Efficient Conversion of Astrocytes to Functional Midbrain
Dopaminergic Neurons Using a Single Polycistronic
Russell C. Addis1*, Fu-Chun Hsu5, Rebecca L. Wright1, Marc A. Dichter2,3, Douglas A. Coulter2,4,5, John D.
1Institute for Regenerative Medicine and Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania,
United States of America, 2Mahoney Institute of Neurological Sciences, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of
America, 3Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America, 4Departments of Pediatrics
and Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America, 5Division of Neurology, Children’s Hospital of
Philadelphia, Philadelphia, Pennsylvania, United States of America
Direct cellular reprogramming is a powerful new tool for regenerative medicine. In efforts to understand and treat
Parkinson’s Disease (PD), which is marked by the degeneration of dopaminergic neurons in the midbrain, direct
reprogramming provides a valuable new source of these cells. Astrocytes, the most plentiful cells in the central nervous
system, are an ideal starting population for the direct generation of dopaminergic neurons. In addition to their potential
utility in cell replacement therapies for PD or in modeling the disease in vitro, astrocyte-derived dopaminergic neurons offer
the prospect of direct in vivo reprogramming within the brain. As a first step toward this goal, we report the reprogramming
of astrocytes to dopaminergic neurons using three transcription factors – ASCL1, LMX1B, and NURR1 – delivered in a single
polycistronic lentiviral vector. The process is efficient, with 18.261.5% of cells expressing markers of dopaminergic neurons
after two weeks. The neurons exhibit expression profiles and electrophysiological characteristics consistent with midbrain
dopaminergic neurons, notably including spontaneous pacemaking activity, stimulated release of dopamine, and calcium
oscillations. The present study is the first demonstration that a single vector can mediate reprogramming to dopaminergic
neurons, and indicates that astrocytes are an ideal starting population for the direct generation of dopaminergic neurons.
Citation: Addis RC, Hsu F-C, Wright RL, Dichter MA, Coulter DA, et al. (2011) Efficient Conversion of Astrocytes to Functional Midbrain Dopaminergic Neurons
Using a Single Polycistronic Vector. PLoS ONE 6(12): e28719. doi:10.1371/journal.pone.0028719
Editor: Alysson Renato Muotri, University of California San Diego, United States of America
Received August 18, 2011; Accepted November 14, 2011; Published December 9, 2011
Copyright: ? 2011 Addis 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 funded by the University of Pennsylvania Institute for Regenerative Medicine and the Pennsylvania Health Research Formula Fund. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Parkinson’s Disease (PD) is marked by progressive loss of
dopaminergic neurons in the ventral midbrain. Although the
somata of these neurons are located in the substantia nigra, it is
their projections to the striatum that release dopamine to mediate
motor control. For this reason, the caudate and putamen regions
of the striatum have been the primary targets for cell replacement
strategies in PD . Restoring dopaminergic tone to the striatum
via the engraftment of dopaminergic neurons has long been a goal
in the field of regenerative medicine, beginning with the
transplantation of fetal mesencephalic tissue [2–6]. Given the
conflicting results of these studies, as well as the difficulty in
obtaining sufficient quantities of fetal tissue, alternative cell sources
have been pursued (well-reviewed in ). Both neural stem cells
 and embryonic stem cells  have shown great promise in their
ability to differentiate into dopaminergic neurons, while the advent
of induced pluripotent stem (iPS) cells made real the possibility of
generating patient-specific stem cell lines . More recently,
direct reprogramming has demonstrated that stem cells may not
be necessary at all – three groups have reported that ectopic
expression of small sets of transcription factors can directly convert
fibroblasts to dopaminergic neurons [11–13]. Astrocytes are an
attractive alternative to fibroblasts as a starting population of cells
for reprogramming to dopaminergic neurons. Previous studies
have demonstrated that astrocytes can be directly reprogrammed
to neurons that form functional synapses [14,15]. Conversion of
astrocytes to dopaminergic neurons would not only provide a new
source of neurons for use in cell-based therapies for PD, but this
approach also raises the possibility of direct in vivo reprogramming
as a novel treatment strategy . Since this virus-based strategy
would require no cellular transplantation, many of the concerns of
immunological rejection in cell replacement therapies would be
negated. Furthermore, there is now considerable evidence that
reprogrammed cells retain an epigenetic memory of their original
cell type [17–19], and iPS cells derived from astrocytes have a
greater propensity for neuronal differentiation than those derived
from fibroblasts . Thus, the developmentally close relationship
of astrocytes to neurons may prove advantageous to effective
reprogramming. In the present study, we report the direct
conversion of astrocytes to dopaminergic neurons via three
transcription factors, with the development of a polycistronic
lentiviral vector to facilitate future efforts at in vivo reprogramming.
