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Directed Differentiation of Dopaminergic Neuronal Subtypes from
Human Embryonic Stem Cells
Yiping Yana,b,d, Dali Yanga,b,d, Ewa D. Zarnowskac, Zhongwei Dua,b,d, Brian Werbeld,
Chuck Valliered, Robert A. Pearcec, James A. Thomsona,e,f, and Su-Chun Zhanga,b,d,f
aDepartment of Anatomy, University of Wisconsin-Madison, Madison, Wisconsin, USA
bDepartment of Neurology, University of Wisconsin-Madison, Madison, Wisconsin, USA
cDepartment of Anesthesiology, University of Wisconsin-Madison, Madison, Wisconsin, USA
dThe Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin, USA
eWisconsin Primate Research Center, University of Wisconsin-Madison, Madison, Wisconsin, USA
fWiCell Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, USA
How dopamine (DA) neuronal subtypes are specified remains unknown. In this study we show a
robust generation of functional DA neurons from human embryonic stem cells (hESCs) through a
specific sequence of application of fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH).
Treatment of hESC-derived Sox1+ neuroepithelial cells with FGF8 and SHH resulted in production
of tyrosine hydroxylase (TH)–positive neurons that were mostly bipolar cells, coexpression with γ-
aminobutyric acid, and lack of midbrain marker engrailed 1 (En1) expression. However, FGF8
treatment of precursor cells before Sox1 expression led to the generation of a similar proportion of
TH+ neurons characteristic of midbrain projection DA neurons with large cell bodies and complex
processes and coexpression of En1. This suggests that one mechanism of generating neuronal
subtypes is temporal availability of morphogens to a specific group of precursors. The in vitro–
generated DA neurons were electrophysiologically active and released DA in an activity-dependent
manner. They may thus provide a renewable source of functional human DA neurons for drug
screening and development of sustainable therapeutics for disorders affecting the DA system.
Neuroepithelial cells; Neural differentiation; Cell replacement; Parkinson's disease
Dopamine (DA) neurons reside in several brain areas, including the midbrain, hypothalamus,
retina, and olfactory bulbs. The most prominent groups of DA neurons are in the substantia
nigra (A9 cell group) and the ventral tegmental area (A10) of the midbrain, both of which
project to the forebrain [1]. DA neurons in the substantia nigra project to the striatum and form
the extrapyramidal motor system that controls postural reflexes and initiation of movement.
Degeneration of these DA neurons underlies Parkinson's disease. The DA neurons in the
© AlphaMed Press
Correspondence: Su-Chun Zhang, M.D., Ph.D., Waisman Center, Room T613, University of Wisconsin, 1500 Highland Avenue,
Madison, Wisconsin 53705, USA. Telephone: 608-265-2543; Fax: 608-263-5267; e-mail: E-mail: firstname.lastname@example.com.
NIH Public Access
Author Manuscript
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Published in final edited form as:
Stem Cells. 2005 ; 23(6): 781–790. doi:10.1634/stemcells.2004-0365.
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tegmental area connect to neurons in the limbic system, and defects in the functioning of these
cells are thus implicated in psychiatric behaviors. DA neurons in other parts of the brain are
mostly interneurons involved in local circuitries. Hence, DA neurons in the midbrain differ
significantly from those in the forebrain in morphology, coexpression of neurotransmitters,
and function [2,3].
The ontogeny of DA neurons is not clear and remains a focus of intensive research. Studies to
date have focused on the genesis of mesencephalic DA neurons. Information available indicates
the involvement of several transcription factors, including Lmx1b, engrailed-1 (En1), Nurr1,
and Ptx3, through independent pathways, most of which are shown to be required for survival
and/or maturation but not specification of DA neurons [4–12]. How the DA neurons are initially
specified from the naive neuroectodermal cells remains largely unknown. Using
neuroepithelial explant cultures, Ye et al. [13] have shown that fibroblast growth factor 8
(FGF8), a morphogen involved in the patterning of isthmus, and sonic hedgehog (SHH), a
ventralizing molecule, can specify a dopaminergic fate from neuroepithelial cells. The same
principle seems to apply to in vitro–generated neuroepithelial cells. Mouse embryonic stem
cells (ESCs), derived from the inner cell mass of a preimplantation blastocyst embryo [14,
15], are first differentiated into neuroepithelial cells, expanded in the presence FGF2, and
further differentiated into DA neurons in response to FGF8 and SHH [16]. Given that the
differentiation of DA neurons in both forebrain and midbrain depends on the signaling of FGF8
and SHH [17,18], it remains unknown how subtypes of DA neurons are differentially specified.
Like their mouse counterparts, human ESCs (hESCs) [19] offer a system for modeling the early
phases of human development, including neural lineage specification. They also provide a
renewable source of specialized cells such as DA neurons for systematic studies of the genesis
of the DA system, pathogenic process affecting the survival and function of DA neurons, and
development of sustainable therapeutics for Parkinson's disease and other disorders affecting
the DA system [20]. We have established a chemically defined system to direct hESCs to
neuroepithelial cells that mirrors in vivo human neuroectoderm development in timing and
neural tube–like structure formation [21]. Using this model system, we have discovered that
FGF8 and SHH efficiently promote the generation of DA neurons from hESCs. However, to
generate DA neurons with midbrain projection neuronal phenotypes, early exposure to FGF8
before precursor cells become Sox1-expressing neuroepithelial cells is necessary. Treatment
of the Sox1-expressing neuroepithelial cells with FGF8 and SHH still results in an efficient
production of DA neurons but with mostly forebrain phenotypes.
hESC Cultures
hESC lines H9 (p21–56) and H1 (p35–40) were propagated weekly on irradiated mouse
embryonic fibroblasts (MEFs) with a daily change of an ESC growth medium that consisted
of Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco, Rockville, MD,
http://www.invitrogen.com), 20% serum replacer (Gibco), 1 mM glutamine (Sigma, St. Louis,
http://sigmaaldrich.com), 0.1 mM nonessential amino acids (Gibco), 2 µg/ml heparin (Sigma),
0.1 mM β-mercaptoethanol (Sigma), and 4 ng/ml FGF2 (R&D Systems, Minneapolis,
http://www.rndsystems.com), as previously described [19]. Differentiated colonies were
physically removed using a curved Pasteur pipette, and the undifferentiated state of ESCs was
confirmed by typical morphology and positive immunostaining for Oct4 and stage-specific
embryonic antigen 4 (SSEA4). Cells from both lines exhibited a similar morphology and
growth rate under the above culture conditions.
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Differentiation and Enrichment of Neuroepithelial Cells
HESC colonies were detached from the MEF layer by treatment of the culture with 0.2 mg/ml
of dispase (Roche Diagnostics, Indianapolis, http://www.roche-diagnostics.com) and grown
as floating cell aggregates (embryoid bodies) for 4 days with a daily change of ESC medium.
They were then grown in an adherent substrate in a neural medium consisting of DMEM/F12
(2:1), supplemented with N2 (Gibco), 0.1 mM nonessential amino acids, and 2 µg/ml heparin,
with a medium change every other day. The ESC aggregates attached and formed individual
colonies at approximately day 6. Neuroepithelial cells, exhibited by columnar cells organizing
into neural tube–like rosettes, were developed at approximately day 14–16 [21]. The neural
rosettes were isolated through differential enzymatic response [21]. Growth factors were added
during the course of differentiation to influence regionalization (see Results).
DA Neuron Differentiation
The enriched neuroepithelial cells were dissociated by 0.025% trypsin and 0.27 mM EDTA in
phosphate-buffered saline (PBS) at 37°C for 10–15 minutes and plated onto 12-mm coverslips
(precoated with 100 µg/ml polyornithine and 10 µg/ml laminin) at a density of 40,000 to 50,000
cells per coverslip. The neuronal differentiation medium consisted of neurobasal medium
(Gibco) supplemented with N2 (Gibco), 0.1 mM nonessential amino acids, 0.5 mM glutamine,
1 µg/ml laminin (Sigma), 1 µM cAMP (Sigma), 200 µM ascorbic acid (Sigma), 10 ng/ml brain-
derived neurotrophic factor (R&D Systems), and 10 ng/ml glial cell line–derived neurotrophic
factor (GDNF) (R&D Systems). The cells were cultured for 3–4 weeks, with medium changes
every other day.
