MEF2C Enhances Dopaminergic Neuron Differentiation
of Human Embryonic Stem Cells in a Parkinsonian Rat
Eun-Gyung Cho1, Jeffrey D. Zaremba1, Scott R. McKercher1, Maria Talantova1, Shichun Tu1, Eliezer
Masliah2, Shing Fai Chan1, Nobuki Nakanishi1, Alexey Terskikh1, Stuart A. Lipton1,2*
1Del E. Webb Center for Neuroscience, Aging, and Stem Cell Research, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America,
2Department of Neurosciences, University of California San Diego, La Jolla, California, United States of America
Human embryonic stem cells (hESCs) can potentially differentiate into any cell type, including dopaminergic neurons to
treat Parkinson’s disease (PD), but hyperproliferation and tumor formation must be avoided. Accordingly, we use myocyte
enhancer factor 2C (MEF2C) as a neurogenic and anti-apoptotic transcription factor to generate neurons from hESC-derived
neural stem/progenitor cells (NPCs), thus avoiding hyperproliferation. Here, we report that forced expression of
constitutively active MEF2C (MEF2CA) generates significantly greater numbers of neurons with dopaminergic properties in
vitro. Conversely, RNAi knockdown of MEF2C in NPCs decreases neuronal differentiation and dendritic length. When we
inject MEF2CA-programmed NPCs into 6-hydroxydopamine—lesioned Parkinsonian rats in vivo, the transplanted cells
survive well, differentiate into tyrosine hydroxylase-positive neurons, and improve behavioral deficits to a significantly
greater degree than non-programmed cells. The enriched generation of dopaminergic neuronal lineages from hESCs by
forced expression of MEF2CA in the proper context may prove valuable in cell-based therapy for CNS disorders such as PD.
Citation: Cho E-G, Zaremba JD, McKercher SR, Talantova M, Tu S, et al. (2011) MEF2C Enhances Dopaminergic Neuron Differentiation of Human Embryonic Stem
Cells in a Parkinsonian Rat Model. PLoS ONE 6(8): e24027. doi:10.1371/journal.pone.0024027
Editor: Cesario V. Borlongan, University of South Florida, United States of America
Received April 20, 2011; Accepted August 4, 2011; Published August 25, 2011
Copyright: ? 2011 Cho et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by CIRM Comprehensive Grant RC1-00125-1, and NIH grants P01 HD29587, P01 ES016738 and R01 NS044326 (to
S.A.L.). The support of facilities by the La Jolla Interdisciplinary Neuroscience Center Cores, NIH Blueprint Grant P30 NS057096, is kindly acknowledged. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: S.A.L. is an inventor on a U.S. patent application filed by his institution related to this work describing the use of MEF2C for neurogenesis
from pluripotent stem cells. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: email@example.com
Human embryonic stem cells (hESC) are not only an important
model system for human neurogenesis [1,2] but are also a
potential source of therapeutic cells to treat neurodegenerative
disorders such as Parkinson’s disease (PD) [3–7], stroke [8,9], and
spinal cord injury [10–12]. Such cell-based therapies require
protocols for differentiating hESCs into neural precursors and
further directing them towards a specialized regional neurotrans-
mitter identity, such as midbrain dopaminergic neurons. However,
current methods for culturing hESCs suffer from contamination
with animal feeder cells or cells of mesodermal and endodermal
origin from embryoid bodies (EB) . Furthermore, hyperpro-
liferation, teratoma formation, and the presence of neuroepithelial
cells are commonly observed after transplantation of hESC-
derived neural cells into disease models [7,14,15]. Hence, in vitro
induction of hESCs into more restricted neural stem/progenitor
cells (hNPCs) prior to transplantation is necessary in order to
promulgate therapeutic use in humans. A method of directed
differentiation into neurons would facilitate transplantation
Along these lines, the transcription factor myocyte enhancer
factor 2 (MEF2) is a central regulator of gene expression in skeletal
and heart muscle , neural crest, lymphocyte development,
vascular integrity , and, as we and others have previously
shown, neuronal differentiation and survival [18–21]. Among
MEF2 family genes in the developing central nervous system
(CNS) [22,23], only MEF2C, initially discovered in our laboratory,
is expressed in a tissue- and region-restricted pattern [20,24–26].
Recently, we and others discovered that MEF2C influences NPC
differentiation and maturation into neurons during embryonic
development , and facilitates plasticity by negatively regulating
synaptic number and function in mature rodents in vivo .
Negative regulation of synaptic morphogenesis has also been
demonstrated by deleting MEF2A or MEF2D in hippocampal
neuronal cultures and rat cerebellar brain slices [21,28].
Furthermore, our previous report demonstrated that MEF2C
directs the differentiation of mouse ESC-derived neural precursors
into neurons and suppresses glial fates . In addition to this
putative instructive role for neurogenesis, we found that MEF2C
promotes cell survival during neuronal differentiation .
Concerning differentiation of hESCs into dopaminergic (DA)
neurons, a simple cis-regulatory element called the DA motif has
recently been reported that binds ETS transcription factors and
controls the expression of virtually all DA-neuron related genes
. In the present study, we noted that there were multiple
MEF2 consensus sites in the promoter of a prominent ETS-family
member, Etv1. Similarly, we found that an important downstream
PLoS ONE | www.plosone.org1August 2011 | Volume 6 | Issue 8 | e24027
transcription factor involved in the DA phenotype, nuclear
receptor related 1 (nurr1) [31,32], also contained several MEF2
binding sites in its promoter region. These observations suggested
to us that MEF2C might be involved in DA neurogenesis. Unlike
Etv1 or nurr1, however, MEF2C also manifests anti-apoptotic,
neuroprotective properties, which could prove useful in keeping
transplanted stem cells alive . Heretofore little was known
about the role of MEF2C in human neurogenesis in general and in
DA neuron development in particular.
Here, we report a feeder- and animal factor-free, sphere-based,
rosette isolation protocol to derive the neural cell lineage from
hESCs with high efficiency and purity. Using these cells, we
demonstrate the ability of MEF2C to drive neurogenesis in vitro
using shRNAs directed at MEF2C or overexpression of a
constitutively active MEF2C (MEF2CA) transgene. Further, we
show the regulatory role of MEF2C in vivo in directing
differentiation of DA neurons and the therapeutic potential of
hESC-derived NPCs engineered with MEF2CA in a rat model of
Feeder-Free Differentiation of hESCs into the Neural
H9 hESCs were passaged by manual microdissection and
monitored for normal karyotype . hESCs were maintained on
Hs27 human fibroblasts but changed to feeder-free conditions
when differentiated to facilitate cell-based therapies in humans
. To obtain high purity hNPCs derived from hESCs (.95%
nestin positive), we established an efficient differentiation method
using a feeder-free, neurosphere-based protocol in N2/B27
medium to isolate rosettes (Figure 1A; see Methods for detailed
protocols). We verified the differentiation of these cells into the
various neural lineages by immunocytochemistry (Figure 1B) and
quantitative RT-PCR (qPCR, Figure S1A and S1B). Specifically,
from hESCs (Figure 1A inset, Oct4+), we derived neuroectoder-
mal spheres (NES), which harbor rosettes that stain for nuclear
Pax6 and Sox2 (Figure 1B). To increase the purity and
homogeneity of cells leading to various neural lineages, NES were
allowed to attach to the substrate, and rosettes were visualized
prior to mechanical isolation. We subsequently dissociated and
plated these rosettes, which are known to contain neural stem cells
(R-NSCs) [1,35,36,37,38], to allow them to develop into
homogeneous NPCs in monolayer cultures (Figure 1A), as
evidenced by their expression of nuclear and cytoplasmic
Musashi1 and cytoplasmic nestin (Figure 1B). These NPCs
(designated hESC-NPCs) were dissociated and replated. Based
on our observations monitoring differentiation of these cells, we
divided development during this final plating into Neural Stage I
(1 to 14 days post plating), Neural Stage II (15 to 28 days post
plating), and Neural Stage III (.28 days post plating). This
protocol produced the various neural lineages (Figure 1B),
corresponding temporally to normal development in vivo. We
found that neurons differentiated first, beginning in Neural Stage I
(as evidenced by immunostaining for doublecortin (DCX) and
microtubule associated protein-2 (MAP2). During Neural Stage II,
we observed more mature neurons, as evidenced by staining for
NeuN, synaptophysin, and postsynaptic density protein 95
(PSD95). By Neural Stage III, neuronal processes displayed
clustering of PSD95 with Synapsin I, suggesting the formation of
functional synaptic contacts (Figure S1C). In this final stage, we
also found evidence for the differentiation of astrocytes (S100b+
cells), and finally oligodendrocytes (29,39-cyclic nucleotide 39-
phosphodiesterase (CNPase)+ cells). Thus, our protocol produced
Figure 1. Neural differentiation of human embryonic stem cells (hESCs). (A) Phase-contrast images were taken during various stages of
differentiation in vitro and represent cell morphology of each developmental step. Arrows indicate rosettes that formed on laminin-coated dishes.
hESC, human embryonic stem cell; NES, neuroectodermal sphere; R-NSC, rosette-neural stem cell; NPC, neural progenitor cell (see Materials and
Methods). (B) Representative cells from the NES, NPC, Neural I, Neural II, and Neural III stages of development were fixed and stained with the
following lineage-specific antibodies. For NPCs: Msi1 (musashi 1) and Nestin. For neurons: DCX, MAP2, NeuN, Synp (synaptophysin), and PSD95. For
astrocytes: S100b. For oligodendrocytes: CNPase. DNA stained with DAPI (blue). Scale bar: 25 mm.
