with
respect to somatic NSCs. (A): Western blotting analysis of IGF-II and
active forms of key components of the pathway. p42/p44 (Erk1/2) and
Akt phosphorylated forms are enriched in ES-derived NSCs. Cell ly-
sates were normalized with respect to their
-actin protein levels. (B):
IGF growth factors sustained ES cell– derived neural proliferation in
culture in the absence of insulin. (C): Functional arrest of IGFR-I
function by a blocking antibody strongly decreased IGF-II– dependent
cell proliferation. Suramin is an inhibitor of both IGF and epidermal
growth factor. (D): Cell proliferation elicited by IGF-II is mediated
mostly by activation of the mitogen-activated protein kinase signal
transduction pathway. Abbreviations: aNSC, adult neural stem cell;
DIV, days in vitro; ES-NSC, embryonic stem–neural stem cell; IGF,
insulin-like growth factor; IGFR-I, insulin-like growth factor receptor I.
831Colombo, Giannelli, Galli et al.
during their proliferating stage. Previous studies revealed an
essential action of IGF-I in maintaining proliferation of somatic
NSCs in cultures [37]. Indeed, all the media used for NSC
cultures include insulin at a concentration of 5–25
g/ml. Its
withdrawal in ES-derived NSC cultures led to a rapid block in
cell proliferation, which is followed by a widespread cell death
even in the presence of the growth factors FGF-II and EGF (Fig.
7B). Similar behavior was described for adult NSCs in a me-
dium not supplemented with insulin growth factors [38]. In
addition, a culture medium deprived of insulin, but supple-
mented with IGF-I or IGF-II (100 nM), sustained the growth of
ES-derived NSCs. Interestingly, the effect of IGF-I on prolifer-
ation was higher than that of IGF-II, although the latter still had
a significant effect.
To identify the receptor through which IGF-II promoted cell
proliferation, we added blocking antibodies for either IGF re-
ceptor I (IGF-IR) or IGF receptor II (IGF-IIR) in conditions in
which cell growth was sustained by IGF-II (see Materials and
Methods). Only cultures treated with anti–IGF-IR, but not anti-
IGF-IIR, exhibited a dramatic decrease in cell proliferation (Fig.
7C). Accordingly, we found a reduction in MAPK phosporyla-
tion in cultures exposed to IGF-RI blocking antibody (Fig. 7C).
These findings suggest that IGF-II elicits cell growth mainly by
signaling via the IGF-IR. Finally, we investigated the intracel-
lular pathway involved in mediating IGF action. Specific inhib-
itors of the MAPK or the PI3K pathways were used, in partic-
ular inhibitors of the ERK kinases such as PD098059, U0126,
and LY294002, to block PI3-kinase [37]. Addition of the
MAPKK inhibitor PD098059 to IGF-II treated cultures abol-
ished the effect of the growth factor on the increased prolifer-
ation in a dose-dependent manner (Fig. 7C). Conversely, a
minor effect was noted on the IGF-II–induced proliferation
upon the addition of the PI3-K inhibitor, LY294002 (Fig. 7C).
These results suggest that IGF-II–induced proliferation is me-
diated mainly by the activation of the MAPK signaling and only
marginally by the PI3K molecular component pathway.
ES Cell–Derived Neural Progenitors Exhibited Main
Properties of Stem Cells
Several protocols suitable for ESC differentiation toward a
neural cell lineage have been established over the years. In
particular, the work originally described by Okabe et al. [1] and
further elaborated by Lee et al. [5] allowed the induction of
neuroepithelial precursors that can be maintained in vitro and
induced to differentiate upon growth factor withdrawal. We
opted for a revision of the general protocol, which included
some important changes in culture mediums and cell-substrate
choices. In our view, these modifications accelerate the entire
procedure and probably maintained the ES cell– derived neural
precursors in a prolonged and stable state of proliferation and
multipotent behavior. Furthermore, we employed a series of
experiments, including clonogenic assays and long-term
growth-rate determination, that enabled us to clearly define the
stem cell features of ES cell– derived neural progenitors. In fact,
we showed that at different cell passages (P3, P7, and P11), and
therefore at different times in culture, ES-derived neural prolif-
erating cells exhibited clonogenic potential and multipotency.