Immunocytochemistry and Cell Quantification
Coverslip cultures were fixed in 4% paraformaldehyde in PBS for 10–20 minutes or methanol
(−20°C) for 5 minutes and processed for immunostaining [21]. The following primary
antibodies were used: mouse anti-SSEA4 (1:40), mouse anti-En1 (1:50), and mouse anti-Pax6
(1:5,000, all from Developmental Studies Hybridoma Bank, Iowa City, IA,
http://www.uiowa.edu/~dshbwww); rabbit anti-Sox1 (1:500), rabbit anti-human nestin
(1:200), rabbit anti-aromatic amino acid decarboxylase (AADC) (1:1,000), rabbit anti-
phenylethanolamine N-methyltransferase (PNMT) (1:200), sheep anti-dopamine β
hydroxylase (DβH) (1:400), mouse anti-synaptophysin (1:500), and rabbit anti-
cholecystokinin octapeptide (CCK8) 1:2,000 (all from Chemicon, Temecula, CA,
http://chemicon.com); mouse anti-tyrosine hydroxylase (TH) (1:1,000), mouse anti–βIII-
tubulin (1:500), rabbit anti–γ-aminobutyric acid (GABA) (1:5,000), and mouse anti-calbindin
(1:400, all from Sigma); rabbit anti-TH (1:500) and rabbit anti-vesicular monoamine
transporter 2 (VMAT2) (1:500, all from Pel-Freez, Rogers, AK, http://www.pel-freez.com);
goat anti-Ret (1:400) and mouse anti-Oct4 (1:1,000, both from Santa Cruz Biotechnology,
Santa Cruz, CA, http://www.scbt.com); rabbit anti-brain factor 1 (Bf1) (1:5,000; gift from
Lorenz Studer, Sloan-Kettering Institute) and Otx2 (1:5,000, gift from F. Vaccarino, Yale
University). Antibody-antigen reaction was revealed by Alexa Flour 488- or 594-conjugated
secondary antibody (1:1,000, Molecular Probes, Eugene, OR, http://probes.invitrogen.com).
Cell nuclei were stained with Hoechst 33342 (Sigma). Staining was visualized with a Nikon
fluorescence microscope. Brain sections from adult rats and E38 embryonic monkeys were
used as positive controls for many of the antibodies against neuronal types and
neurotransmitters. Negative controls were also set by omitting the primary or secondary
antibodies in the immunostaining procedures. Cell counting was achieved blindly by using a
reticule on an eyepiece and a ×40 objective. The cells in 10 visual fields were randomly selected
and counted from each coverslip. Data were presented as mean ± SD. Values of p< .05 were
considered significant by the unpaired Student's t-test.
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Reverse Transcription–Polymerase Chain Reaction
Total RNA was extracted from cultured cells using RNA Stat-60 (Tel-Test, Friendswood, TX,
http://www.isotexdiagnostics.com), followed by the treatment with DNase I (DNA-free,
Ambion, Austin, TX, http://www.ambion.com). Synthesis of cDNA was carried out with the
Superscript First-Strand Synthesis System for reverse transcription–polymerase chain reaction
(RT-PCR) (Invitrogen Corporation, Carlsbad, CA, http://www.invitrogen.com) according to
the manufacturer's directions. PCR amplification was performed using a standard procedure
with Taq Polymerase (Promega, Madison, WI, http://www.promega.com). The number of
cycles varied from 25 to 35 cycles depending on the particular mRNA abundance with
denaturation at 94°C for 15 seconds, annealing temperatures at 55°C or 60°C for 30 seconds
according to the primers, and elongation at 72°C for 45 seconds. Negative control was achieved
by omitting transcriptase during RT or cDNA sample during PCR. The primers and product
lengths were as follows:
TCCACCACCCTGTTGCTGTA-3', 450 bp)
Nurr1 (5'-CGATGCCTTGTGTTCAGGCGCAG-3', 5'-
AGCCTTTGCAGCCCTCACAGGTG-3', 858 bp)
Ptx3 (5'-GTGGGTGGAGAGGAGAACAA-3', 5'-
TTCCTCCCTCAGGAAACAATG-3', 175 bp)
Lmx1b (5'-GGGATCGGAAACTGTTACTGC-3', 5'-
GTAGTCACCCTTGCACAGCA-3', 218 bp)
En1 (5'-CCCTGGTTTCTCTGGGACTT-3', 5'-GCAGTCTGTGGGGTCGTATT-3', 162
Bf1 (5'-ACTCAAAACTCGCTGGGCAAC-3', 5'-
CGTGGGGGAAAAAGTAACTGG-3', 226 bp)
Gbx2 (5'-CACCACGTCTACGGGCAAGAAC-3', 5'-
AGCTGCTGATGCTGACTTCTGA-3', 308 bp)
339 bp)
Nkx6.1 (5'-ACACGAGACCCACTTTTTCCG-3', 5'-
TGCTGGACTTGTGCTTCTTCAAC-3', 335 bp)
Wnt1 (5'-CGGGCAACAACCAAAGTCG-3', 5'-GACAGTTCCAGCGGCGATTC-3',
321 bp)
Pax2 (5'-ATGTTCGCCTGGGAGATTCG,-3' 5'-GCAAGTGCTTCCGCAAACTG-3',
361 bp).
DA Measurement
After 21 days of DA neuronal differentiation, media conditioned for 48 hours were collected.
Activity-dependent DA release from the cultured cells was measured by first conditioning
cultured cells in Hanks' balanced salt solution (HBSS) for 15 minutes and then replacing it
with HBSS containing 56 mM KCl for 15 minutes at 37°C. DA in the culture media or in HBSS
was stabilized by adding 20 µl stabilization buffer (900 mg EGTA and 700 mg gluthatione in
10 ml of 0.1 M NaOH), and samples were stored at −80°C. A high-performance liquid
chromatography ( HPLC) kit (Chromsystems, München, Germany,
http://www.chromsystems.de) was used to extract monoamines. The levels of monoamines
were determined by HPLC (Model 508 autosampler and model 118 pump, Beckman Coulter,
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Fullerton, CA, http://www.beckmancoulter.com) coupled to an electrochemical detector
(Coulochem II, ESA Inc., Chelmsford, MA, http://www.esainc.com) by using MD-TM mobile
phase (ESA Inc.). The cultures in each group were triplicated, and data were collected from
three separate experiments.
Electrophysiological Recording
Electrophysiological properties of the hESC-generated DA neurons were investigated using
whole-cell patch-clamp recording techniques [22]. Pipettes were filled with intracellular
solutions containing (in mM) KCl 140 or K-gluconate 140, Na+-HEPES 10, BAPTA 10, and
Mg2+-ATP 4 (pH 7.2, 290 mOsm, 2.3–5.0 MΩ). Biocytin (0.5%, Sigma) was added to the
recording solution, and subsequent labeling with streptavidin-Alex Fluor 488 (1:1,000,
Molecular Probes) and an antibody against TH was used to identify DA neurons. The bath
solution contained (in mM) NaCl 127, KH2PO4 1.2, KCl 1.9, NaHCO3 26, CaCl2 2.2,
MgSO4 1.4, and glucose 10 and 95% O2/5% CO2 (pH 7.3; 300 mOsm). For some experiments,
tetrodotoxin (TTX) (1 µM; Sigma) was applied in the bath solution to block voltage-gated
sodium currents.
Current-clamp and voltage-clamp recordings were performed using a MultiClamp 700A
amplifier (Axon Instruments, Inverurie, Scotland, U.K., http://www.moleculardevices.com).
Signals were filtered at 4 kHz, sampled at 10 kHz using a Digidata 1322A analogue-digital
converter (Axon Instruments), and acquired and stored on a computer hard disk using
commercially available software (pClamp9, Axon Instruments). Access resistance was
typically 8–18 MΩ and was compensated by 50%–80% using amplifier circuitry. Voltages
were corrected for liquid junction potential of +13 mV [23]. Vrest and action potentials (APs)
were examined in current-clamp mode. Spontaneous excitatory (inward) and inhibitory
(outward) synaptic currents were characterized in voltage-clamp mode using K-gluconate–
based pipette solution and Vhold = −40 mV. Synaptic events were detected using a template
detection algorithm (Mini Analysis Program 5.6.28, Synaptosoft, Decatur, GA,
http://www.synaptosoft.com), and deactivation phase was fitted to a biexponential function
using the Levenberg-Marquardt algorithm. Data were presented as mean ± SE.
hESC-Derived Neuroepithelial Cells Display a Forebrain Character
ESC colonies, detached from a feeder layer, were cultured in suspension as aggregates for 4
days in the ESC growth medium and then grown in an adhesive culture dish in a chemically
defined neural medium containing FGF2 (20 ng/ml). Cells in the colony center developed a
columnar morphology and lined up in a rosette formation around days 8–10 (Fig. 1A). These
columnar cells were positive for Pax6 but negative for the pan-neural transcription factor Sox1
[24], indicative of early neuroepithelial cells. Over another 4–6 days (days 14–16), the
columnar cells expanded and organized into neural tube–like rosettes (Fig. 1B) and expressed
Sox1 (Fig. 1C), a transcription factor expressed by definitive neuroepithelial cells during neural
tube closure [25]. They were positive for Otx2 [24] and Bf1, transcription factors expressed
by forebrain cells [26], but negative for En1 (Fig. 1D), a transcription factor expressed by
midbrain cells [27,28], suggesting a forebrain identity of the in vitro–generated neuroepithelial
Induction of Midbrain Progenitors Requires Early Action of FGF8
For specification of a ventral midbrain fate, neuroepithelial cells in the neural tube–like rosettes
were enriched through differential enzymatic and adhesion treatment [21], expanded for 2 days
as aggregates in suspension with FGF2, and then plated onto a laminin substrate and treated
with SHH (50–200 ng/ml) and FGF8 (20–100 ng/ml) for 6 days (Fig. 2A).