MEF2C Enhances DA Neuron Differentiation of hESCs
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all three neural lineages from highly homogenous and expandable
MEF2 Proteins Show Differential Expression during
Neural Differentiation from hESCs
To begin to dissect the role of the various isoforms of MEF2
during neural differentiation of hESCs in vitro, we performed
qPCR at various time points (Figure 2A). Expression of MEF2C
message was detected at low levels in hESCs. As neural
differentiation proceeded, the expression of MEF2C gradually
increased through the NPC stage, declined as neurons began to
appear (Neural Stage I), and thereafter increased again. Com-
pared to MEF2C expression, MEF2D increased later, beginning at
the R-NSC stage, and then more dramatically in mature neurons
(Neural Stage II). Though widely expressed in adult brain,
expression of MEF2A remained relatively quite low during our
differentiation protocol. These data are consistent with the notion
that MEF2C is potentially important early in neurogenesis from
We next examined the relative protein expression by immuno-
blot analysis of the MEF2 family of transcription factors during
differentiation (Figure 2B). The neuronal postsynaptic protein
PSD95 was highly expressed in mature neurons (Neural Stage II),
and was hence used as a putative marker for this stage. We
detected MEF2C and MEF2D proteins at the NES stage, with a
gradual increase through development of NPCs. MEF2C showed
relatively higher expression in R-NSCs and NPCs, while MEF2D
showed the highest level later at Neural Stage II. MEF2A protein
was barely detectable. Protein expression closely matched that of
mRNA as assessed by qPCR.
Knockdown of MEF2C at the R-NSC Stage Causes Decline
or Delay in Neurogenesis
Because we had found that MEF2C expression was highest in
hNPCs in monolayer cultures, we hypothesized that MEF2C
might be causal in triggering neurogenesis at this stage (an effect
distinct from the reported effect of MEF2 transcription factors on
synapse formation by mature neurons) [21,28]. To test this
premise, we introduced lenti-shRNAs against MEF2C (lenti-
shMEF2C) into R-NSCs prior to the development of NPCs. Each
lenti-shMEF2C construct was validated using a target reporter
containing the coding region and 39 UTR of MEF2C. Lenti-
shMEF2C-1 exhibited stronger suppression of MEF2C than lenti-
shMEF2C-2 (Figure S2A and S2B). Given that MEF2C is also a
known cell-survival factor [29,39] and that smaller-than-normal
neurospheres were observed after infection with lenti-shMEF2C-1
virus (Figure S2C), we initially quantified cell death using the
TUNEL assay at different time points after lenti-infection. At 14
days post infection (dpi, during the NPC stage), lenti-shMEF2C-
1—infected cells displayed an ,2-fold higher level of cell death
than scrambled control-infected cells or lenti-shMEF2C-2—
infected cells (Figure 3A), consistent with an early effect of
MEF2C on cell viability. However, by 33 dpi (during Neural Stage
II), the degree of cell death after lenti-shMEF2C-1 infection was
no different than with the other constructs.
As an index of neurogenesis, we therefore examined expression
of MEF2C and the neuronal marker MAP2 at 33 dpi when cell
viability was stable. We found a significant reduction in both
MEF2C and MAP2 mRNA expression and protein levels after
infection with lenti-shMEF2Cs (Figure 3B and 3C). Next, we
MEF2C knockdown. Scrambled control-infected cells manifested
substantial numbers of MAP2+ neurons under conditions fostering
terminal differentiation, while the vast majority of lenti-shMEF2C-
1-infected cells did not show neuronal morphology (Figure 3D).
Quantitative assessment of these results showed that MAP2+
neurons, co-stained with GFP to indicate lenti-infection, under-
went far less neuronal differentiation if the cells were infected with
lenti-shMEF2C-1- or -2 compared to control infection (Figure 3E).
As expected since shMEF2C-1 is more effective than shMEF2C-2
in knocking down MEF2C, we observed a dose-dependent effect
of knocking down MEF2C with regard to inhibiting neurogenesis.
Additionally, scrambled control-infected cells displayed long
Figure 2. Endogenous expression of MEF2 gene products during neural differentiation. Total RNA or cell lysate was prepared from cells at
the hESC, NES, isolated rosette, R-NSC, NPC monolayer, Neural I, or Neural II stages for quantitative RT-PCR (A) or immunoblot analysis (B). Arrowhead
indicates endogenous MEF2C. Anti-PSD95 antibody was used to assess neuronal differentiation. Histogram values are mean + SEM, n=3; *p,0.001
compared to hESC by ANOVA.
MEF2C Enhances DA Neuron Differentiation of hESCs
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dendritic processes and clearly-defined dendritic spines, whereas
lenti-shMEF2C-1-infected neurons manifested shortened den-
drites and a virtual lack of spines (Figure 3F, boxed regions
magnified below), as verified by quantitative assessment (Figure 3G
and 3H). These data are consistent with the notion that MEF2C is
required for the full program of neuronal differentiation and
maturation from hESC-derived R-NSC/NPCs.
Constitutively Active MEF2C (MEF2CA) Increases
Neurogenesis in hESC-Derived R-NSC/NPCs
Genetic manipulation of transcription factors has been suggest-
ed previously as a strategy to generate sufficient numbers of
appropriately directed hESC-derived cells for transplantation
[40,41]. In a prior report, we showed that murine ESC-derived
NPCs differentiate virtually exclusively into neurons and are
protected from apoptosis when stably transfected with MEF2CA
. Therefore, we asked if MEF2CA expression would also direct
or accelerate neurogenesis from NPC-derived hESCs and increase
survival of the neurons thus generated. For this purpose, using our
in vitro protocol we transduced cells at the R-NSC stage with
control or lenti-MEF2CA viral vectors (Figure S3A-S3C), placed
the infected cells under terminal neural differentiation conditions,
and examined the cells at four subsequent time points (Figure
S3D). As a further control, R-NSCs were transduced with the anti-
apoptotic construct lenti-Bcl-xL to allow us to distinguish between
the pro-survival and neurogenic functions of MEF2C. Our
infection efficiency in these experiments was 35–45% and not
statistically different among the various test groups, as determined
by anti-GFP antibody staining (Figure S3E). We initially examined
the effect of MEF2CA on neuronal differentiation by co-staining
Figure 3. Negative effect of MEF2C knockdown on neurogenesis. (A) TUNEL assay on NPCs at 14 dpi and on Neural Stage II cells at 33 dpi
with control or lenti-shMEF2C shRNAs. Lenti-shMEF2C-1-infected cells show increased apoptotic cell death at 14 but not 33 dpi. Values are mean +
SEM, n=6; ***p,0.001 by ANOVA. dpi: days post infection. (B, C) Endogenous MEF2C and MAP2 expression at 33 dpi by qPCR and immunoblot.
Values are mean + SEM, n=3; **p,0.01, ***p,0.001 by ANOVA. (D) Fewer MAP2+ (red) neurons after lenti-MEF2C-1 shRNA versus scrambled shRNA
(GFP+/green) at 33 dpi indicate absent or delayed neurogenesis at Neural Stage II. (E) Percentage of MEF2C shRNA-transduced R-NSCs (total GFP+
cells) differentiated into neurons (MAP2+/GFP+ cells). Values are mean + SEM, n$140 cells scored for each condition; **p,0.01, ***p,0.001 by
ANOVA. (F) Shorter dendrites, fewer dendritic spines in lenti-shMEF2C-1-infected cells versus scrambled control. shRNA-infected cells are GFP+
(green), neuronal dendrites are MAP2+ (red); co-labeling (yellow) indicated by asterisks; dendrites in boxes magnified to show spines. DAPI (blue).
Scale bars: 25 mm. (G) Dendritic length using Neuron J software (dendrites scored if $2-fold longer than diameter of cell body). Values are mean +
SEM, n=32 cells; **p=0.003 by t-test. (H) Numbers of dendritic spines for lenti-MEF2C-1 shRNA-transduced neurons and scrambled control (spines
scored along entire length of dendrite). Values are mean + SEM, n=17 cells; ***p,0.0001 by t-test.