Such functional features are critical attributes of cultured stem
cells and are probably a consequence of their ability to self-
renew. Therefore, these cells displayed all cardinal properties of
stem cells. Recently, Barberi et al. provided a different in vitro
method based on a coculture system in which stromal feeder
cells were able to prompt a neural commitment of naı¨ve ES cells
[7]. A series of clonogenic assays allowed the authors to em-
phasize the stem cell nature of the induced ES cells. All these
results indicate that different strategies may be applied to derive
NSCs from more primitive totipotent cells. This suggests that
further studies are required to understand the differences among
various NSC-inducing systems and the developmental potential
of derived cells.
Cellular and Molecular Phenotypes of Somatic and
ES-Derived NSCs
The stem cell features of these ES cell–derived populations
prompted us to investigate their relationships with somatic
NSCs. We used embryonic NSCs derived from the forebrain
regions as a basis of comparison with the ES-derived NSCs,
although in some circumstances adult NSCs isolated from the
subependymal zone were added in the analysis.
ES-derived NSCs showed an in vitro behavior completely
different from that displayed by somatic NSCs. In fact, the
former grew attached to a substrate, whereas the latter formed
clonal aggregates known as neurospheres. Our large-scale gene
transcription analysis, based on GeneChip technology, provided
some possible explanations for these dissimilar behaviors. In
fact, we found ES-derived NSCs to highly express the genes
coding for Fibronectin, Fibrillin-1 and -2, and various forms of
Collagens. All of them are important components of the extra-
cellular matrix; Fibronectin, in particular, allows the cells to
strongly adhere, linking the substrate to the cell membrane and
to the intracellular cytoskeleton via
-1 integrin. Undiffer-
entiated ES cells show a highly organized extracellular matrix,
forming focal adhesions in specific circumstances [39]. Inter-
estingly, naı¨ve ES cells and ES-induced NSCs exhibit a very
similar behavior on different cell substrates (data not shown).
Therefore, it is tempting to speculate that some specific cellular
features of ES cells are maintained during their conversion to a
neural fate and that extracellular matrix organization may be one
of those. On the other hand, these data provide evidence that
stem cell potential is partially independent from the physical
composition of the extracellular matrix. Despite this diversity,
ES-derived and somatic NSCs exhibited a large variety of
common features. In fact, they share the main properties of stem
cells—self-renewal and multipotency activities—as tested by
clonogenic assays. Furthermore, we showed here that their
transcriptional profiles are highly overlapping with only a few
hundred genes enriched in either one of the two cell populations.
Many genes known to play a pivotal role in somatic NSC are
also present in ES-derived NSCs with a similar transcriptional
level. We should emphasize that this analysis offers some clues
for the identification of novel genes with an unexplored role in
NSCs. Indeed, genes commonly expressed in both proliferating
cell populations, but not in their differentiated progenes and in
ES cells, may represent good candidates. For instance, FoxM1 is
among the strongest expressed transcription factors in both cell
lines. FoxM1 has been implicated in regulating the cell cycle in
hepatocytes [40], but its expression was reported also in prolif-
erating NSCs [41]. Interestingly, the gene is overexpressed at
832 Characterization of ES-Derived Neural Stem Cells
various degrees in different forms of astrocytoma and/or glio-
blastoma [42]. Among the chromatin architectural proteins, the
Hmgb proteins may reveal unsuspected functions in NSCs. In
fact, it has been reported that Hmgb1 protein acts in specific
cellular contexts as a secreted cytokine [43] and that it is able to
trigger proliferation and migration of vessel-associated stem
cells (mesoangioblasts) [44]. Thus, a similar function may be
supposed in NSCs, where it shows a high level of expression.
