Download Neural differentiation of mouse embryonic stem cells

Signalling the Future
Neural differentiation of mouse embryonic
stem cells
M.P. Stavridis and A.G. Smith1
Institute for Stem Cell Research, University of Edinburgh, The King’s Buildings, West Mains Road, Edinburgh EH9 3JQ, U.K.
Abstract
Pluripotent embryonic stem cells can give rise to neuroectodermal derivatives in culture. This potential could
be harnessed to generate neurons and glia for cell-replacement therapies in the central nervous system and
for use in drug discovery. However, current methods of neural differentiation are empirical and relatively
innefficient. Here, we review these methodologies and present new tools for quantification, analysis and
manipulation of embryonic stem cell neural determination.
Introduction
Mouse embryonic stem (ES) cells are pluripotent cell lines
derived from the inner cell mass of the blastocyst. Their
properties include a stable diploid karyotype, the ability to
participate fully in foetal development upon re-introduction
into a blastocyst and the capacity to differentiate into a
plethora of cell types in vitro. The maintenance of the
undifferentiated state of ES cells in vitro is dependent on selfrenewing cell divisions in the presence of cytokines which
signal through the gp130 receptor, e.g. leukaemia inhibitory
factor (LIF) (reviewed in [1]). Upon withdrawal of a selfrenewal stimulus, ES cells rapidly lose their undifferentiated
phenotype and differentiate. These properties have made ES
cells a popular system both to introduce specific genetic
alterations (gene targeting) into animals and to study gene
function during differentiation in vitro [2,3]. To date, the
best-studied mode of ES cell differentiation is the formation
in suspension culture of multicellular aggregates called
embryoid bodies (EBs) [4]. Within these aggregates, complex
interactions between heterologous cell types result in the
induction of differentiation of stem cells to derivatives of
all three embryonic germ layers [5]. Plating of the embryoid
bodies causes further differentiation and outgrowth.
Neural differentiation in EBs
The generation of ES cell-derived neural cells, especially
neurons [6–8], is a much-studied example of ES cell
differentiation. Neural differentiation of ES cells has been
achieved by several different protocols, some of which are
strikingly different. In the protocols that were published
first, EBs were treated with retinoic acid at different time
windows and then plated on to laminin [6], gelatin [7] or tissue
Key words: in vitro neurogenesis, neuron, neural progenitor.
Abbreviations used: BMP, bone morphogenetic protein; EB, embryoid body; ES, embryonic stem;
FACS, fluorescence-activated cell sorting; FGF, fibroblast growth factor; GFP, green fluorescent
protein; LIF, leukaemia inhibitory factor; Shh, Sonic hedgehog.
1
To whom correspondence should be addressed (e-mail [email protected]).
culture plastic [8]. Cells with overt neuronal morphology
appeared after plating, and were found to express neuronspecific genes such as neurofilament light chain, microtubuleassociated proteins 2 and 5, synaptophysin and others. These
cells were found to respond to a range of neurotransmitters
and depolarizing currents, confirming that they were indeed
excitable neurons. Glial cell types also appeared in such
differentiated cultures, as judged both by morphology and
expression of specific glial markers. The majority of glial cells
produced were astrocytes, but oligodendrocytes have also
been generated and selectively expanded from EB cultures
[9].
A variant protocol for neural differentiation of ES cells involved the formation of EBs without retinoic acid treatment,
but depended on the subsequent culture of the attached EBs in
a selective, serum-free medium to eliminate non-neural cells.
Culture of the EB-derived cell pool in this selective medium
results in a dramatic decrease in cell number, as the majority of
the cells do not survive these culture conditions. The surviving
portion is enriched for nestin-positive neural progenitor cells
[10], which can be expanded and induced to differentiate into
neurons with high efficiency [11]. The majority of neurons
generated from ES cells following differentiation in EBs
are GABAergic (where GABA stands for γ -aminobutyric
acid), with some glutamatergic neurons also generated at
lower frequency. In order to generate dopaminergic neurons
(which are potentially useful for treating Parkinson’s disease)
and serotonergic neurons, Lee et al. [12] treated ES cellderived neural precursors with Sonic hedgehog (Shh) and
fibroblast growth factor (FGF)-8. Both of these molecules are
important for patterning the ventral midbrain and hindbrain
[13], where such neurons are generated in vivo. More recently,
another group successfully generated spinal cord motor
neurons from ES cells by treating differentiating ES cells
with a posteriorizing (retinoic acid) and a ventralizing (an
Shh agonist) molecule in order to impart on them the ventral
spinal identity of motor neuron precursors [14]. The fact that
the neural precursors generated from ES cells can respond
to morphogenetic cues suggests that they are (at least to
C 2003
Biochemical Society
45
46
Biochemical Society Transactions (2003) Volume 31, part 1
some degree) malleable, and parallels can be drawn between
neural differentiation of ES cells and neural development
in vivo.