MEF2C Enhances DA Neuron Differentiation of hESCs
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with neuronal-specific anti-DCX antibody (Figure 4A), and we
found that the MEF2CA-infected cells produced 3.2-fold as many
neurons as the control groups by 32–35 dpi (during Neural Stage
II; Figure 4B). Additionally, these lenti-MEF2CA—infected cells
manifested long dendritic processes (Figure 4A, right-hand panel),
with a 1.7-fold increase in mean dendritic length over control or
Bcl-xL—expressing neurons (Figure 4C). For comparison, we
obtained similar effects on human fetal brain-derived neural
progenitors infected with lenti-MEF2CA (4.4-fold increase in
neuronal marker expression and 2.8-fold longer dendritic
processes; Figure S4A–S4D). We further evaluated the effect of
MEF2CA on hESC-derived NPCs by examining the level of
several neuronal-specific proteins. Compared to controls, lenti-
MEF2CA-infected cells manifested increased levels of MAP2ab,
MAP2c, and tau (Figure 4D).
To characterize the functional capabilities of neurons derived
from lenti-MEF2CA expression in R-NSCs, we made electro-
physiological recordings on cells five to nine weeks after infection
(Neural Stage III). During whole-cell recordings with patch
electrodes, these MEF2CA-programmed cells manifested tetrodo-
Figure 4. Enrichment in neuronal markers and electrophysiological activity of cells derived from MEF2CA-expressing R-NSC/NPCs.
(A) Lentiviral-infected cells plated on poly-L-ornithine/laminin. Anti-GFP antibody identifies infected cells and anti-DCX antibody identifies early
neurons. Scale bars: 25 mm. (B) Neuronal enrichment engendered by MEF2CA (n=4 experiments; scheme for infection and analysis shown in Figure
S3D). In each experiment, ,300 GFP-positive cells were scored. Values are mean + SEM, n$1200 cells counted; ***p,0.001 by ANOVA. (C) Longest
neuronal process per cell at 33 dpi during Neural Stage II, measured with Neuron J software. Values are mean + SEM, n=61; ***p,0.0001 by ANOVA.
(D) Neuronal-specific proteins in lenti-MEF2CA-infected cells by immunoblot during Neural Stage II. Note the much stronger GFP expression in
control cells because GFP was expressed from a single gene construct rather than in tandem with MEF2CA and IRES . MEF2CA increased
expression of neuronal proteins compared to control infection. (E) Whole-cell recordings of lenti-MEF2CA-infected cells with patch electrodes
revealed sodium currents evoked by 100 ms depolarizing steps from 260 to +80 mV in 20 mV increments following 300 ms prepulse to 290 mV
(n=5); these currents were inhibited by tetrodotoxin (TTX). (F) Application of 10 pA current steps resulted in depolarization and generation of a
‘‘train’’ of action potentials during current-clamp recordings in n=3 of 8 (37.5%) MEF2CA-infected cells. (G, H) GABA-evoked currents were observed
in n=5 of 7 cells (71.4%) and NMDA-evoked currents in n=3 of 3 cells recorded (100%).
MEF2C Enhances DA Neuron Differentiation of hESCs
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toxin-sensitive sodium currents under voltage clamp (Figure 4E).
During current-clamp recording, application of a 10 pA current
step resulted in depolarization and generation of a ‘‘train’’ of
action potentials (Figure 4F). Additionally, these neurons displayed
ligand-gated currents evoked by GABA (Figure 4G) or NMDA
plus co-agonist glycine (Figure 4H). These data indicate that the
MEF2CA-programmed neurons were electrophysiologically func-
tional and could respond to neurotransmitters.
MEF2CA Increases DA Neuron-Specific Markers In Vitro
In the absence of exogenous MEF2CA, we found that our
protocol for differentiating R-NSC/NPCs resulted in increased
expression of endogenous MEF2C (Figure 2A) in addition to some
degree of expression of TH+ dopaminergic neuronal markers in
vitro (Figure 5A). For example, DA neuron-related mRNAs
encoding nurr1, engrailed-1 (EN1) and TH began to increase at
the R-NSC stage (Figure 5B). With further development, hESC/
Figure 5. Dopaminergic characteristics of control vs. MEF2CA-expressing hESC-derived neuronal cells. (A) hESC-derived cells at Neural
Stage III stained for MAP2 (red/neurons), tyrosine hydroxylase (TH/green, DA phenotype; lower panel magnified). Some TH+ cells did not express
MAP2 . Scale bars: 25 mm. (B) Relative mRNA levels of DA-specific genes at various stages. Values: mean + SEM, n=3; *p,0.05 by ANOVA. EN1,
engrailed-1. (C) EN1+ or TH+ cells expressed VMAT2 (vesicle monoamine transporter 2) and DAT (dopamine transporter). Scale bars: 25 mm. (D)
Expression of GIRK2/DAT+ and CD28k/TH+ cells. (E) Control vector- and MEF2CA-infected cells (GFP+/green) stained for TH (red, left-hand panels) or
EN1 (red, right-hand panels), with double-labeled cells (yellow) predominating in the MEF2CA group. Scale bars: 50 mm for TH; 25 mm for EN1. (F)
Percentage of MEF2CA/R-NSCs vs. control cells (GFP+) differentiated to DA neurons during Neural Stage I at 17 dpi (TH+, left graph) and Neural Stage
II at 32 dpi (EN1+, right graph). Values: mean + SEM, n$1200 TH cells, n$500 EN1 cells; **p=0.001, ***p=0.0007 by t-test. (G) qPCR shows relative
amount of DA gene expression during Neural Stage I at 13 dpi. Values: mean + SEM, n=3; **p,0.01, ***p,0.001 by ANOVA; endo MEF2C:
endogenous MEF2C. (H) qPCR shows expression of Etv1 at different stages. Values: mean + SEM, n=3; **p,0.01 by ANOVA. (I) MEF2C activation of
2.1 kb Etv1 promoter-luciferase construct in HeLa cells. Values: mean + SEM, n=6; *p,0.05, ***p,0.001 by ANOVA. MEF2DN: dominant negative
MEF2C; MEF2C: full-length MEF2C; MEF2CA: constitutively active MEF2C. (J) ChIP analysis of MEF2C association with the Etv1 promoter. Values: mean
+ SEM, n=3; ***p,0.001 by t-test.
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NPC-derived TH+ neurons achieved a degree of functional
maturation in culture, as evidenced by their expression of vesicle
monoamine transporter 2 (VMAT2) and dopamine transporter
(DAT), although the expression of DAT was weak and observed
only in a minority of cells (Figure 5C). Additionally, during
neuronal maturation we found evidence at both the message and
protein levels for expression of G-protein-gated inwardly rectifying
K+channels (GIRK2) and calbindin-D28k (CD28k), which are
known to be present in DA/TH+ neurons in the midbrain
substantia nigra (A9) and ventral tegmental area (A10), respec-
tively (Figures 5D and S5A) . Under our culture conditions, by
Neural Stage III 54.663.0% of the MAP2+ cells were also TH+, a
comparable or somewhat higher proportion than obtained with
previously published protocols [6,7,43].
Because the level of endogenous MEF2C expression in R-
NCS/NPCs correlated with the subsequent development of DA
markers, we next tested if overexpression of MEF2CA would
further enhance the DA phenotype. We found that infection with
lenti-MEF2CA significantly increased the expression of these DA
neuron-related genes and the proportion of anti-EN1/TH—
labeled neurons. For example, during Neural Stage I at 17 dpi
(Figure 5E, left-hand panels), we observed that lenti-MEF2CA
infection yielded 2.4-fold more TH+ neurons than control-
infected cells (Figure 5F, left graph). During Neural Stage II at
32 dpi (Figure 5E, right-hand panels), the proportion of EN1+
cells increased approximately four-fold in lenti-MEF2CA—
infected cells compared to control infection (Figure 5F, right
graph). To further characterize the effect of MEF2CA, we
performed qPCR for DA-related gene products (Figure 5G).
During Neural Stage I at 13 dpi, MAP2 expression was increased
in lenti-MEF2CA—infected cells by 6.2-fold, and EN1 and TH
mRNAs were upregulated by 4.1- and 3.2-fold, respectively. By
Neural Stage III at 40 dpi, lenti-MEF2CA-infected cells also
manifested an increase in nurr1 expression (Figure S5B);
endogenous MEF2C increased as well, probably because of the
presence of multiple MEF2 sites in its own promoter (Figure
S5C). Taken together, these data indicate that feeder-free/
neurosphere-based neural differentiation of hESCs can generate
DA neurons spontaneously, and MEF2C expression can signi-
ficantly enrich this process.