ES-Derived NSCs Exhibit a High Proliferative Index
and Sustained IGF Signaling
ES-derived NSCs displayed a higher proliferation rate in com-
parison with both embryonic and adult somatic NSCs. Seeking
the molecular basis of this behavior, we used the microarray
analysis to identify the enriched expression of IGF-II in the
ES-derived cell population. IGFs are widely expressed mole-
cules that regulate proliferation, survival, and differentiation. In
particular, several studies have revealed their essential role in
supporting proliferation of NSCs either in culture or during
neural development [36, 38]. Thus, IGF signaling could repre-
sent a good candidate to explain the different proliferation rates
between the two cell populations. Indeed, in addition to the
increased IGF-II expression detected both by microarray and
semiquantitative RT-PCRs, Western blotting analysis contrib-
uted to show an effective upregulation of the entire IGF pathway
by scoring the amount of activated forms of key components of
the intracellular transduction pathway, represented by phosphor-
ylated MAPK and AKT proteins. Importantly, we showed that
IGF factors modulate ES-derived NSC growth, in particular,
through activation of the MAPK pathway. Interestingly, recent
data described that somatic NSC cultures also need an activated
MAPK signaling to sustain proliferation and neurosphere for-
mation [45]. Thus, these and other data suggest that MAPK acts
as a primary signaling in regulating proliferation in ES-derived
and somatic NSCs, reinforcing the view of a general homoge-
neity between the two NSC cell populations. Taken together,
these findings represent an initial step toward the molecular
characterization of different types of NSCs, highlighting spe-
cific features for each cell line. Furthermore, these character-
izations will reveal new molecules able to control cell differen-
tiation and cell lineage choices.
We thank L. Cornaghi and T. Veneroso for expert technical
assistance, Drs. A. Faedo and A. Bulfone for initial support in
the microarray analysis, and Dr. A. Nagy for providing YC5-ES
cells. This research was supported by Istituto Superiore di Sanita´
grant CS-71 to V.B.
The authors indicate no potential conflicts of interest.
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See www.StemCells.com for supplemental material available online.
834 Characterization of ES-Derived Neural Stem Cells
    • "NPCs derived from ESCs were cultured in medium containing 20 ng/mL EGF and 10 ng/mL FGF2 as previously described (Colombo et al. 2006). Zrf1 and Pax6 re-expression was obtained by electropoating pCBA Zrf1 3xFlag and pCBA Pax6 3xFlag (Amaxa neural stem cell kit), and selected with neomycin for 6 d. "
    [Show abstract] [Hide abstract] ABSTRACT: The molecular mechanisms underlying specification from embryonic stem cells (ESCs) and maintenance of neural progenitor cells (NPCs) are largely unknown. Recently, we reported that the Zuotin-related factor 1 (Zrf1) is necessary for chromatin displacement of the Polycomb-repressive complex 1 (PRC1). We found that Zrf1 is required for NPC specification from ESCs and that it promotes the expression of NPC markers, including the key regulator Pax6. Moreover, Zrf1 is essential to establish and maintain Wnt ligand expression levels, which are necessary for NPC self-renewal. Reactivation of proper Wnt signaling in Zrf1-depleted NPCs restores Pax6 expression and the self-renewal capacity. ESC-derived NPCs in vitro resemble most of the characteristics of the self-renewing NPCs located in the developing embryonic cortex, which are termed radial glial cells (RGCs). Depletion of Zrf1 in vivo impairs the expression of key self-renewal regulators and Wnt ligand genes in RGCs. Thus, we demonstrate that Zrf1 plays an essential role in NPC generation and maintenance.