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Immunocytochemical analyses revealed that most of the neural progenitors remained positive
for Bf1 and Otx2 but not for En1 (not shown). In long-term differentiation cultures, En1+ cells
were observed (see below).
The failure of FGF8 to induce Sox1+ neuroepithelial cells to express En1 suggests that the
Sox1-expressing neuroepithelial cells may be refractory to patterning signals. Because the
Sox1-expressing cells are generated 2 weeks after differentiation of hESCs (equivalent to a 6-
day-old embryo) [19] and form neural tube–like structures, they may correspond to the
neuroepithelial cells at the neural tube closure, during which neuroepithelial cells express Sox1
and are regionally specified [29]. This led us to hypothesize that FGF8 may promote midbrain
specification before neuroepithelial cells express Sox1. We thus applied FGF8 (100 ng/ml) at
the time when the cells in the colony center became columnar at day 10 (Fig. 2A). After 6 days,
cells in the colony center developed neural tube–like formations, as seen in the presence of
FGF2. These neuroepithelial cells were similarly enriched and treated with FGF8 and SHH
(200 ng/ml) for 6 days on the laminin substrate (Fig. 2A). Under this culture condition, En1
expression was observed in the nestin-expressing neural progenitors, often in the form of
rosettes (Fig. 1E), although there were still cells that expressed Bf1 (Fig. 1F). RT-PCR analyses
revealed a more effective induction of midbrain-related transcription factors En1, Pax2, and
Wnt1 by early FGF8 exposure (Fig. 1G). Early exposure to FGF8 also induced the expression
of SHH mRNA and a more robust mRNA expression of the ventral gene Nkx6.1 (Fig. 1G).
Thus, neuroepithelial cells are more efficiently regionalized to a ventral midbrain fate by
exposure to FGF8 before precursor cells become Sox1+.
Neural Progenitors Differentiate to DA Neurons in the Presence of FGF8 and SHH
The neural progenitors, treated with FGF8 before (at day 10) or after (day 18) Sox1 expression,
were dissociated and differentiated in a neuronal differentiation medium (Fig. 2A). The
enriched neural progenitors were essentially nestin+ (>95%) and did not express SSEA4 (Fig.
2B), a glycoprotein highly expressed by undifferentiated hESCs. The disaggregated neural
progenitors, initially distributed evenly, reformed rosettes 3–5 days after plating. They then
extended processes and exhibited polar morphology (Fig. 2C). At 3 weeks after differentiation,
approximately one third of the total differentiated cells were positive for TH (Fig. 2D; 31.8%
± 3.1% in the early FGF8 treatment group, 17,965 cells counted from four experiments; 29.8%
± 1.6% in the late FGF8 treatment group, 7,954 cells counted from three experiments). All of
the TH+ cells were stained positively for a neuronal marker βIII-tubulin (Figs. 3A, 3B), among
which 50%–60% were TH+. Morphologically, the TH-expressing neurons in the early FGF8
treatment group were 10–20 µm in diameter. They exhibited multipolar morphology, with
differentiable axons and dendrites (Fig. 3A). In the late FGF8 treatment group, the TH+ neurons
were relatively uniform, with a size of approximately 8–13 µm. Most of them displayed a
bipolar morphology (Fig. 2B). A similar proportion of TH+ neurons was also obtained from
the H1 hESC line.
In the biosynthesis of monoamines, TH hydroxylates tyrosine to levodopa, which is
subsequently decarboxylated by AADC to become DA. Two other enzymes, DβH and PNMT,
transform DA to norepinephrine and catalyze norepinephrine to epinephrine, respectively.
Immunostaining showed that all TH+ neurons were AADC+, although some AADC+ cells were
negative for TH (Figs. 2E–2G). These TH+ cells were negative for DβH (Fig. 2D) and PNMT
(not shown), although DβH strongly stained noradrenergic neurons in the adult rat and
embryonic monkey brainstem (inset in Fig. 2D). These suggest that the TH-expressing neurons,
whether they were generated with early or late FGF8 treatment, possess both enzymes that are
necessary for DA synthesis and that these neurons are DA neurons rather than noradrenergic
or adrenergic neurons.
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DA Neurons Produced with FGF8 Treatment Before or After Sox1 Expression Display
Different Regional Identities
In addition to morphological difference of the TH+ neurons, immunocytochemical analyses
indicated that most TH+ cells with multiple processes in the early FGF8 treatment group
coexpressed the midbrain marker En1 in the nuclei (Fig. 3C). In contrast, few TH+ neurons
coexpressed En1, although En1 was expressed by some cells in the late FGF8 treatment group
(Fig. 3D). Thus, DA neurons generated with the early FGF8 treatment possess a midbrain
positional identity.
DA neurons in the forebrain, especially those in the olfactory bulb, often coexpress GABA
[2,3]. Double immunostaining of TH and GABA indicated that most of the DA neurons in the
early FGF8 treatment group were negative for GABA, although GABA+ neurons were found
in the culture (Fig. 3E). Among all TH+ cells, 8% (8.7% ± 3.9%, 6,520 TH+ cells counted from
four experiments) of TH+ cells coexpressed GABA. Most of these double-positive cells were
small bipolar cells. However, 22.5% ± 8.9% of the TH+ neurons in the late FGF8 treatment
group (5,032 TH+ cells counted from four experiments, p< .05 compared with the early FGF8
treatment group) coexpressed GABA (Fig. 2F). Thus, DA neurons generated in the late FGF8
treatment group are likely forebrain DA neurons.
Some midbrain DA neurons, especially those in the ventral tegmental area, contain CCK8 or
calbindin [30,31]. Immunohistochemical analyses indicated that the TH+ neurons generated
after early FGF8 treatment were not labeled with calbindin, although calbindin-expressing
neurons were observed (Fig. 3G). These calbindin neurons were mostly small cells. No CCK8-
positive cells were detected in the cultures. RT-PCR analyses indicated that the transcription
factors, such as Lmx1b, Nurr1, and Ptx3, which are involved in midbrain DA neuron
development [4–7,9,10,12], were expressed in the DA neuron cultures that were induced with
early FGF8 treatment (Fig. 3H). The forebrain transcription factor Bf1 was detectable in the
DA culture (Fig. 3H)
hESC-Generated Midbrain DA Neurons Are Biologically Functional
The above studies indicate that the DA neurons generated after the early neuroepithelial cells
are exposed to FGF8 and SHH are likely midbrain DA neurons. Further analyses showed that
all of these TH+ neurons expressed c-Ret, a component of the receptor for GDNF (Figs. 4A–
4C). Most of the TH+ cells, especially those with branched neurites, expressed VMAT2 (Figs.
4D–4F), which is responsible for packaging DA into subcellular compartments in monoamine
neurons [32]. In addition, TH+ neurons expressed synaptophysin (Figs. 4G–4I), a membrane
glycoprotein essential to synapse formation.
DA release is a functional hallmark of DA neurons. HPLC analyses revealed the presence of
DA in the medium of DA neuron differentiation cultures (230.8 ± 44.0 pg/ml). In the control
cultures without the presence of SHH and FGF8, few TH+ neurons (3%–5%) were generated
[21]. DA was detectable in the control cultures but at a lower level (46.3 ± 9.2 pg/ml) (Fig.
5A). When cultured cells were washed and incubated in HBSS for 15 minutes, the DA level
was similar between the two cultures (Fig. 5A). However, depolarization of the cultured
neurons by 56 mM KCl in HBSS significantly increased the amount of DA (35.8 ± 9.2 and
111.0 ± 15.0 pg/ml in the control and DA differentiation cultures, respectively; Fig. 5A). These
observations suggest that the in vitro–generated DA neurons can secrete DA and that the release
of DA is activity-dependent.