Next, we characterized the molecular pathway whereby MEF2
drives the DA phenotype. The activity of ETS transcription factors
has been associated with the DA phenotype [30,44]. Interestingly,
we noted that Etv1, an ETS factor proven to be related to DA
neurogenesis in the mouse brain in vivo , has several MEF2
binding sites in its enhancer/promoter (Figure S5C). Hence, we
investigated whether MEF2C can increase Etv1 transcription,
accounting, at least in part, for the involvement of MEF2C in DA
neurogenesis. We found that Etv1 expression increased 2.1-fold in
NPCs after lenti-MEF2CA transduction, but not in cells at Neural
Stage I or Neural Stage III (Figure 5H). This finding suggests that
MEF2C affects Etv1 at an early time point. Therefore, we
examined if MEF2C directly regulates Etv1 expression. When
MEF2C constructs were transiently co-expressed with an Etv1
promoter-driven luciferase reporter construct, we observed that
full-length MEF2C and MEF2CA increased Etv1 expression by
1.7- and 10-fold, respectively (Figure 5I). We then confirmed the
direct association of MEF2C with the Etv1 promoter region by
chromatin immunoprecipitation (ChIP, Figure 5J). Additionally,
we noted that the promoters of several other DA-related genes,
including nurr1, EN1 and TH, all possess consensus MEF2
binding motifs (Figure S5C). We found that MEF2C bound to the
nurr1 promoter and dramatically up-regulated nurr1 expression at
the transcriptional level (Figure S5D and S5E).
Parkinsonian Rats Transplanted with MEF2CA-Expressing
R-NSCs Manifest Improved Motor Function
Since we found that MEF2C drives the development of the DA
phenotype in vitro, we next asked if hESC-derived cells that had
been programmed with MEF2CA would prove more beneficial in
vivo than unprogrammed hESC-derived cells when used in a
transplantation paradigm to improve motoric function in a rat
model of PD. For this purpose, we injected MEF2CA-expressing
R-NSCs into the striatum in an attempt to improve behavioral
deficits due to 6-hydroxydopamine (6-OHDA)-induced lesions.
hESC-derived R-NSCs, prepared as described above, were first
dissociated and infected with control- or lenti-MEF2CA viral
constructs (Figure 6A). The infected R-NSCs were then grown for
7–10 days as nestin/musashi1-positive neurospheres and dissoci-
ated just prior to transplantation. Aliquots of the neurospheres
were plated onto dishes to evaluate infection efficiency. Under our
conditions, control- and lenti-MEF2CA/R-NSCs displayed 75.6
6 4.6% and 66.9 6 5.3% GFP+ cells, respectively (Figure S6A
and SB). The fact that infection efficiency was less than 100% in
this paradigm was useful because uninfected cells served as an
internal control in the transplantation experiments.
The ipsilateral striatum of 6-OHDA-lesioned rats was implant-
ed with 500,000 control lentiviral- or lenti-MEF2CA—infected R-
NSCs (n=14). At various time points, starting two weeks after
transplantation, the rats were challenged with apomorphine to
induce rotation for behavioral/motor testing. Over time, rats
receiving control-infected/R-NSCs exhibited a decrease in
apomorphine-induced rotations compared with pre-transplanta-
tion values (Figure 6B, Control). Notably, with time rats
transplanted with MEF2CA/R-NSCs exhibited an increasing
reduction in the number of apomorphine-induced rotations
compared to rats transplanted with control stem cells, an effect
that reached statistical significance by eight weeks (p#0.035;
Figure 6B). Therefore, axial function significantly improved in
Parkinsonian rats after transplantation of lenti-MEF2CA/R-NSCs
compared to control-infected R-NSCs. We also conducted a
second motor test to confirm these findings. In this case, the
‘cylinder asymmetry paw use test’  was performed 9 weeks post
transplantation to measure forelimb preference during vertical
exploration. Lenti-MEF2CA/R-NSC—transplanted rats exhibit-
ed a significantly lower asymmetry score compared to control-
infected R-NSCs (p,0.03; Figure 6C). Hence, rats transplanted
with MEF2CA/R-NSCs showed significantly less preference for
use of the paw on the non-lesioned side than control rats.
Together, these behavioral paradigms indicate that the PD rats
receiving MEF2CA/R-NSCs were significantly improved com-
pared to controls.
Engrafted MEF2CA-Derived R-NSCs Exhibit Increased
Survival and Greater Numbers of DA Neurons in
Next, we sought histological evidence for improvement after
transplantation of lenti-MEF2CA/R-NSCs vs. control-infected R-
NSCs. After neurobehavioral testing, four rats were chosen at
random from each group, sacrificed, and their brains analyzed in
detail for survival, integration, and differentiation of the
transplanted cells. After transplantation of control-infected/R-
NSCs, we observed extensive engraftment into the host rat brain,
as shown by the presence of GFP+ cells (Figure 6D). Compared to
host cells, the engrafted cells appeared smaller in size with a more
irregular arrangement upon hematoxylin and eosin staining
(Figure 6D, inset). Under epifluorescence microscopy, the identity
of the transplanted cells was verified by the presence of human
nuclear antigen (HNA) and expression of GFP (Figure 6E and 6F).
MEF2C Enhances DA Neuron Differentiation of hESCs
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We next examined if MEF2CA-programmed vs. control R-
NSCs could generate neurons of DA phenotype in vivo after
transplantation. First, we examined the grafts by staining with anti-
TH antibody using peroxidase immunohistochemistry, and we
observed more TH+ cell bodies and processes in the MEF2CA/R-
NSC—transplants vs. control-transplants (Figure 6G). Not only
were there more TH+ cells in the MEF2CA/R-NSC group,
consistent with the notion that the implanted stem cells had
become DA neurons, but there were also more TH+ fibers in the
adjacent host tissue (Figure 6G, blue box, and Figure S7). Since
no GFP+ fibers (representing transplanted cells) were seen outside
of the graft (Figure 6D), these TH+ fibers adjancent to the
transplant must have been of host origin. This latter finding
suggests a non-cell autonomous or trophic effect of transplanting
MEF2CA/R-NSCs on nearby host cells. Because the implanted
grafts also contained uninfected cells, in addition to staining with
anti-TH antibody, we co-stained with anti-GFP to identify the
infected cells, and then counted the number of TH+/GFP+ DA
neurons (Figure 6H). The infected MEF2CA/R-NSCs contained
15.1 6 1.7% TH+ neurons (representing, in absolute numbers,
20,535 6 3,630 TH+/mm3), whereas control-infected/R-NSCs
contained only 1.9 6 0.9% TH+ neurons (2,605 6 1,116 TH+/
mm3) (Figure 6I).
We then further analyzed the cellular phenotypes of the
engrafted/infected cells. We found that 47.4 6 5.9% of control-
infected/R-NSCs versus 67.0 6 5.7% of MEF2CA/R-NSCs
differentiated into human neuronal protein-positive (HuC/D+)
neurons in the rat brain within twelve weeks of transplantation
(Figure 7A and 7B). Additionally, 22.8 6 5.2% versus 9.7 6 2.7%
of control- and MEF2CA-infected cells became GFAP+ astrocytes,
respectively (Figure 7C, and 7D). These results indicate that
MEF2CA expression was significantly better than control (p,0.03)
in enhancing differentiation into neurons over astrocytes in vivo.
Most importantly, over a greater than six-month period we did
not observe cellular hyperproliferation, hESC-derived teratomas,
or Oct4/alkaline phosphatase-positive cells developing from
MEF2CA/R-NSCs transplanted into the rat brain. Using anti-
proliferating cell nuclear antigen (PCNA) staining as an index of
proliferation, we observed ,1% proliferation of these engrafted
cells twelve weeks after transplantation (Figure S6C), less than that
previously reported for growth factor-induced TH+ differentiation
from hESCs at this time point [6,7]. In contrast, twelve weeks after
Figure 6. Functional recovery of 6-OHDA-lesioned rats and generation of DA neurons after MEF2CA-derived R-NSC transplants.
(A) Infection/transplantation scheme (cf. Fig. 1A). (B) Apomorphine-induced rotations in 6-OHDA-lesionsed rats after R-NSC transplantation into the
striatum. Values are mean + SEM, n=14. Rats receiving MEF2CA/R-NSCs show increasing improvement versus controls (*p#0.035 by ANOVA with
planned comparisons post-hoc test). (C) Cylinder asymmetry test 9 weeks post-transplant. Forelimb use in transparent cylinder for 10 min. For score
calculation, see Text S1. Values are mean + SEM, n=11; *p,0.03 by t-test. (D) Transplanted control R-NSCs (GFP+/green); sagittal brain sections along
mediolateral axis. Hoechst dye-stained DNA (blue). Scale bar: 1 mm. Inset: Hematoxylin/eosin (H&E) stain shows xenograft/host boundary (dashed
line). Xeno: xenograft; STR: striatum; LV: lateral ventricle. (E) Human origin of cells verified by human nuclear antigen (HNA). Scale bar: 25 mm. (F)
Infected cells (GFP+) co-expressing HNA (red) to yield merged yellow fluorescence (remaining GFP positivity represents cell debris). Scale bar: 25 mm.