    Full-text · Article · Jan 2014
    • "In this project we chose to use iPSCs, which overcome many limitations of other sources; for example, ethical issues, limited accessibility, and restricted genetic backgrounds. In addition, iPSCs seem to be more stable, with a higher neuronal diff erentiation effi ciency than, for example, somatic stem cells [26,27]. However, generating iPSCs is challenging. "
    [Show abstract] [Hide abstract] ABSTRACT: This project aims to establish and characterize an in vitro model of the developing human brain for the purpose of testing drugs and chemicals. To accurately assess risk, a model needs to recapitulate the complex interactions between different types of glial cells and neurons in a three-dimensional platform. Moreover, human cells are preferred over cells from rodents to eliminate cross-species differences in sensitivity to chemicals. Previously, we established conditions to culture rat primary cells as three-dimensional aggregates, which will be humanized and evaluated here with induced pluripotent stem cells (iPSCs). The use of iPSCs allows us to address gene/environment interactions as well as the potential of chemicals to interfere with epigenetic mechanisms. Additionally, iPSCs afford us the opportunity to study the effect of chemicals during very early stages of brain development. It is well recognized that assays for testing toxicity in the developing brain must consider differences in sensitivity and susceptibility that arise depending on the time of exposure. This model will reflect critical developmental processes such as proliferation, differentiation, lineage specification, migration, axonal growth, dendritic arborization and synaptogenesis, which will probably display differences in sensitivity to different types of chemicals. Functional endpoints will evaluate the complex cell-to-cell interactions that are affected in neurodevelopment through chemical perturbation, and the efficacy of drug intervention to prevent or reverse phenotypes. The model described is designed to assess developmental neurotoxicity effects on unique processes occurring during human brain development by leveraging human iPSCs from diverse genetic backgrounds, which can be differentiated into different cell types of the central nervous system. Our goal is to demonstrate the feasibility of the personalized model using iPSCs derived from individuals with neurodevelopmental disorders caused by known mutations and chromosomal aberrations. Notably, such a human brain model will be a versatile tool for more complex testing platforms and strategies as well as research into central nervous system physiology and pathology.
    Full-text · Article · Dec 2013
    • "While there have been few direct comparisons of potentially therapeutic NSCs and MSCs [50]–[52], differences in migration and other behaviors may exist as consequences of underlying signaling pathways that will vary between stem cells of different lineages. For example, subtle distinctions have been noted between embryonic stem cell-derived and somatic NSCs in differentiation potential and proliferation [53], and MSCs of different origins differ in proliferation, differentiation potential and tumor-homing capabilities [54]–[56]. In addition, a difference in the capacity of NSC and MSC lines to deliver a therapeutic oncolytic adenovirus payload has been reported [52]. "
    [Show abstract] [Hide abstract] ABSTRACT: Pathotropic neural stem and/or progenitor cells (NSCs) can potentially deliver therapeutic agents to otherwise inaccessible cancers. In glioma, NSCs are found in close contact with tumor cells, raising the possibility that specificity of NSC contact with glioma targets originates in the tumor cells themselves. Alternatively, target preferences may originate, at least in part, in the tumor microenvironment. To better understand mechanisms underlying NSC interactions with glioma cells, we examined NSC-target cell contacts in a highly simplified 3-dimensional peptide hydrogel (Puramatrix) in which cell behaviors can be studied in the relative absence of external cues. HB1.F3 is an immortalized clonal human NSC line extensively characterized in preclinical investigations. To study contact formation between HB1.F3 NSCs and glioma cells, we first examined co-cultures of eGFP-expressing HB1.F3 (HB1.F3.eGFP) NSCs and dsRed-expressing U251 glioma (U251.dsRed) cells. Using confocal microscopy, HB1.F3.eGFP cells were observed contacting or encircling U251.dsRed glioma cells, but never the reverse. Next, examining specificity of these contacts, no significant quantitative differences in either percentages of HB1.F3 NSCs contacting targets, or in the extent of target cell encirclement, were observed when HB1.F3.eGFP cells were presented with various potential target cells (human glioma and breast cancer cell lines, patient-derived brain tumor lines, non-tumor fibroblasts, primary mouse and human astroglial cells, and primary adult and newborn human dermal fibroblasts) except that interactions between HB1.F3 cells did not progress beyond establishing contacts. Finally cytoskeletal mechanisms employed by HB1.F3.eGFP cells varied with the substrate. When migrating in Puramatrix, HB1.F3 NSCs exhibited intermittent process extension followed by soma translocation, while during encirclement their movements were more amoeboid. We conclude that formation of contacts and subsequent encirclement of target cells by HB1.F3 NSCs is an intrinsic property of these NSCs, and that preferential contact formation with tumor cells in vivo must therefore be highly dependent on microenvironmental cues.
    Full-text · Article · Dec 2012
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