Electrophysiological recordings were used to determine whether ESC-generated DA neurons
were physiologically active. In cells differentiated in culture for 30–38 days (n = 14), the resting
membrane potential (Vrest) ranged from −32–−72 mV (−54 ± 2.9 mV), cell capacitance (Cm)
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ranged from 11–45 pF (21 ± 2.7 pF), and input resistance (Rm) ranged from 480–3,500 MΩ
(1,506 ± 282 MΩ). Depolarizing current steps (0.2 nA × 200–500 ms) usually elicited single
APs, but in several cases, decrementing trains of APs were observed (Figs. 5Bi, 5Bii). AP
threshold ranged from −26–−5.2 mV (−17.4 ± 2.1 mV) and peaked at −9.6–30 mV. APs up to
50.2 mV were observed (32 ± 2.8 mV). AP duration ranged from 3–20.6 ms (7.2 ± 1.3 ms).
Spontaneous firing was observed in two cells (Fig. 5C).
In voltage-clamp mode, both inward and outward currents were observed in all cells (not
shown), but their relative magnitudes varied considerably. Inward currents were activated
rapidly (<1 ms) and peaked within 1–3 ms. Activation threshold was −30 ± 1 mV, maximal
peak current amplitude was obtained at a mean voltage of −13 ± 1.9 mV, and currents were
completely blocked by TTX (n = 3). These properties are consistent with the presence of
voltage-gated sodium channels that underlie AP generation. In three cells, we observed
spontaneous transient currents that had the characteristics of synaptic currents, including a
rapid rise and slower decay phase. One of these recordings was made with a K-gluconate–
based pipette solution, and holding this cell at −40 mV allowed us to observe both outward
(inhibitory) and inward (excitatory) currents (Figs. 5Di, 5Dii). Although all 14 cells were
injected with biocytin, only five cells were recovered after the completion of the subsequent
immunostaining procedures. However, all of the 5 biocytin-filled cells were TH+ (Figs. 5E–
Neuronal subtype specification depends on interplay of the intrinsic program of precursor cells
and extrinsic morphogens that are present in the surroundings [33]. Although the intrinsic
control of dopaminergic fate specification remains to be clarified, FGF8 and SHH play an
instructive role [17,18]. FGF8 and SHH efficiently promote differentiation of DA neurons from
primary [13] and ESC-derived neuroepithelia [16,34,35]. We have demonstrated in the present
study that DA neurons with either midbrain or forebrain characters can be efficiently generated
from hESCs in response to a simple administration of FGF8 and SHH. By characterizing the
process of neuroepithelial differentiation, we have discovered that it is the time window of
FGF8 and SHH application or the intrinsic program of the precursor cells that determines the
specification of midbrain versus forebrain DA neurons. Early exposure to FGF8 during the
process of neuroepithelial specification directs the precursors preferentially to a midbrain fate
and subsequent differentiation to midbrain DA neurons. Exposure of FGF2-expanded
neuroepithelial cells to FGF8 and SHH promotes differentiation of DA neurons but mostly to
cells with a forebrain phenotype.
Mouse ESCs have been differentiated to neuroepithelial cells, expanded with FGF2,
regionalized or specified with FGF8 and SHH, and subsequently differentiated into DA
neurons [16]. We hypothesized that the same principle should apply to human primates. Indeed,
we are able to generate a large number of DA neurons by differentiating hESCs into
neuroepithelial cells that express Sox1 and organize into neural tube–like rosettes in the
presence of FGF2 [21], treating the neuroepithelial cells with FGF8 and SHH to induce a ventral
midbrain fate and finally differentiating the progenitors to DA neurons. However, most of the
DA neurons generated in this way lack some of the key characteristics of midbrain projection
DA neurons such as large size with complex morphology and expression of midbrain
transcription factors at the protein level. Given that Sox1 is usually expressed by neuroepithelial
cells at the neural tube closure stage in animals [25] and that it is expressed by cells in the
neural tube–like rosettes that correspond to the neural tube formation stage in human
development [20], we reasoned that the Sox1-expressing neuroepithelial cells are regionally
specified and are thus less responsive to morphogens for generating DA neurons with midbrain
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Our hypothesis that FGF8 may instruct the early precursors to adopt a midbrain identity is
confirmed by the efficient induction of its downstream transcription factors, such as En1, Wnt1,
Pax2, and Gbx2, all of which are crucial in patterning the mid-hindbrain junction [36,37], and
by the generation of DA neurons that possess characteristics of projection neurons such as large
cell bodies with complex processes and expression of midbrain markers. Developmentally,
FGF8 is first expressed by epiblasts or ectodermal cells during gastrulation with a proximal
boundary around the primitive streak (E6.5 in mouse). After the neural plate/tube forms (E8.5–
9), FGF8 is present in the telencephalic commissural plate, the ventral midline of the
hypothalamus, and the optic stalks in addition to the mid-hindbrain boundary (isthmus) [38].
Another major event during gastrulation is the formation of the notochord, which produces the
ventralizing morphogen SHH. Hence, from a pure mechanical view, this would mean that both
FGF8 and SHH are made available first to the ectodermal or perhaps primitive neuroectodermal
cells in the mid-hindbrain region. Consequently, these progenitors differentiate into DA
neurons with early born, large-projection neuron phenotypes. This is in line with our
observation that early application of FGF8 and SHH induces predominantly midbrain DA
neurons. Theoretically, SHH needs to be present at the same time. However, we found that
early addition of SHH did not increase the production of DA neurons, which may be due to
the induction of endogenous SHH by FGF8 at this particular window. Alternatively, but not
mutually exclusive, the intrinsic program of the neuroepithelial cells, such as expression of
Pax6 but not Sox1, may endow the precursors with the machinery to respond to FGF8 and
SHH for production of large-projection DA neurons. This notion parallels our recent finding
that only the Pax6+ but not the Sox1+ early neuroepithelial cells can be efficiently instructed
to differentiate into spinal motor neurons by retinoic acid and SHH [24]. Therefore,
specification of early born projection neurons such as midbrain DA neurons and spinal motor
neurons requires morphogen instruction while the neuroectodermal fate is still being
It is presently not clear why FGF2-expanded mouse ESC-derived, but not human ESC-derived,
neuroepithelial cells can be efficiently specified to a ventral midbrain fate. There is recent
evidence that the dorsal or ventral identity of neural progenitors isolated from mouse spinal
cord may be deregulated upon culture, especially in the presence of FGF2 [39], which may
partially account for the capability of expanded mouse ESC-derived neuroepithelial cells to be
respecified. We have observed that the hESC-derived neuroepithelial cells, after expansion in
FGF2, can still generate cells that express En1 but rarely coexpress TH after further
differentiation in the presence of SHH and FGF8. This suggests that human neuroepithelial
cells may not be as plastic as their mouse counterparts to be regionally respecified. Human
neural stem cells retain their regional phenotypes after long-term expansion in the presence of
mitogens, including FGF2 [40], whereas those from mouse brain seem to be respecified, at
least from the gene expression standpoint [41]. Technically, most differentiation protocols
including our present one generate mixed progenies that include undifferentiated or partially
differentiated precursors [42]. These precursors may respond to morphogens at a later stage.
Besides, FGF2 can also caudalize neuroepithelial cells. Due to a much shorter cell-cycle time
in mouse cells, these newly specified progenitors might readily take over the differentiated
population, thus masking the true mechanism. In our present study, undifferentiated stem cells
are removed during the neuroepithelial enrichment process, which minimizes the chance of
the less-differentiated precursors being regionally specified at a later stage.
The differentiation of DA neurons from hESCs has recently been reported by using a coculture
system with stromal cells [35,43,44] or in an aggregated suspension culture system [45]. The
signals associated with stromal cells are strong inducers of neuroectodermal and dopaminergic
differentiation [46]. However, the nature of the signals remains unknown. In contrast, the entire
differentiation process in our present study is achieved under a chemically defined system.
This allows delineating environmental factors that influence cell-fate decision at each
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individual step along the pathway. It is because of this chemically defined system and the well-
characterized neuroectodermal induction process that we are able to differentiate the
requirements for production of midbrain versus forebrain DA neurons. From the application
standpoint, coculture with cell lines that are often tumorigenic introduces risk components as
well as unknown factors, whereas our chemically defined system will allow direct translation
to clinical application should new hESC lines be created without contact with animal products.