(G) Density of TH+ neurons in MEF2CA/R-NSC xenografts (xeno) close to the ventral area of the striatum was higher than for control/R-NSCs (boxed
areas enlarged in right panels). Arrowheads: TH+ neuronal cell bodies; arrows: neuronal processes; dashed lines: graft/host boundary based on H&E
staining; blue dashed box: outlines endogenous host TH+ fibers (quantified in Figure S7). (H) MEF2CA/R-NSC versus control/R-NSC DA/TH+ neurons.
Transplanted cells are GFP+; TH+ cells are red. Scale bar: 25 mm. (I) TH+ neurons in grafts as percent of GFP+ cells (upper panel). Density of TH+/GFP+
cells in grafts as absolute cell number (lower panel). Values are mean + SEM, n=19; ***p,0.0001 by t-test.
MEF2C Enhances DA Neuron Differentiation of hESCs
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transplantation, control-infected R-NSCs frequently manifested
features of hyperproliferation such as rosette formation. These
changes were visualized as a radial arrangement of cells after
staining with either Hoechst dye or hematoxylin/eosin, and these
rosettes contained control-infected/GFP-positive cells (Figure 7E,
Control; rosettes marked by asterisks). Although we also observed
rosettes in brains injected with MEF2CA-infected/R-NSCs,
MEF2CA-infected/GFP-positive cells were never found within
the rosettes (Figure 7E, MEF2CA; asterisks). As stated above, the
brains transplanted with MEF2CA-infected (GFP-positive)/R-
NSCs also received a percentage of uninfected (GFP-negative)/
R-NSCs; it was in fact these uninfected R-NSCs that formed the
hyperproliferating rosettes in the grafts. These data indicate that
R-NSCs not containing MEF2CA retained an undifferentiated
phenotype after transplantation and underwent hyperprolifera-
tion, unlike R-NSCs expressing MEF2CA.
Parkinson’s disease (PD) is currently treated with dopamine
agonists, although fetal cell-based therapy has also been attempted
[46–48]. Pharmacological agents treat symptoms but do not
restore DA neurons in PD patients. In addition, long-term
treatments with dopamine agonists such as
dyskinesias and eventually become ineffective. To overcome these
disadvantages, cell therapy using fetal mesencephalic brain tissue
has been employed, but the results have been mixed and largely
unsatisfactory. One reason for the failure of such transplants is
graft-induced dyskinesias; in fact, many of these grafts contain
more serotonin and GABAergic neurons than DA neurons. Both
pulsatile delivery of DA agonists and upregulation of GABAA
receptors have been shown to contribute to dyskinesias in primates
, so we reasoned that improved production of DA neurons
Figure 7. Neuronal differentiation of MEF2CA-infected R-NSCs in the striatum of 6-hydroxydopamine—lesioned rats. (A, C) Twelve
weeks after transplantation, infected (GFP+) cells were analyzed with cell-type specific antibodies (anti-HuC/D for neurons and anti-GFAP for
astrocytes, although neural progenitors can also be labeled with this marker). (B, D) Quantification of neuronal and astrocytic markers in control- and
MEF2CA-infected cells. Ten random fields were selected in multiple sections at the same distance from the Bregma for each rat (n=4). Values are
mean + SEM, n=10; *p,0.03 by t-test. (E) In transplanted brains, several regions manifested rosette structures (asterisks), consistent with
hyperproliferation. For control-infected/R-NSC transplants (left-hand and middle panels), the GFP+ cells (arrows) were located within rosettes.
MEF2CA/R-NSC transplants contained both MEF2CA-expressing cells (GFP+, green) as well as uninfected R-NSCs, but GFP+ cells (indicated by arrows)
were located exclusively outside of the rosettes (right-hand panel). All scale bars: 25 mm.
MEF2C Enhances DA Neuron Differentiation of hESCs
PLoS ONE | www.plosone.org9 August 2011 | Volume 6 | Issue 8 | e24027
might improve this situation. An additional problem has been
extensive cell death in the grafts . Therefore, strategies to
enrich for DA neurons, especially of the A9 subtype rather than
other neuronal types, and to prolong cell survival are likely to
result in improved cell-based therapies for PD.
hESC-derived DA neuronal precursors have been considered as
an alternative source for cell-based therapies in PD. Although this
approach holds promise, hESC-based therapies face several
hurdles, including death of engrafted cells, failure of migration/
incorporation into host brain, lack of differentiation into
appropriate neuronal cell types, and formation of tumors after
transplantation. One technical advance that we propose here to
overcome these problems is the forced expression of a transcrip-
tion factor that drives more restricted neuronal lineage choices
from ESCs, while also fostering survival of the differentiated cells.
In this study, we present evidence that MEF2C represents a
transcription factor that can accomplish these goals. Our feeder-
free/neurosphere-based protocol for isolating rosettes followed by
transduction with MEF2CA addresses many of the issues
associated with the production of human stem cells for therapeutic
use in the brain. This approach is particularly well suited for PD
since, unlike previous methods , a substantial number of
dopaminergic neurons can be generated in the absence of
hyperproliferating cell types. Previously, we and others have
shown that the MEF2 transcription factors, with MEF2C the
predominant isoform during early brain development, represent a
family of activity-dependent neurogenic effectors that enhance
survival and neuronal differentiation [18–20,50]. Moreover, we
have recently shown that conditional knockout of Mef2c in mice
during very early brain development – at the NPC stage – impairs
neurogenesis, migration, and synaptogenesis in vivo, resulting in a
behavioral phenotype resembling Autism-spectrum disorders .
Here, we initially investigated the function of MEF2C during
early neurogenesis of hESCs in vitro by knocking down endogenous
MEF2C using shRNAs at the R-NSC/NPC stage. At this stage,
MEF2C expression was maximal, and cells homogenously
expressed nestin and musashi1. As a result, we found a reduction
in the number of hESC-derived neurons and dendritic/synaptic
spines on those neurons, fitting well with our previous in vivo results
in mice when we knocked out MEF2C at the NPC stage .
Conversely, when we overexpressed MEF2CA in vivo, we drove
neurogenesis from hESC-derived R-NSCs/NPCs, specifically
producing an enrichment of the dopaminergic phenotype. We
found that MEF2C-mediated neurogenesis is not simply due to
neuronal survival. For example, to distinguish neurogenic vs.
survival effects of MEF2CA, we included Bcl-xL as a control and
found that it did not significantly increase neuronal differentiation.
Previously, both growth factors and transcription factors have
been used to increase the percentage of NSCs that express
neuronal and dopaminergic phenotypes [5,51], but no method has
proven to be totally effective. In this regard, a critical
consideration is that if 100% of the hESCs do not become
terminally differentiated as neuronal cells, then hyperproliferation
can occur with possible tumorigenic potential. In contrast, with
our approach, the great majority of cells receiving MEF2CA
became neuronal, which can overcome this conundrum. Thus, our
findings suggest that MEF2C is an effective driver of neurogenesis
and in the proper context its expression prevents hyperprolifera-
tion. Additionally, our results show that under our conditions
expression of MEF2C produces substantial enrichment in the
dopaminergic phenotype. We show that the mechanism for this
dopaminergic effect includes up-regulation by MEF2C of two
additional transcription factors, nurr1 and the ETS family
member Etv1. In the future, with more efficient transduction of
MEF2CA or sorting of MEF2CA-positive cells, coupled with
transient expression of MEF2CA only during the critical
neurogenesis stage, this approach may provide an even more
valuable source of human neural progenitor cells that are
programmed to become dopaminergic neurons after transplanta-
tion in PD patients.
Our in vivo experiments using 6-OHDA-lesioned Parkinsonian
rats and implanted lenti-MEF2CA/R-NSCs revealed good
survivability for at least 6 months after transplantation, possibly
because of the anti-apoptotic properties in addition to the
neurogenic effect of MEF2CA [18,29]. Most importantly, we
observed significant neurobehavioral/motoric improvement after
transplantation of MEF2CA/R-NSCs in this PD animal model
compared to control/R-NSCs. By comparing control/R-NSCs vs.
MEF2CA/R-NSCs in the same experimental model, we con-
trolled for nonspecific transplantation-related effects on behavior.