Stromal cells
A distinct system for differentiation of ES cells into
dopaminergic neurons has been described [15]. This system
differs from the other methods for neural differentiation in
that the differentiation process happens not in aggregates,
but via the co-culture of ES cells on monolayers of bone
marrow-derived stromal cells (PA-6 cells). Retinoic acid is
not used to induce neural fate and Shh or FGF-8 are not
added to the cultures to induce dopaminergic neurons. This
has been interpreted in favour of an inductive mechanism
for the production of neurons from ES cells, although the
molecular nature of the inducing signal(s) remains elusive.
It is noteworthy that PA6-mediated neural differentiation
is suppressed in the presence of serum or bone morphogenetic protein (BMP)-4 which direct cells to alternative
fates.
Defined media
The first indication that ES cell neural determination is under
inhibitory control and may resemble ‘default’ amphibian
neural induction [16,17] came from experiments in serum-free
medium. Using EBs generated in chemically defined serumfree medium, Wiles and Johansson [18] showed that neural
markers were expressed unless signals of the transforming
growth factor superfamily (including BMPs and activin)
were present. In the presence of activin A, an inducer of
the mesoderm in the frog, mesodermal as well as neural
markers appeared, along with a strong induction of the
activin antagonist, follistatin [18]. Treatment with BMP-4
resulted in the loss of neural cell markers, as is seen with
frog ectodermal explants [19]. BMP-4 also suppressed neural
differentiation of ES cells in a related system [20]. In this
case, ES cells were selected in serum-free conditions in the
presence of LIF, and at a frequency of 0.1% gave rise to
nestin-positive cell aggregates (‘neurospheres’) which could
be then expanded in FGF-2 or induced to differentiate
into neurons, astrocytes or oligodendrocytes. ES cells that
lack a component of the BMP signalling pathway gave rise
to 3.5-fold more spheres, suggesting that differentiation in
this system is under inhibitory control by BMPs [20]. Sphere
formation was inhibited by blocking antibodies to FGF-2,
possibly due to a requirement of FGF signalling for expansion
of neural cells. The low efficiency of colony formation under
these conditions suggests a strongly selective mechanism that
precludes the drawing of any conclusions concerning neural
determination.
Lineage selection
A hurdle in the study of neural cell generation, expansion
and differentiation using ES cells as a model system is that,
C 2003
Biochemical Society
even with the best available protocols, differentiation is not
uniquely neural. All of the available protocols for neural
differentiation result in the generation of multiple cell types
(often of more than one germ layer) and the progression
from ES cell via a committed neural precursor to a fully
differentiated, post-mitotic neural cell is determined post hoc
either by antibody staining or gene expression profiling. The
presence of alternative differentiation hampers such analyses,
as it is not possible to determine if the effect of added
factors is direct or via other differentiated cell types in
the culture. Furthermore, one potential use of ES-derived
neural precursors is as material for transplantation studies for
the treatment of neurodegenerative diseases such as Parkinson’s or Alzheimer’s disease, or neural damage following
stroke or injury. For such studies it is important that any
ES cells are eliminated from the transplanted tissue because
they can generate teratocarcinomas [21–23]. We [24] have
used the technique of lineage selection on differentiating ES
cells to purify neural precursors (Figure 1a). This technique
relies on the introduction of a reporter/selection cassette
into a locus with restricted expression in the desired cell
type by homologous recombination. The application of
a selection drug eliminates cells that do not express the
gene, resulting in a pure population of Sox2+ cells. A
drawback of our original study was that the gene used for
selection (Sox2) is expressed not only by neural precursors,
but also by ES cells. Consequently, selection for Sox2+
cells does not eliminate undifferentiated ES cells from the
culture. This is significant because persistence of ES cells
at some level is a common feature of current differentiation
methods.
To address this we have engineered an ES cell line
(46C) in which one copy of the pan-neural gene Sox1
[25,26] has been replaced by the dual selection/reporter
cassette egfpIRESpac, which confers cell-autonomous green
fluorescence and puromycin resistance to cells that express
Sox1 (Figure 1b). Sox1 belongs to the same family of
high-mobility group transcription factors as Sox2, but its
expression is restricted to proliferating neuroectodermal cells
and the lens [25]. In addition to the restricted expression of the
reporter, 46C cells have the advantage of a vital label (green
fluorescent protein, GFP) meaning that activation of Sox1
can be monitored in living cells. As Sox1 is not expressed
in ES cells, undifferentiated 46C ES cells are not fluorescent.