The early requirement of morphogen action on neuroepithelial cells for production of
projection neurons like midbrain DA neurons may explain why stem cells or progenitors
expanded from embryonic and adult mammalian brains do not produce projection neurons
[47–49]. Our study also indicates a slow maturation process of the human DA neurons in vitro,
as revealed by the lack of typical electrophysiological properties of midbrain DA neurons
[50] and the poor DA uptake activity (unpublished observation). This may partly reflect the
nature of human cells as well as culture environment that lacks targets and further signals
important for full maturation. Nonetheless, the electrophysiological properties and the activity-
dependent DA release do indicate that the in vitro–generated DA neurons are functional. The
ability to efficiently generate functional human DA neurons from a renewable source offers a
system for toxicological and pharmaceutical screening for chemicals and drugs that may affect
human DA neurons as well as potential cell therapy in the future.
By examining the neuroectoderm differentiation process, we have shown that DA neurons with
the forebrain or midbrain phenotypes can be differentially specified under a chemical-defined
culture system. Although DA phenotype can be specified by exposure to FGF8 and SHH, it is
the time window in which precursors are exposed to the morphogens that determines the
regional identity. Exposure of the hESC-derived Sox1+ neuroepithelial cells to FGF8 and SHH
results in the generation of DA neurons with a forebrain identity such as bipolar morphology,
coexpression with GABA, and absence of midbrain marker En1 expression. Early application
of FGF8 and SHH during the process of neuroectoderm specification leads to the differentiation
of midbrain neuroepithelial cells and subsequent production of midbrain DA neurons, which
have large cell bodies and complex processes and express En1. The in vitro–generated DA
neurons are biologically functional and may provide a renewable source for developing
sustainable therapies for disorders that affect the DA system, such as Parkinson's disease.
This project was supported by The Michael J. Fox Foundation, NIH/NINDS U01 (NS 046587), and partly by a core
grant to the Waisman Center from the National Institute of Child Health and Human Development (P30 HD03352).
We thank Lorenz Studer for suggestions in HPLC analysis and the Bf1 antibody. We are grateful to Elizabeth Lyons
and Patricia Moreno for the help with hESC cultures.
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Figure 1.
Specification of midbrain neural progenitors. Columnar cells (A) appeared in the
differentiating human embryonic stem cell colony at days 8–10, (B) formed neural tube–like
rosettes at day 14, and (C) expressed Sox1. (D): The neuroepithelial cells in FGF2-treated
cultures expressed Bf1 (red) but not En1 (green). (E): En1 (green) expression was observed
in the nestin+ (red) neural progenitors after 6 days of treatment with FGF8 (100 ng/ml) at day
10, expansion in FGF8 for 2 days, and subsequent treatment with FGF8 and SHH (200 ng/ml)
for another 6 days on laminin substrate. (F): These En1+ cells (green) were negative for Bf1
(red). The cell nuclei were stained with Hoechst (bluein C, D). Bar = 50 µm. (G): Reverse
transcription–polymerase chain reaction showed a higher expression level of Pax2, En1, Wnt1,
and Gbx2, as well as Nkx6.1 and SHH mRNAs, when the embryonic stem cell (lane 1)–derived
early neuroepithelial cells (lane 2) were treated with FGF8 followed by FGF8 and SHH (lane
4) compared with the cells that were treated with FGF2 followed by FGF8 and SHH for the
same period (lane 3). Abbreviations: FGF, fibroblast growth factor; SHH, sonic hedgehog.
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Figure 2.
Differentiation of DA neurons. (A): Schematic procedures of DA neuron differentiation.
hESCs were differentiated to early neuroepithelial cells in the form of early rosettes at 8–10
days, followed by formation of neural tube–like rosettes for another week in the presence of
either FGF2 (late FGF8 treatment) or FGF8 (early FGF8 treatment). They were then
differentiated to NPs in the presence of FGF8 and SHH for 6 days before proceeding to DA
neurons in the next 2–3 weeks after withdrawal of morphogens. (B): The neural progenitors
derived form hESCs were positive for nestin (red) and negative for SSEA4 (green), which was
expressed in the undifferentiated hESCs (inset). (C): The dissociated NPs reformed rosettes
and began to extend neurorites 5 days after plating. (D): Approximately one third of the
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text:
Matthew Sweet
Modern Inventors
World Cafe Live Philadelphia
3025 Walnut Street
Philadelphia, PA, 19104
Doors 6:30 PM / Show 8:00 PM
This event is all ages
During a career that stretches back to the mid-'80s, Matthew Sweet has never followed trends, though his landmark 1991 album Girlfriend was responsible for starting one—its bone-dry, caterwauling sonics opening up a wild and picturesque new terrain for restless singer/songwriters to inhabit and explore. Two decades later, Sweet has once again swung for the fences—and connected—with the boldly experimental yet still deeply personal Modern Art.
Modern Inventors have devised their own unique musical niche in a staggeringly short amount of time, but Matt Kass and Josh Benus are not in the business of cheap thrills. The Philadelphia-based duo, whose debut album "Trains & Aeroplanes" will be self-released in December 2012, craft songs as if they were pieces of abstract art that patiently blossom into view; carefully rendered arrangements, heartbreaking harmonies and dense turns of phrases slowly creep up into the foreground upon repeated listens. The single "Hello," for instance, is an ode to suburban teenage angst inspired by acclaimed American painter Eric Fischl. "Goodbye Northbrook Drive" segues a rustic folk composition into a massive crescendo that prods at feelings of emotional paralysis. And then there's the group's pop-infused cover of Paul McCartney's "Every Night" — which has earned praise from Macca himself.
$23.50 - $27.00
All sales are final. Ticket prices do not include processing fees. There are no refunds or exchanges. Cameras & recording devices are not permitted. Showtime and supporting acts are subject to change.
Mezzanine tickets are assessed an additional service fee. For more information, please read the Mezzanine FAQ.
Dining is available at World Cafe Live. Reservations are accepted for Upstairs Live, our full service restaurant; we recommend scheduling a reservation 1½ to 2 hours before show time. Downstairs Live offers a full service bar, and a limited food menu is available for most shows. For more information, please read the Dining FAQ.
WHEELCHAIR ACCESSIBLE SEATS are located on all seated levels of this theatre. For more information, please contact us at email@example.com or call 215-222-1400.
Upcoming Events
World Cafe Live Philadelphia
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The Last Bison
Matthew Sweet with Modern Inventors
Monday, August 26 · Doors 6:30 PM / Show 8:00 PM at World Cafe Live Philadelphia

subset: commoncrawl


text: Stayed at Wendy's B&B whilst my husband was in hospital. First visit had to be postponed so contacted Wendy and explained and was told no problem just let us know when you need the room. Two months later I emailed and rebooked a room for the following month. Such a lovely lady and Leanne her assistant is equally lovely. When... More


text:
People You Know, October 2008 to Jan. 2009
Dr. Jim Corey, longtime professor of English at New Mexico Tech, and his wife Lonna, celebrated their 50th anniversary on Dec. 27, 2008. As a gift from their children, spouses, and 10 grandchildren, they took a trip to San Juan, Puerto Rico, in February 2009. Their children are: Ray Corey (85, BS, environmental engr.) of Kennewick, Wash.; Jeff Corey (84, BS, petroleum engr.) of Durango, Colo.; Craig Corey (92, BS, environmental engr.) of Albuquerque; Glen Corey of Albuquerque; and Janine Corey of Kalispell, Mont.
Jim Doty (50, BS, geophysics) recently wrote to us, reminiscing about being a School of Mines student in the years after World War II.
"In those days, everybody took the same courses for the first two years except that mining engineers didn't take quantitative analysis. One day in my sophomore year, I was sitting out in front of Fitch Hall (the dining room was in the basement), and a guy came up and said, 'They're going to offer a course in geophysics.' With my classy act, I said, 'What the hell is geophysics?' He said, 'I don't know, but I hear you have to be smarter than hell and they make big money.' I said, 'I'm going to be a geophysicist.' As I recall, when I graduated in 1950, there were about 150-300 students in the School of Mines, and our graduating class was about 50-100. With that class, New Mexico School of Mines was really back in business, and obviously has never even thought about going out of business."
"In my senior year, on Friday, January 13, 1950, I married the love of my life and we never parted. I graduated in May, and we had about 30-40 bucks. I got a job with Shell in August and we embarked on as wonderful a life as anybody could have. Leta and I lived on four continents, I worked on six (over 40 countries). Along the way I picked up an MBA and became a CPA and a registered Professional Geophysicist. I worked as a geophysicist, a management consultant and a computer programmer for a missile motor company. Right now, I am almost 83 and I am working on being a professional golfer. I doubt I'll make it, but, what the hell, I might."