Prior studies had observed comparable improvement in motor
function in the same Parkinsonian rat model after transplanting
hESCs that had been co-cultured with telomerase-immortalized
human mesencephalic astrocytes to enhance differentiation of the
stem cells into dopaminergic neurons; however, this improvement
was accompanied by hyperproliferation, heralding potential tumor
formation . Our approach using MEF2CA-transduced stem
cells can avoid this major deterrent to transplantation.
In conclusion, our results demonstrate that MEF2C restricts
hESCs to the neuronal lineage and that this attribute can be used
to generate neurons and avoid tumor formation when used for
cell-based therapies. Furthermore, hESC/NPCs programmed to
become neurons via MEF2C activity were protected from
apoptosis and enriched for the dopaminergic phenotype. This
approach could potentially provide a limitless supply of stem cells
for therapeutic application in PD. Our technique represents a
unique approach for the production of cells for regenerative
medicine, while at the same time avoiding apoptosis and
tumorigenesis by promoting directed differentiation with a pro-
survival/neurogenic transcription factor.
Materials and Methods
The Institute’s Animal Care and Use Program is accredited by
the AAALAC International and a Multiple Project Assurance
A3053-1 is on file in the OLAW DHHS. Animal Usage Form 08–
054, ‘‘Rat Model for Parkinson’s Disease,’’ approval date April 16,
hESC Culture and Neural Induction
Undifferentiated H9 hESCs (WiCell Research Institute) of
passage 48–69 were cultured on a feeder layer of c-irradiated
human foreskin fibroblasts (Hs27, ATCC)/0.1% gelatin in growth
medium (DMEM/F12, 20% knockout serum replacement, 1 mM
non-essential amino acids, 0.1 mM b-mercaptoethanol (Invitro-
gen)) supplemented with 8 ng bFGF/ml (Sigma). Cells were
subcultured once a week, and the medium changed everyday. For
neural induction, hESCs were incubated in neural induction
medium (NIM; DMEM/F12:Neurobasal (1:1), 2% B27, 1% N2
(Invitrogen)) for 24 h and dissociated into small clumps by
mechanical scraping. Small clumps were transferred to a bacterial
Petri dish in NIM for an additional 3 days. The small clumps,
termed neuroectodermal spheres (NES), were transferred to a new
Petri dish for a 6-day incubation in neural proliferation medium
(NPM; DMEM/F12:Neurobasal (1:1), 1% B27, 0.5% N2, 20 ng
bFGF/ml, and 20 ng EGF/ml (R&D)). These NES were replated
onto laminin (LN, 10 mg/ml)-coated cell culture dishes in NPM for
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2–3 days to form rosettes. Under a stereomicroscope (Leica),
rosettes were isolated with a needle, transferred to a bacterial Petri
dish and cultured in NPM for 3 days to 1 month. At this rosette
stage, cells were designated rosette-neural stem cells (R-NSCs).
These R-NSCs were dissociated into a single cell suspension with
Accutase (Chemicon) and plated onto poly-L-ornithine (PLO,
10 mg/ml)/LN (1 mg/ml)-coated cell culture plates in NPM. At
this stage, cells were designated neural progenitor cells (NPCs) and
formed a monolayer. One week later, the cells were dissociated
again and replated onto PLO (100 mg/ml)/LN (10 mg/ml) in
terminal differentiation medium (TDM; DMEM/F12:Neurobasal
(1:1), 1% B27, 10 ng BDNF/ml (R&D), and 10 ng GDNF/ml
(R&D)). During the first two weeks after this plating, the cells
began to stain for immature neuronal markers and were therefore
designated Neural Stage I. Cells differentiating for 15 to 28 days
after plating were designated Neural Stage II, and greater than 28
days, Neural Stage III. During these stages, we examined the
expression of neuronal, astrocytic and oligodendrocytic markers.
Total RNA and cell lysates were collected from each representa-
tive stage for quantitative PCR and immunoblotting.
Lentiviral Constructs and Cell Infection
Lentiviral transfer vectors were transfected into HEK293 cells
to generate each lentivirus (see Text S1). For infection of hESC-
derived cells, we used cells at the R-NSC stage. Infected R-NSCs
were dissociated and plated onto PLO (10 mg/ml)/LN (1 mg/ml)-
coated plates in NPM in order to grow hNPCs in monolayer
culture for in vitro experiments. At confluence, approximately 14
days after infection, these hNPCs were dissociated, plated onto
glass coverslips coated with PLO (100 mg/ml)/LN (10 mg/ml), and
terminally differentiated in TDM. The differentiated cells were
fixed at various stages for immunostaining. For the in vivo
transplantation experiments, infected R-NSCs were allowed to
differentiate for 7–10 days as nestin/musashi1-positive neuro-
spheres, and then dissociated just prior to injection into the
Quantitative RT- PCR
Total RNA (500 ng) obtained from cells at various time points
were used to make cDNA. The expression level of each gene was
normalized to endogenous GAPDH. Fold change in gene
expression was calculated using the Pfaffl equation . For
detailed information concerning RNA isolation, reverse transcrip-
tion, and qPCR, see Text S1.
R-NSCs were infected with lenti-scrambled control shRNA or
lenti-shMEF2Cs and differentiated in TDM in vitro. Cells were
fixed at 14 or 33 dpi, and apoptotic cells were labeled with the
ApopTag In Situ Apoptosis Detection Kit (Chemicon; see Text S1).
Cells in culture were fixed and permeabilized for staining.
Parkinsonian rat brains were sectioned at 15 mm by cryostat,
treated with Antigen unmasking solution (Vector), and permeabi-
lized before staining with primary antibodies and fluorophore-
conjugated secondary antibodies. Detailed information related to
fixation, antibodies, dilution, and image analysis are listed under
In Vitro Electrophysiology
GFP-labeled NPCs were sorted by FACS to provide a pure
population of lenti-infected cells for subsequent electrophysiolog-
ical recording. The cells were differentiated in TDM for at least 5
weeks, and then analyzed for neuronal electrophysiological
properties by patch-clamp recording (see Text S1).
Chromatin Immunoprecipitation (ChIP) Assay
R-NSCs/NPCs were dissociated into single cells, cross-linked,
lysed, and sonicated as described in the Text S1. The supernatant
was used for immunoprecipitation with IgG or anti-MEF2
antibody. DNA fragments were purified and used for qPCR (see
6-Hydroxydopamine (6-OHDA) Lesions, Transplantation,
Immunosuppression, and Behavioral Tests
Sprague-Dawley rats with unilateral 6-OHDA lesions of the
nigrostriatal pathway were monitored by apomorphine-induced
rotations after transplantation with MEF2CA-programmed R-
NSCs or control cells. Rats were housed and handled in
accordance with the guidelines of Institutional Animal Care and
Use Committee of Sanford-Burnham Medical Research Institute.
For detailed information on behavioral tests and cell transplan-
tation, see Text S1.
Data are reported as mean 6 SEM. Statistical tests in each
experiment are listed in the figure legends or text. All data were
analyzed using the Prism 5 program (GraphPad Software, Inc.).
Statistical significance between two experimental groups was
assessed with a one-tailed Student’s t-test. For analysis of data from
three or more groups with a single independent factor that was
variable, a one-way ANOVA with post hoc Tukey’s multiple
comparison test was used. A two-way ANOVA with planned
comparisons was used for analysis of data among multiple pairs
with two independent factors.
Supplemental methods and references.
genes during neural differentiation monitored by qPCR.
(A, B) Total RNA was isolated from cells at the hESC, NES, R-
NSC, NPC, Neural I, Neural II, and Neural III stages for
quantitative RT-PCR. Note that the RNA levels of myelin basic
protein (MBP, from oligodendrocytes) and S100b (from astrocytes)
increased at Neural Stage III, at which time neuronal maturation
was also occurring. Syn I, Synapsin I. Values are mean + SEM,
n=3; *p,0.05 compared to hESC by ANOVA. (C) Neurons from
Neural Stage III stained with anti-PSD95 and -Synapsin I (Syn I)
antibodies. Arrows indicate the clusters showing juxtaposition of
PSD95 to Synapsin I. Scale bar: 5 mm.
Endogenous expression of stage-specific
DNA constructs of the target reporter and shRNAs for MEF2C.
(B) Scrambled or shRNAs directed against MEF2C (shMEF2C-1,
-2, or -3) were co-transfected with a target reporter into HEK293
cells. Four days later, cells were harvested for immunoblot using
anti-GFP (to detect the target reporter), anti-turbo GFP (for
scrambled or shMEF2Cs), or anti-actin (as a loading control). An
siRNA against MEF2C (siMEF2C) was used as a positive control.
(C) hESC-derived R-NSCs were infected with scrambled, lenti-
shMEF2C-1 or -2. Fluorescent images were taken at 20 days post
Validation of shRNAs against MEF2C. (A)
MEF2C Enhances DA Neuron Differentiation of hESCs
PLoS ONE | www.plosone.org11 August 2011 | Volume 6 | Issue 8 | e24027
and scheme for the infection of hESC-derived R-NSCs.