Upon neural differentiation (in EBs treated with retinoic acid,
on PA6 or in serum-free medium), Sox1 is activated and
the cells produce green fluorescence. This property of 46C
cells enables us to purify both neural and non-neural cells
generated during differentiation and analyse their phenotype.
We used the activation of Sox1–GFP as a marker for neural
cells, and measured it by flow cytometry during 46C ES cell
differentiation. This enables us to measure the proportion
of neural cells at any point during differentiation (Figure 2).
Another advantage of the Sox1–GFP reporter is that it enables
time-lapse observation of the differentiation process with
minimum perturbation of the culture. This method of lineage
identification is sensitive enough to detect slight changes in
Signalling the Future
Figure 1
Sox1 lineage selection
(a) Diagrammatic representation of the lineage selection scheme. Endodermal and mesodermal cells derived from ES cells
are eliminated either by antibiotic selection or flow cytometry, while neurectodermal cells can be amplified and induced to
differentiate to neurons, astrocytes and oligodendrocytes. The inset shows ES cell-derived neurons visualized by antibody
staining for the neuronal marker βIII-tubulin. Scale bar, 50 µm. (b) Sox1 targeting scheme. The entire Sox1 open reading
frame has been replaced by the selection/reporter cassette egfpIRESpac. A cassette for selection in ES cells (Hy-TK) containing
its own promoter (arrow) flanked by site-specific recombination sites (Lox; arrowheads) is included, as Sox1 is not expressed
by undifferentiated ES cells, but is removed by a transient transfection with a recombinase (Cre)-expressing plasmid.
the efficiency of neural determination or the kinetics by which
this process is taking place, making it ideal for the study of the
molecular requirements for neural differentiation of ES cells.
Figure 2 Quantification of neural differentiation of 46C ES cells
ES cells were allowed to form EBs in the absence of LIF. On day 4,
10−6 M all-trans retinoic acid (RA) was added to half of the plates
for a further 4 days. A sample from each culture was analysed for
Sox1–GFP fluorescence by flow cytometry every 2 days. Data points are
means ± S.D. of three experiments (data courtesy of Dr Meng Li).
Specific regulatory molecules or inhibitors can be introduced
into the system and their effect on neural determination can
be measured accurately.
We have used this system to develop a defined culture
system for efficient neural differentiation without aggregation
or co-culture [27]. This provides a platform for dissecting
the molecular mechanisms underlying neural lineage specification. Furthermore, because the GFP reporter enables the
use of cell sorting, it is not necessary to kill off non-neural
cell types by drug selection in order to purify the culture.
Use of fluorescence-activated cell sorting (FACS) allows for
the isolation of both Sox1–GFP-positive and -negative cells
for further culture or analysis. Purified populations of Sox1–
GFP-positive neural progenitors will allow the study of
growth factors, growth factor inhibitors and morphogens on
a neural progenitor population, without intermediate effects
from non-neural cells. This may enable further study of
the factors that direct neural precursors to specific fates,
with a view to using such ES cell-derived cell types for
treatment of specific neurological conditions. FACS-purified
populations can also be used to construct subtractive cDNAs
and examine microarray expression profiling in order to
C 2003
Biochemical Society
47
48
Biochemical Society Transactions (2003) Volume 31, part 1
Figure 3 Identification of novel genes involved in neural determination
After ES cell differentiation, populations of Sox1–GFP+ and Sox1–GFP− cells are isolated by FACS. Following mRNA extraction,
subtraction, cDNA synthesis and library generation, array analyses can be used to identify candidate genes. After bioinformatic
analysis and obtaining in vivo expression data, candidate genes can be introduced into ES cells for functional analysis of their
effect on neural differentiation [28].
identify genes involved in mammalian neural determination
(Figure 3) [27].
Concluding remarks
Generation of differentiated cell lineages from ES cells is a
very promising tool for both the study of development and
the generation of transplantation material for the treatment of
a wide range of degenerative disorders and lesions. In order for
this potential to be realized, the differentiation of ES cells must
be harnessed to produce suitable cells for the treatment of
each condition, eliminate unwanted or harmful cell populations and optimize the conditions for successful graft
integration and survival. These objectives highlight the
importance of understanding and directing initial lineage
choice so that we can make desired cell types with maximum
efficiency and reproducibility. Experience to date with
generation of neural cell types suggests that addressing all of
these issues successfully will involve a combination of both
instructive and selective approaches.