William B. Sayer (54, BS, mineral engr.) writes, "I retired in 1989 from the United States Department of Energy, Albuquerque Operations Office, as director of the Engineering Division. My duties involved the management of the facilities and construction program for nine nationwide sites comprising the DOE nuclear weapons complex. After that retirement, I became an engineering management consultant to several major corporations. In 2005, I retired from consulting and moved to Del Mar, Calif., with my wife."
Fred Beck (83, BS, metalurgical engr.) writes, "I work on a United States Navy submarine launched Nuclear Weapons program that is 40 years old, that has plans out till 2046 and beyond! So the job is stable, considering the current recession. I get to travel different places throughout the country, and occasionally to the UK (3 times so far in 22 years). My program actually does some testing from time to time out at TERA (now EMRTC) at Tech, (blowing stuff up of course!) and White Sands as well as the Nevada Test site. I work with a great group of engineers that would be right at home at Tech so... All is very well!"
Delilah Vega-Walsh (97, BS, psychology) is the new Socorro County manager. Walsh has 12 years experience in banking, most of that time at First State Bank. She served as county assessor for two years in the 1990s. She was also a student member of the New Mexico Tech Board of Regents.
Enrique Herrera (02, BS, electrical engr.) lives in Rio Rancho and works for Stirling Energy Systems as a controls software engineer. He is working on the control systems firmware for the SunCatcher, a solar power system designed to track the sun, focus solar heat, and convert it to grid-quality electricity. Enrique welcomes everyone to follow him on his twitter account, https://twitter.com/EnriqueH73.
Chewbacca Actor Featured During Spring Fling
SOCORRO, N.M. March 24, 2015 – This year's Spring Fling features a series of special Comic Con type of events, culminating with appearances by four personalities from the Star Wars franchise. Most ...
Research Symposium Features 90 Projects
SOCORRO, N.M. March 13, 2015 – The fourth annual Student Research Symposium at New Mexico Tech features a wide range of innovative projects, including 48 posters, 20 oral presentations and 22 three-...
Nelson Illusions: 'Smoke & Mystery' At Macey Center
SOCORRO, N.M. March 17, 2015 – Magic at its best, from a woman sawed in half to incredible death-defying stunts, will take center stage at New Mexico Tech's Macey Center at 7:30 p.m. Friday, March 27,...
Legendary Professor Dr. Cormack Passes Away
SOCORRO, N.M. March 16, 2015 – Dr. Robert Cormack, professor of psychology, passed away Saturday, March 14, 2015, in Socorro. A service of remembrance will be held on campus later in the semester. ...
Tech Welcomes H.S. Counselors To Campus
SOCORRO, N.M. March 12, 2015 – Counselor Day is for high school counselors and college advisors to come visit the New Mexico Tech campus Wednesday, April 8. This is the first such event that Tech ha...


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UNFCCC Publishes Deadlines for Submission of Information and Views [viewed]
9 February 2010: The UNFCCC Secretariat has published a message (ODES/COP15/10) to parties on the early submission of information and views.
9 February 2010: The UNFCCC Secretariat has published a message (ODES/COP15/10) to parties on the early submission of information and views.
8 February 2010: UN Secretary-General Ban Ki-moon has released a report titled "Social Dimensions of the New Partnership for Africa's Development (NEPAD)," which reviews the state of progress in the implementation of NEPAD, as well as its social dimensions within the context of the converging global food, financial and energy crises, and climate change (E/CN.5/2010/3).
5 February 2010: The UNFCCC Secretariat has published the report (FCCC/ARR/2009/SVN) of the individual review of the annual submission of Slovenia submitted in 2009.
8 February 2010: The UN Food and Agriculture Organization (FAO) has created a new website on assisted natural regeneration (ANR) of forests, an effective and low cost forest and biodiversity restoration and rehabilitation method.
5 February 2010: The UNFCCC Secretariat has published the report of the tenth session of Ad Hoc Working Group on Further Commitments for Annex I Parties under the Kyoto Protocol (AWG-KP) (FCCC/KP/AWG/2009/17) and the report of the eighth session of the Ad Hoc Working Group on Long-term Cooperative Action (AWG-LCA) (FCCC/AWGLCA/2009/17), which were held in Copenhagen, Denmark, from 7-15 December 2009.
1 February 2010: The UN World Tourism Organization (UNWTO) has released the February 2010 issue of its e-Bulletin Sustainable Development of Tourism.
November 2009: As part of its flagship publication, the State of the World Population Report, the UN Population Fund (UNFPA) has released the fourth edition of the youth supplement, which focuses on young people and climate change.
4 February 2010: The Intergovernmental Panel on Climate Change (IPCC) has published a report of the expert meeting on Detection and Attribution Related to Anthropogenic Climate Change.
3 February 2010: The International Energy Agency (IEA) has released a report titled "Energy Policies of IEA Countries – Italy," which evaluates both thematic policy issues such as energy efficiency and climate change, as well as sectoral policies, as it delves into renewables, electricity, natural gas, oil, coal and nuclear energies.
2 February 2010: The Secretariat of the Convention on Biological Diversity (CBD) has released a brochure titled "Case Studies Illustrating the Socio-economic Benefits of Ecological Networks," highlighting climate change adaptation as one of the benefits.


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Union Station Apartments for Rent
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Finding an apartment in Union StationThe apartments for rent in Union Station are a lot of the best in Denver, that wonderful kitchen (or whatever your dream interior looks like) is waiting for you on MyNewPlace! MyNewPlace's apartment search can help you look for that perfect Union Station apartment. Browse through the above 'popular rentals' listings in your new neighborhood in Denver! Good luck finding your new place!
Want the inside scoop on Union Station? Check out our Union Station Local Talk for local tips and information about Union Station. You can also get answers to your Union Station questions from our local experts by using the "Ask a Question" feature.
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There are many great communities and cities nearby Union Station to also consider:
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There are many great communities and cities nearby Denver, CO to also consider:
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There are many great communities and cities nearby Union Station to also consider:
Featured Apartment Rentals Near Union Station.
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Universities Near Union Station
- Bel-Rea Institute of Animal Technology Off Campus Apartments
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Map & Local Information
Local Talk
Get answers about Union Station from local experts or tell us what you love about it.
What are the best schools in Union Station?
There are several outstanding schools in the Union Station (LoDo) area of Denver that you could look into. The Polaris and Bromwell Elementary Schools are extremely highly rated. Most parents rave about how good they are!
Hi, I am Rhonda and moving from Indiana to Colorado. Is Park Hill a nice decent clean place to live? Thanks.
Park Hill is a nice place to live. There is a golf club, a park and a zoo nearby. It is a quiet place and the distance to the city's center is 5 to 7 miles.


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We've just come from Michael Bastian's S/S 2013 show, where the designer took us on a swanky, seventies-feeling trip to the beach. The bathing suits were short, and usually euro-style briefs, paired with popped-collar sport shirts and beautiful knits, often in white and other pastel collars. Especially loved the sweaters with the geometric shapes, and others that carried forward Bastian's rugby-stripe from last spring season had us wishing we were on vacation as the weather cooled down. Instead, onward to the rest of New York Fashion Week. Stay tuned.
Michael Bastian Has Us Wanting Summer Back
Can Kanye and Damon Dash Save Karmaloop?
Why one expert thinks making Karmaloop an exclusive, upscale retailer might save it from bankruptcy.
Drake + Toronto Raptors = Pure Win
With partners like Drake and Mitchell & Ness, the Raptors's 20th anniversary capsule collection can't miss.
Behind the Harlem Store that Outfits Hip Hop Royalty
After outfitting the Notorious B.I.G. for his funeral and dressing NBA stars like LeBron James, Guy and Sharene Wood are breaking out with...
5 Sleek New Minimalist Runners
Fun Run: the new rules of runners say the simpler the sneaker, the sweeter the shoe.
More From Style


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Press and Medias
STIJN CELIS ADDS ANOTHER UNFORGETTABLE WORK TO THE REPERTOIRE OF LES GRANDS BALLETS THE WORLD PREMIERE OF TRANSFIGURED NIGHTPress Release PDF Les Grands Ballets are thrilled to announce the premiere of another commissioned choreography by Stijn Celis, a long-time collaborator and friend of the company. His new work, Transfigured Night, will be performed for the first time by our dancers at Théâtre Maisonneuve of Place des Arts on May 15, 16, 17, 22, 23 and 24, 2014. Inspired by a poem written by Richard Dehmel in 1896 and the innovative musical composition of the same name by Arnold Schoenberg that followed in 1899, Transfigured Night opens with a man and a woman walking in a dark forest. The woman reveals to her new lover that she is pregnant with another man's child. Each of the movements that follows this announcement evokes the radiant transfiguration of two (three!) becoming one, glowing whole. The work—and the music—is divided into five distinct sections, reflecting the five stanzas of Dehmel's poem and the profound emotions expressed in each.