(A) Diagram of the lentiviral transfer vector harboring PGK
promoter-MEF2CA-IRES2-GFP. (B) GFP expression level was
measured by immunoblot to monitor the efficacy of infection of
SH-SY5Y cells with lenti-control, -Bcl-xL or -MEF2CA viruses at
different multiplicities of infection (MOI). Actin served as a loading
control. (C) Transcriptional activity of lenti-MEF2CA was
measured using a MEF2 RE-MHC-luciferase reporter gene.
Values are mean + SEM, n=3; ***p,0.001 by ANOVA. (D)
Scheme for infection of R-NSCs with lenti-MEF2CA or control
constructs and analysis of resulting cells (numbers indicate days in
culture; refer to Figure 1A and Materials and Methods for details).
(E) Infection efficiency of R-NSCs by each lentiviral construct was
calculated by counting the ratio of GFP+ to total DAPI+ cells.
Values are mean + SEM, n=9. MEF2CA, constitutively active
MEF2C; IRES, internal ribosome entry site; MEF2 RE, MEF2
Construction of lenti-MEF2CA viral construct
brain-derived neural progenitor cells (hFB-NPCs). (A)
Schematic diagram showing the two differentiation procedures
used here. (B) Fluorescent images of cells infected by lenti-control
or -MEF2CA virus. For assessment of neuronal markers, hFB-
NPCs were differentiated by the upper protocol shown in (A). Cells
were double labeled with an anti-GFP to identify viral-infected
cells and anti-TuJ1 to identify newly generated neurons, or with
anti-GFAP to label neural precursor cells or astrocytes. Scale bar:
25 mm. (C) Quantification of fluorescent marker data after
differentiation of control-infected and lenti-MEF2CA—infected
cells. Plots show TuJ1+ and GFAP+ versus total cells (left), and
TuJ1+ and GFAP+ versus GFP+/infected cells (right). Values are
mean + SEM, n=10; **p,0.01, ***p,0.001 by ANOVA. (D) The
longest neuronal process per cell was measured with Neuron J
software. Values are mean + SEM, n=50 cells counted for each
Neurogenic effect of MEF2CA on human fetal
promoter analysis of DA neuron-related genes in
MEF2CA-infected cells derived from R-NSCs. (A) Relative
mRNA levels of GIRK2 and CD28k were assessed throughout
development in vitro. Values are mean + SEM, n=3; *p,0.001 for
values greater than in Neural Stage I by ANOVA. (B) Endogenous
expression of MEF2C and nurr1 in MEF2CA-infected cells were
analyzed by qPCR during Neural Stage III at 40 dpi. Values are
mean + SEM, n=3; **p,0.003, ***p,0.0002 compared to
respective control by t-test. (C) Schematic diagram of putative
Enrichment of DA neuronal markers and
MEF2 binding sites in the enhancer/promoter of various DA
neuron-related genes. (D) Effects of various MEF2C constructs on
nurr1 promoter activity. HeLa cells were cotransfected with empty
vector, dominant negative MEF2C (MEF2DN), full-length wild-
type MEF2C, or constitutively active MEF2C (MEF2CA) plus a
nurr1 promoter (1.3 kb)-luciferase construct. Values are mean +
SEM, n=3; ***p,0.001 by ANOVA. (E) ChIP analysis of
MEF2C association with the nurr1 promoter. After chromatin
immunoprecipitation with anti-MEF2 antibody, qPCR primers
detected the MEF2C response element in the nurr1 promoter
region. Values are mean + SEM, n=3; **p,0.01 by t-test.
tor and lenti-MEF2CA in hESC-derived R-NSCs. (A)
Control- or MEF2CA-infected R-NSCs were grown as neuro-
spheres for one week in preparation for their transplantation. Note
that the MEF2CA construct bears an IRES, which results in
weaker expression of GFP. (B) Infection efficiency of control-
lentiviral vector and lenti-MEF2CA. To count infected cells, an
aliquot of R-NSCs was plated onto poly-L-ornithine/laminin-
coated coverslips and stained with anti-GFP antibody. Values are
mean + SEM, n=9. (C) Twelve weeks after transplantation,
0.960.15% of the engrafted MEF2CA/R-NSCs (green) expressed
PCNA (red). Arrows indicate PCNA+ cells among transplanted
cells; n=13 experiments with 2,200 cells scored (quantified in
histogram at right). Scale bar: 25 mm.
Infection efficiency of control-lentiviral vec-
neuropil in host tissue adjacent to graft. Tissue sections
were stained for tyrosine hydroxylase (TH), and the intensity of
staining in the neuropil adjacent to the graft was measured.
Random fields (n=7 randomly chosen fields for each of 4 animals)
were interrogated and adjusted for background staining by
subtracting the intensity of a similar field distal to the graft.
Values are mean + SEM, *p,0.0001 by t-test.
Increase in tyrosine hydroxylase-positive
We thank members of the Lipton laboratory for helpful suggestions and
J. Cui for aid with the initial experiments.
Conceived and designed the experiments: EGC SRM SAL. Performed the
experiments: EGC JDZ MT ST EM SFC. Analyzed the data: EGC JDZ
MT ST EM SFC SAL. Contributed reagents/materials/analysis tools: NN
AT. Wrote the paper: EGC SRM SAL.
1. Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA (2001) In vitro
differentiation of transplantable neural precursors from human embryonic stem
cells. Nat Biotechnol 19: 1129–1133.
2. Reubinoff BE, Itsykson P, Turetsky T, Pera MF, Reinhartz E, et al. (2001)
Neural progenitors from human embryonic stem cells. Nat Biotechnol 19:
3. Ben-Hur T, Idelson M, Khaner H, Pera M, Reinhartz E, et al. (2004)
Transplantation of human embryonic stem cell-derived neural progenitors
improves behavioral deficit in Parkinsonian rats. Stem Cells 22: 1246–1255.
4. Chiba S, Lee YM, Zhou W, Freed CR (2008) Noggin enhances dopamine
neuron production from human embryonic stem cells and improves behavioral
outcome after transplantation into Parkinsonian rats. Stem Cells 26: 2810–
5. Cho MS, Lee YE, Kim JY, Chung S, Cho YH, et al. (2008) Highly efficient and
large-scale generation of functional dopamine neurons from human embryonic
stem cells. Proc Natl Acad Sci U S A 105: 3392–3397.
6. Yang D, Zhang ZJ, Oldenburg M, Ayala M, Zhang SC (2008) Human
embryonic stem cell-derived dopaminergic neurons reverse functional deficit in
parkinsonian rats. Stem Cells 26: 55–63.
7. Roy NS, Cleren C, Singh SK, Yang L, Beal MF, et al. (2006) Functional
engraftment ofhuman EScell-deriveddopaminergic neurons enriched by coculture
with telomerase-immortalized midbrain astrocytes. Nat Med 12: 1259–1268.
8. Hicks AU, Lappalainen RS, Narkilahti S, Suuronen R, Corbett D, et al. (2009)
Transplantation of human embryonic stem cell-derived neural precursor cells
and enriched environment after cortical stroke in rats: cell survival and
functional recovery. Eur J Neurosci 29: 562–574.
9. Daadi MM, Maag AL, Steinberg GK (2008) Adherent self-renewable human
embryonic stem cell-derived neural stem cell line: functional engraftment in
experimental stroke model. PLoS One 3: e1644.
10. Hatami M, Mehrjardi NZ, Kiani S, Hemmesi K, Azizi H, et al. (2009) Human
embryonic stem cell-derived neural precursor transplants in collagen scaffolds
promote recovery in injured rat spinal cord. Cytotherapy. pp 1–13.
MEF2C Enhances DA Neuron Differentiation of hESCs
PLoS ONE | www.plosone.org12August 2011 | Volume 6 | Issue 8 | e24027
11. Nistor GI, Totoiu MO, Haque N, Carpenter MK, Keirstead HS (2005) Human Download full-text
embryonic stem cells differentiate into oligodendrocytes in high purity and
myelinate after spinal cord transplantation. Glia 49: 385–396.
12. Keirstead HS, Nistor G, Bernal G, Totoiu M, Cloutier F, et al. (2005) Human
embryonic stem cell-derived oligodendrocyte progenitor cell transplants
remyelinate and restore locomotion after spinal cord injury. J Neurosci 25:
13. Schwartz PH, Brick DJ, Stover AE, Loring JF, Muller FJ (2008) Differentiation
of neural lineage cells from human pluripotent stem cells. Methods 45: 142–158.
14. Schulz TC, Noggle SA, Palmarini GM, Weiler DA, Lyons IG, et al. (2004)
Differentiation of human embryonic stem cells to dopaminergic neurons in
serum-free suspension culture. Stem Cells 22: 1218–1238.