References
1 Smith, A.G. (2001) Annu. Rev. Cell. Dev. Biol. 17, 435–462
2 Guan, K., Schmidt, M.M., Ding, Q., Chang, H. and Wobus, A.M. (1999)
Altex 16, 135–141
3 O’Shea, K.S. (1999) Anat. Rec. 257, 32–41
4 Doetschman, T.C., Eistetter, H., Katz, M., Schmidt, W. and Kemler, R.
(1985) J. Embryol. Exp. Morphol. 87, 27–45
C 2003
Biochemical Society
5 Martin, G.R., Wiley, L.M. and Damjanov, I. (1977) Dev. Biol. 61,
230–244
6 Bain, G., Kitchens, D., Yao, M., Huettner, J.E. and Gottlieb, D.I. (1995)
Dev. Biol. 168, 342–357
7 Strubing, C., Ahnert-Hilger, G., Shan, J., Wiedemann, B., Hescheler, J. and
Wobus, A.M. (1995) Mech. Dev. 53, 275–287
8 Fraichard, A., Chassande, O., Bilbaut, G., Dehay, C., Savatier, P. and
Samarut, J. (1995) J. Cell Sci. 108, 3181–3188
9 Liu, S., Qu, Y., Stewart, T.J., Howard, M.J., Chakrabortty, S., Holekamp, T.F.
and McDonald, J.W. (2000) Proc. Natl. Acad. Sci. U.S.A. 97,
6126–6131
10 Lendahl, U., Zimmerman, L.B. and McKay, R.D.G. (1990) Cell 60,
585–595
11 Okabe, S., Forsberg-Nilsson, K., Spiro, A.C., Segal, M. and McKay, R.D.
(1996) Mech. Dev. 59, 89–102
12 Lee, S.H., Lumelsky, N., Studer, L., Auerbach, J.M. and McKay, R.D. (2000)
Nat. Biotechnol. 18, 675–679
13 Ye, W., Shimamura, K., Rubenstein, J.L., Hynes, M.A. and Rosenthal, A.
(1998) Cell 93, 755–766
14 Wichterle, H., Lieberam, I., Porter, J. and Jessell, T. (2002) Cell 110, 385
15 Kawasaki, H., Mizuseki, K., Nishikawa, S., Kaneko, S., Kuwana, Y.,
Nakanishi, S., Nishikawa, S.I. and Sasai, Y. (2000) Neuron 28,
31–40
16 Hemmati-Brivanlou, A. and Melton, D. (1997) Cell 88,
13–17
17 Hawley, S.H., Wunnenberg-Stapleton, K., Hashimoto, C., Laurent, M.N.,
Watabe, T., Blumberg, B.W. and Cho, K.W. (1995) Genes Dev. 9,
2923–2935
18 Wiles, M.V. and Johansson, B.M. (1999) Exp. Cell. Res. 247,
241–248
19 Wilson, P.A. and Hemmati-Brivanlou, A. (1995) Nature (London) 376,
331–333
20 Tropepe, V., Hitoshi, S., Sirard, C., Mak, T.W., Rossant, J. and
van der Kooy, D. (2001) Neuron 30, 65–78
21 Bjorklund, L.M., Sanchez-Pernaute, R., Chung, S., Andersson, T., Chen, I.Y.,
McNaught, K.S., Brownell, A.L., Jenkins, B.G., Wahlestedt, C., Kim, K.S.
and Isacson, O. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 2344–2349
Signalling the Future
22 Zhang, S.C., Wernig, M., Duncan, I.D., Brustle, O. and Thomson, J.A.
(2001) Nat. Biotechnol. 19, 1129–1133
23 Deacon, T., Dinsmore, J., Costantini, L.C., Ratliff, J. and Isacson, O. (1998)
Exp. Neurol. 149, 28–41
24 Li, M., Pevny, L., Lovell-Badge, R. and Smith, A. (1998) Curr. Biol. 8,
971–974
25 Pevny, L.H., Sockanathan, S., Placzek, M. and Lovell-Badge, R. (1998)
Development 125, 1967–1978
26 Wood, H.B. and Episkopou, V. (1999) Mech. Dev. 86, 197–201
27 Ying, Q.-L., Stavridis, M., Griffiths, D., Li, M. and Smith, A. (2003)
Nat. Biotechnol., in the press
28 Aubert, J., Dunstan, H., Chambers, I. and Smith, A. (2002)
Nat. Biotechnol. 20, 1240–1245
Received 6 September 2002
C 2003
Biochemical Society
49