About Stijn CelisAmong the most active contemporary choreographers working today, Belgian-born Stijn Celis first gained attention as a dancer for the Royal Ballet of Flanders, Zurich Ballet, Cullberg Ballet, Bern Ballet and Ballet du Grand Théâtre de Genève. His creative talents first came to light in 1991 during a choreographic workshop with the Grand Théâtre, then under the artistic direction of Gradimir Pankov. Since 1997, he has focused exclusively on choreography and set design. Stijn's works have been staged by Ballet Gulbenkian, Ballet Mainz, Ballet Wiesbaden, Ballet Nürnberg and Cullberg Ballet and Bern Ballet. In 2002, Les Grands Ballets Canadiens de Montréal commissioned him to create Noces, a vibrant treatise on the rites of marriage set to Stravinsky's famous score and in 2003, Les Grands called on his choreographic talents once more for the creation of Cinderella (The Lost Shoe). Transfigured Night is his third work for the company.
Media contact
Aléna Jeangrand
Relationniste / Publicist
514-849-8681 # 263
Relationniste / Publicist
514-849-8681 # 263


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Foals Album Stream
always had the urge to check them out. this seems to finally be the best time to ACTUALLY check them out.
Album rules, no lie.
you're right, it does
Definitely the correct time to check them out. Superb album.
But why does Rosco's rating on this news piece say 2.5?
Digging: Too Close To Touch - Nerve Endings
Glitch. Happens a lot with news posts.
Damn, this is really good.
Its alright Davey, mine's a 5
7.5 divided by 2. Nope, that average isn't high enough.
Great album, recommended to anyone.
This sounds awesome according to Davey Boy's review.
It's really good, oh man.
This Is Awesome (clap, clap, clap, clap, clap)
I just started listening but I can already tell I'm gonna love this.
Yep, should happen about 20 seconds in... Except for mutatedfreek, who doesn't like 'Blue Blood'! ;-)
I love that track...it reminds me of Antidotes but all grown up *Tear*
Can't wait to give this a hear
"I love that track...it reminds me of Antidotes but all grown up"
Such a spot on comment. I described it as the perfect bridge between albums.
I'm diggin this album a lot. So much variation, it's refreshing.
Uranium... Sandcastles... Digging. There's a theme going on there.
• Slint guitarist survives suicide ..• Tool + Mastodon supergroup• Scarlett Johansson forms band• Muse unleash new single• Nickelback get groovy • New Godspeed You! Black Emperor a..• New Muse song• Steven Wilson premieres new tune• New Incubus Song• Kendrick Lamar unveils new single
• Plain White T's enjoy Lipton• Waxahatchee streams Ivy Tripp• Asa creates indiegogo campaign• Frank Carter unveils new band• Lost Salt Blood Purges streams Vo..• Nils Frahm issues out demos• Sickening Horror stream new LP• Flower Jesus drop new song• Portico stream new album• Arcturus post new song• John Renbourn passes away• The U.S. hates Bieber, Nickelback• Alesana release new song• Bjork releases free live album• Weedeater stream new tune» see all news
Bands: A B C D E F G H I J K L M N O P Q R S T U V W X Y Z


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The Truth and Realities of E-Learning
The latest feature from E-Learning 24/7 provides insight into what is happening with some LMS vendors along with mini takes (reviews) on lesser known systems (coming in May).
We use mobile devices every day and yet each and every day we fail to see their potential. Their potential for learning that is.
The e-learning and distance learning conference in Saudi Arabia, brought a whole new perspective on what is taking place with e-learning in the Kingdom. It was enlightening and exciting.
The latest on the LMS scene, plus some terms you will need to know.
Mobile the final frontier. These are the voyages of mobile learning. To seek out new devices. To boldly go where no e-learning vendor has gone before, specifically LMS vendors who are now starting to get it. To, ah forget it you now the rest, but what you may not know …
I'm back. Well, back from being sick that is (hence the one week delay). And with that delay, came my delay of the LTUK15 review. So, with out any further notice, let's dive right in. Overall Impressions Generally speaking, I really came away quite impressed with the show. As with …
Select any channel below to view all posts within that channel:
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Craig Weiss is an e-learning analyst, consultant and advisor whose firm E-Learning 24/7 provides services to buyers and suppliers in the e-learning industry.
He has been named the most influential person in the world for e-learning (2015).
Craig writes the E-Learning 24/7 blog which is read weekly in 154 countries, territories and colonial territories. .
His latest report, "Top 50 LMSs for 2015", will be available for purchase on Jan. 20th. If you are interested in pre-ordering, please contact Craig today.
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Ball Don't Lie
Stan Van Gundy is getting tired of playing so many back-to-backsBen Rohrbach at Ball Don't Lie2 hrs ago
Scheduling imbalance has been a hot-button NBA issue ever since lottery-bound Western Conference teams began regularly winning more games than the East's lower playoff seeds more than a decade ago, and the disparity between the two conferences may have reached an all-time high this season.
While NBA commissioner Adam Silver has pledged "to make a change" to the league's playoff format, no such adjustment is expected until at least 2016-17, but there is another scheduling quirk that could be addressed even sooner: The discrepancy between the number of back-to-back games each team faces.
Anthony Davis says he'd vote for James Harden as the NBA's Most Valuable PlayerDan Devine at Ball Don't Lie5 hrs ago
You know how you can tell Anthony Davis is a good egg? Given the chance to name himself as the person he'd vote for as the NBA's 2015 Most Valuable Player — a case that's certainly got its merits, what with the league-leading Player Efficiency Rating that's tied for the , the insane "clutch" numbers, the elite shot-blocking and top-notch rim protection, the fact that the only two bigs in NBA history this productive this young were Shaq and Bob McAdoo, and so on — he didn't do it. (What a nice young man.)
[DraftKings: FREE entry to huge cash Fantasy Basketball Contest with first deposit]
Instead, during a Monday visit to "The Dan Patrick Show," the New Orleans Pelicans big man cast his hypothetical MVP ballot for Houston Rockets shooting guard James Harden.LiveSacramento3 - 13HoustonFollow Game
Blake Griffin wishes Clippers fans got louder: 'Home-court advantage is just not there for us'Dan Devine at Ball Don't Lie6 hrs ago
The Golden State Warriors beat the Los Angeles Clippers on Tuesday night, despite having nothing significant to play for after clinching the No. 1 seed in the Western Conference over the weekend, despite sitting starting power forward Draymond Green and despite a 40-point, 12-rebound, five-assist explosion by Blake Griffin in his best game since returning from elbow surgery. The Warriors came back from a 17-point first-half deficit to win their 10th straight game, improve their league-best record to 61-13, and move to a sterling 27-11 record away from the friendly confines of Oracle Arena.Wed, Apr 110:00 PM EDTLA Clippers at PortlandPreview Game
Lionel Hollins: 'I don't think managing your minutes lengthens your career by one minute'Kelly Dwyer at Ball Don't Lie9 hrs ago
The standard superstar level for minutes per game is 36, and Tim Duncan hasn't played more than 36 minutes a game in a season since 2003-04. This is part of the reason he's still a dominant force despite the fact that he'll turn 39 later in April and despite the knowledge that he played his first college game the same month Michael Jordan retired from basketball for the first time.
[DraftKings: FREE entry to huge cash Fantasy Basketball Contest with first deposit]
LeBron James averaged over 41 minutes an NBA contest during the same stretch of ages that Duncan played college basketball, leading the NBA in total minutes twice during that span, and he has to take entire weeks off in order to rest his body just in the days following his 30th birthday. LeBron James eclipsed Larry Bird's combined careerregular season and postseason total minutes earlier this season, and while we're not going to blame his minutes buildup for his bout of leg cramps in last season's NBA Finals, let's just say .
NBA Playoff Picture Update: Brooklyn begins to separate from a crowded fieldEric Freeman at Ball Don't Lie17 hrs ago
With just a few weeks remaining until the NBA postseason, every night can impact the standings. NBA Playoff Picture keeps you up to date on all the most important news for all 16 berths and seeds.