15. Brederlau A, Correia AS, Anisimov SV, Elmi M, Paul G, et al. (2006)
Transplantation of human embryonic stem cell-derived cells to a rat model of
Parkinson’s disease: effect of in vitro differentiation on graft survival and
teratoma formation. Stem Cells 24: 1433–1440.
16. Gossett LA, Kelvin DJ, Sternberg EA, Olson EN (1989) A new myocyte-specific
enhancer-binding factor that recognizes a conserved element associated with
multiple muscle-specific genes. Mol Cell Biol 9: 5022–5033.
17. Potthoff MJ, Olson EN (2007) MEF2: a central regulator of diverse
developmental programs. Development 134: 4131–4140.
18. Li Z, McKercher SR, Cui J, Nie Z, Soussou W, et al. (2008) Myocyte enhancer
factor 2C as a neurogenic and antiapoptotic transcription factor in murine
embryonic stem cells. J Neurosci 28: 6557–6568.
19. Li H, Radford JC, Ragusa MJ, Shea KL, McKercher SR, et al. (2008)
Transcription factor MEF2C influences neural stem/progenitor cell differenti-
ation and maturation in vivo. Proc Natl Acad Sci U S A 105: 9397–9402.
20. Leifer D, Krainc D, Yu YT, McDermott J, Breitbart RE, et al. (1993) MEF2C, a
MADS/MEF2-family transcription factor expressed in a laminar distribution in
cerebral cortex. Proc Natl Acad Sci U S A 90: 1546–1550.
21. Flavell SW, Cowan CW, Kim TK, Greer PL, Lin Y, et al. (2006) Activity-
dependent regulation of MEF2 transcription factors suppresses excitatory
synapse number. Science 311: 1008–1012.
22. Lyons GE, Micales BK, Schwarz J, Martin JF, Olson EN (1995) Expression of
mef2 genes in the mouse central nervous system suggests a role in neuronal
maturation. J Neurosci 15: 5727–5738.
23. Leifer D, Golden J, Kowall NW (1994) Myocyte-specific enhancer binding factor
2C expression in human brain development. Neuroscience 63: 1067–1079.
24. Zhu B, Ramachandran B, Gulick T (2005) Alternative pre-mRNA splicing
governs expression of a conserved acidic transactivation domain in myocyte
enhancer factor 2 factors of striated muscle and brain. J Biol Chem 280:
25. Zhu B, Gulick T (2004) Phosphorylation and alternative pre-mRNA splicing
converge to regulate myocyte enhancer factor 2C activity. Mol Cell Biol 24:
26. McDermott JC, Cardoso MC, Yu YT, Andres V, Leifer D, et al. (1993)
hMEF2C gene encodes skeletal muscle- and brain-specific transcription factors.
Mol Cell Biol 13: 2564–2577.
27. Barbosa AC, Kim MS, Ertunc M, Adachi M, Nelson ED, et al. (2008) MEF2C,
a transcription factor that facilitates learning and memory by negative regulation
of synapse numbers and function. Proc Natl Acad Sci U S A 105: 9391–9396.
28. Shalizi A, Gaudilliere B, Yuan Z, Stegmuller J, Shirogane T, et al. (2006) A
calcium-regulated MEF2 sumoylation switch controls postsynaptic differentia-
tion. Science 311: 1012–1017.
29. Okamoto S, Krainc D, Sherman K, Lipton SA (2000) Antiapoptotic role of the
p38 mitogen-activated protein kinase-myocyte enhancer factor 2 transcription
factor pathway during neuronal differentiation. Proc Natl Acad Sci U S A 97:
30. Flames N, Hobert O (2009) Gene regulatory logic of dopamine neuron
differentiation. Nature 458: 885–889.
31. Jankovic J, Chen S, Le WD (2005) The role of Nurr1 in the development of
dopaminergic neurons and Parkinson’s disease. Prog Neurobiol 77: 128–138.
32. Perlmann T, Wallen-Mackenzie A (2004) Nurr1, an orphan nuclear receptor
with essential functions in developing dopamine cells. Cell Tissue Res 318:
33. Buzzard JJ, Gough NM, Crook JM, Colman A (2004) Karyotype of human ES
cells during extended culture. Nat Biotechnol 22: 381–382; author reply 382.
34. Amit M, Margulets V, Segev H, Shariki K, Laevsky I, et al. (2003) Human
feeder layers for human embryonic stem cells. Biol Reprod 68: 2150–2156.
35. Elkabetz Y, Studer L (2008) Human ESC-derived neural rosettes and neural
stem cell progression. Cold Spring Harb Symp Quant Biol 73: 377–387.
36. Elkabetz Y, Panagiotakos G, Al Shamy G, Socci ND, Tabar V, et al. (2008)
Human ES cell-derived neural rosettes reveal a functionally distinct early neural
stem cell stage. Genes Dev 22: 152–165.
37. Nat R, Cretoiu D, Popescu LM (2007) Neurogenic neuroepithelial and radial
glial cells generated from six human embryonic stem cell lines in serum-free
suspension and adherent cultures. Glia 55: 385–399.
38. Ying QL, Stavridis M, Griffiths D, Li M, Smith A (2003) Conversion of
embryonic stem cells into neuroectodermal precursors in adherent monoculture.
Nat Biotechnol 21: 183–186.
39. Mao Z, Bonni A, Xia F, Nadal-Vicens M, Greenberg ME (1999) Neuronal
activity-dependent cell survival mediated by transcription factor MEF2. Science
40. Li JY, Christophersen NS, Hall V, Soulet D, Brundin P (2008) Critical issues of
clinical human embryonic stem cell therapy for brain repair. Trends Neurosci
41. Kim DW, Chung S, Hwang M, Ferree A, Tsai HC, et al. (2006) Stromal cell-
derived inducing activity, Nurr1, and signaling molecules synergistically induce
dopaminergic neurons from mouse embryonic stem cells. Stem Cells 24:
42. Thompson L, Barraud P, Andersson E, Kirik D, Bjorklund A (2005)
Identification of dopaminergic neurons of nigral and ventral tegmental area
subtypes in grafts of fetal ventral mesencephalon based on cell morphology,
protein expression, and efferent projections. J Neurosci 25: 6467–6477.
43. Yan Y, Yang D, Zarnowska ED, Du Z, Werbel B, et al. (2005) Directed
differentiation of dopaminergic neuronal subtypes from human embryonic stem
cells. 23: 781–790.
44. De Val S, Anderson JP, Heidt AB, Khiem D, Xu SM, et al. (2004) Mef2c is
activated directly by Ets transcription factors through an evolutionarily
conserved endothelial cell-specific enhancer. Dev Biol 275: 424–434.
45. Schallert T, Fleming SM, Leasure JL, Tillerson JL, Bland ST (2000) CNS
plasticity and assessment of forelimb sensorimotor outcome in unilateral rat
models of stroke, cortical ablation, parkinsonism and spinal cord injury.
Neuropharmacology 39: 777–787.
46. Hedlund E, Perlmann T (2009) Neuronal cell replacement in Parkinson’s
disease. J Intern Med 266: 358–371.
47. Arenas E (2010) Towards stem cell replacement therapies for Parkinson’s
disease. Biochem Biophys Res Commun 396: 152–156.
48. Allan LE, Petit GH, Brundin P (2010) Cell transplantation in Parkinson’s
disease: problems and perspectives. Curr Opin Neurol 23: 426–432.
49. Calon F, Goulet M, Blanchet PJ, Martel JC, Piercey MF, et al. (1995) Levodopa
or D2 agonist induced dyskinesia in MPTP monkeys: correlation with changes in
dopamine and GABAA receptors in the striatopallidal complex. Brain Res 680:
50. McKinsey TA, Zhang CL, Olson EN (2002) MEF2: a calcium-dependent
regulator of cell division, differentiation and death. Trends Biochem Sci 27:
51. Friling S, Andersson E, Thompson LH, Jonsson ME, Hebsgaard JB, et al. (2009)
Efficient production of mesencephalic dopamine neurons by Lmx1a expression
in embryonic stem cells. Proc Natl Acad Sci U S A 106: 7613–7618.
52. Pfaffl MW (2001) A new mathematical model for relative quantification in real-
time RT-PCR. Nucleic Acids Res 29: e45.
53. Hellen CU, Sarnow P (2001) Internal ribosome entry sites in eukaryotic mRNA
molecules. Genes Dev 15: 1593–1612.
54. Kim JY, Koh HC, Lee JY, Chang MY, Kim YC, et al. (2003) Dopaminergic
neuronal differentiation from rat embryonic neural precursors by Nurr1
overexpression. J Neurochem 85: 1443–1454.
MEF2C Enhances DA Neuron Differentiation of hESCs
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