[DraftKings: FREE entry to huge cash Fantasy Basketball Contest with first deposit]
Stuck with the Nets: Among the four squads vying for the East's eighth and final playoff spot, the Brooklyn Nets stand out as the team that most needs to reach the postseason to avoid a crushingly disappointing season. Perhaps it should be no surprise, then, that they are in the best form of any of those contenders.
[Follow Dunks Don't Lie on Tumblr: The best slams from all of basketball]
Brooklyn continued their ascent to mediocrity on Tuesday with a very important home game with the Indiana Pacers, the conference's 10th-place team that still could have swapped spots with the Nets with a win. Yet the hosts controlled the game most of the way and iced the game with this Joe Johnson three-pointer with 15 seconds remaining in regulation:LiveBrooklyn41 - 33New YorkFollow Game
Kevin Durant likes the idea of spending his whole career with one teamEric Freeman at Ball Don't Lie19 hrs ago
Kevin Durant is out for the year due to ongoing foot issues, but he remains near the center of the NBA universe. With just one more season remaining on his contract until the 26-year-old superstar becomes an unrestricted free agent during the largest salary-cap increase ever, many teams and interested observers are keeping an eye on everything KD says or does for potential insight into his pending decision. Every statement counts when so much is at stake.
[DraftKings: FREE entry to huge cash Fantasy Basketball Contest with first deposit]
Gregg Popovich's reaction to Mario Chalmers' beyond-half-court buzzer-beater was perfectDan Devine at Ball Don't Lie21 hrs ago
The San Antonio Spurs opened up their nationally televised matchup with the Miami Heat playing sound defense, holding Erik Spoelstra's club without a made field goal for the first 6 1/2 minutes of Tuesday's contest. The Spurs didn't capitalize early, though, committing seven turnovers that turned into 10 Miami points and kept the Heat up after 12 minutes.
[DraftKings: FREE entry to huge cash Fantasy Basketball Contest with first deposit]
Things tightened up considerably in the second, as San Antonio coughed it up just twice while cranking up the ball movement and offensive effectiveness to outpace the Heat by nine points heading into the final seconds of the half ... only to see the Heat stay within a handful of points at intermission thanks to a wing-and-a-prayer, buzzer-beating, 53-foot 3-pointer by point guard Mario Chalmers:
[Follow Dunks Don't Lie on Tumblr: The best slams from all of basketball]
The best thing about Chalmers' 3? How Spurs coach Gregg Popovich reacted to it, naturally:
LeBron James has so few friends that only his kids will play video games with himKelly Dwyer at Ball Don't Lie1 day ago
(We're joking, people.)
Assured of his status as the East's second seed and rightfully confident in his Cleveland Cavaliers' ability to wipe the floor with the Eastern Conference come playoff time, LeBron James has now taken to destroying his kids at video games.
[DraftKings: FREE entry to huge cash Fantasy Basketball Contest with first deposit]
From James' Facebook page on Tuesday:
Possibly playing as himself on a Cavalier off-day, James gave his sons LeBron Jr. and Bryce fair warning prior to their time with the controller, while letting daughter Zhuri and wife Savannah off the hook.
[Follow Dunks Don't Lie on Tumblr: The best slams from all of basketball ]
The welcome respite came toward the end of another rather event-filled day for James, who disclosed on Monday evening that, shock horror, he doesn't consider himself to be great friends the whole of the NBA. From Joe Vardon's feature at Northeast Ohio Media Group:
Kevin Durant posts a post-surgery picture from his hospital bedKelly Dwyer at Ball Don't Lie1 day ago
March turns into April on Wednesday, the month that the NBA playoffs begin. The days are getting longer, and warmer, and the NBA is gearing up for what could be a killer postseason that will once again feature the Oklahoma City Thunder.
[DraftKings: FREE entry to huge cash Fantasy Basketball Contest with first deposit]
Oklahoma City Thunder fans were hoping not to see Instagram like this on March 31:
Here are Kevin Durant's thoughts, from the same Instagram page:LiveDallas16 - 12Oklahoma CityFollow Game
The NBA has a tanking problem because the NBA used to employ a lot of problemsKelly Dwyer at Ball Don't Lie1 day ago
On Monday evening, the Los Angeles Lakers downed the Philadelphia 76ers on a last-second shot from rookie Jordan Clarkson. Hands were rung and eyes rolled, as a significant portion of NBA fandom could hardly recall what number Clarkson wore prior to this professional event, and yet they could recite the two teams' 2015 NBA draft odds by rote: Los Angeles keeps its pick unless it either continues to win, or in case bad lottery luck pushes the team out of the top five. If it falls out of the top five, the lowly Sixers takes in its own high end lottery pick, and the Lakers' selection.
[DraftKings: FREE entry to huge cash Fantasy Basketball Contest with first deposit]LivePhiladelphia42 - 65WashingtonFollow Game


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For more details visit our Book Festival page at www.maynardpubliclibrary.org/mbf2015
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Museum passes are available to anyone who holds a library card for the Minuteman Library Network. Passes should be reserved in advance at the Circulation Desk or by calling 978-897-1010.
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Download free music you can keep with Freegal!
Freegal Music is a free music service from your library. All you need is your library card number and some libraries utilize a PIN. Freegal offers access to about 8 million songs, including Sony Music's catalog of legendary artists. In total the collection is comprised of music from over 28,000+ labels with music that originates in over 80 countries.
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New E-Resource Added! Consumer Reports Online
Consumer Reports Online
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9 spring-y things to enjoy way past break
This sweetie sure knows how to make a girl feel special
Dear Carol: How can I turn my attitude around?
Get your heart racing with The Walls Around Us
Feel Good Friday: Adorable photo series helps local dogs from a Utah shelter get adopted
The only spring sandal you'll want to wear this season
4 steps to perfectly thick 'n' long lashes
5 strappy sports bras you need in your life
Share your thoughts on our April/May 2015 music issue! What did YOU love?
Nfinity Champions League 2: The Biggest Night in Cheerleading
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Camila Cabello celebrates her birthday with all her famous friends
As of yesterday, Fifth Harmony's Camila Cabello is officially eighteen. Happy birthday, girl! In order to celebrate becoming a legal adult, Camila rounded up a bunch of her favorite people – including Taylor Swift, Selena Gomez, Hailee Steinfeld and Sarah...
Fash face off: Golden Globes
After the People's Choice Awards last week and the Golden Globes this past Sunday, it's clear that awards season has officially started. And you know what that means, right? Our faves are getting all glam and fancy to rock the...
The biggest OMG celeb moments of the week
December is right around the corner. How did that happen? We feel like 2014 just started and now there's only one month left until 2015! We love this time of year, and so do all of our fave celebs. Read...
Friday is here, which means another week has flown by! Staying in-the-know with everything our fave celebs do is difficult when you have an English test and bio project due on the same day. That's why we're taking over the...
Whew! Another week is over and we're here to give you the 4-1-1 on what's happening with your fave celebs. Who broke up, who's making a major comeback and what was the biggest surprise of them all? The only way...
Another week has passed, which means there have been seven days for our fave celebs to make jaw-dropping news. From surprise cancellations to amazing previews, this week had it all. Get the scoop on what's been happening with the stars...
Fash face off: 2014 Billboard Music Awards
The 2014 Billboard Music
Awards rocked Las Vegas this past weekend. Did you get a chance to tune in?
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Try our simple formal updo how-to
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Sarah Hyland goes natural on the red carpet and we totally love it
Sarah Hyland's makeup look at the 2014 Golden Globes is the perfect way to look flawless while staying natural. She pairs a halo braid with dewy skin and bold brows for an ultra chic look and boy, did she nail...
Fash face off: The ultimate red carpet looks of 2013
2013 was loaded with awards shoes and premieres, which means there were hundreds of red-carpet looks. From gorgeous gowns and pretty patterns to cringe-worthy creations, we've seen it all. We're putting our favorite looks from the past year head to...
Design your dress with Duck Tape for a chance to win $10,000
Wanna stick out at prom? Check out Duck Tape's #StuckAtProm scholarship contest for a chance to show off your ah-mazing design skills! More than $50,000 in scholarships is up for grabs—CLICK HERE for deets.


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Now, I have to admit… I do like tart. You'll never offend me with a tart apple or a lemony lemonade. So a case might be made that I'm not the one to talk to about tart desserts.
And slicing up a whole lemon and throwing it in a pie might sound a little crazy, but bear with me here, because this unassuming little dessert might surprise you.
When Meyer lemons are used in place of their more lip puckering companions, I find it hard to believe that anyone could turn up their nose at this tasty Shaker pie.