PLoS ONE | www.plosone.org1December 2011 | Volume 6 | Issue 12 | e28719
Transcription factor screen and polycistronic vector
To identify a combination of transcription factors that is
sufficient to mediate reprogramming to dopaminergic neurons,
twelve transcription factors known to play critical roles in
midbrain dopaminergic neuron development and/or maintenance
[21–23] were cloned into the doxycycline-inducible lentiviral
vector FU-tetO-Gateway (Figure 1A). Seventy-four unique
combinations of these vectors were used to transduce mouse
embryonic fibroblasts for the initial factor screen. RNA of
transduced cells was harvested after 7 days of doxycycline-induced
factor expression and assayed via RT-PCR for expression of
tyrosine hydroxylase (Th) and DOPA decarboxylase (Ddc), the
enzymes of dopamine synthesis. All experiments were performed
in triplicate. The three-factor combination of ASCL1, LMX1B,
and NURR1 resulted in the most robust expression of Th and Ddc
(Figure 1A–B). This combination was selected for further analysis.
In order to increase the efficiency of cells receiving all three
transcription factors, as well as to reduce variability resulting from
different ratios of the factors reaching individual cells, we
constructed a polycistronic vector. The open reading frames of
ASCL1, LMX1B, and NURR1 were linked via recombinant PCR
such that viral 2A peptide sequences  separate the three genes,
as shown in Figure 1C. A glycine-serine-glycine (GSG) linker was
included upstream of each 2A sequence to facilitate protein
cleavage. The ASCL1-P2A-LMX1B-T2A-NURR1 cassette (here-
after referred to as ALN) was inserted into FU-tetO-Gateway to
produce the tetO-ALN vector. Cleavage at the 2A sites was
validated by performing in vitro transcription and translation using
the T7 promoter located at the start of the Gateway recombina-
tion site in the presence of biotinylated lysine. A streptavidin-HRP
western blot confirmed complete cleavage at both the T2A and
P2A sites (Figure 1D). The tetO-ALN lentiviral vector delivered
ALN to both astrocytes and fibroblasts at .99% efficiency, as
determined by immunocytochemistry for the C-terminal V5 tag
on NURR1 (data not shown).
Characterization of gene and protein expression in
induced dopaminergic neurons
Astrocytes were transduced with FUdeltaGW-rtTA (the reverse
tetracycline transactivator, ) and tetO-ALN. The following
day, doxycycline was added to astrocyte medium to induce ALN
expression (Day 0). After four days in astrocyte medium with
doxycycline, transduced cells were switched to NB27G neuronal
medium. Doxycycline was removed on Day 10. After 14 days from
the initial ALN induction, 35.161.5% of cells expressed type III
beta-tubulin (clone TUJ1), a neuronal marker. 50.963.3% of
TUJ1+ cells were also positive for tyrosine hydroxylase, yielding an
overall conversion rate of 18.261.5% (Figure 2A). Quantification
was performed by counting a total of 3357 cells in three
independent reprogramming experiments.
We constructed a lentiviral reporter vector in which the cell
surface marker, CD4, is expressed under the control of the
neuron-specific MAP2 gene promoter. To further characterize the
induced dopaminergic neurons, we sorted ALN-derived neurons
Figure 1. Transcription factor screen and polycistronic vector construction. (A–B) Evaluation of transcription factor combinations to induce
reprogramming. RT-PCR results for a subset of the 74 transcription factor combinations tested for their ability to induce expression of DOPA
decarboxylase (Ddc) and tyrosine hydroxylase (Th) in mouse embryonic fibroblasts after 7 days of factor expression. 0F: uninfected control; A: ASCL1;
B: BRN2; L: LMX1B; N: NURR1; P: PITX3. The combination of ASCL1, LMX1B, and NURR1 (ALN) was the only combination to give robust expression of
both Ddc and Th. (C) Polycistronic vector containing open reading frames of human ASCL1, LMX1B, and NURR1 linked by viral 2A sequences. (D)
Complete cleavage at 2A peptide sites confirmed by in vitro transcription and translation (TnT) of lentiviral plasmids in the presence of biotinylated
lysine. Streptavidin-HRP Western blot shows all newly synthesized protein. Lane 1: No DNA TnT control. Lanes 2–4: TnT performed on original single-
factor plasmids. Lane 5: TnT for polycistronic ALN plasmid. Band intensity is proportional to the number of lysine residues present in each protein
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on Day 14 using the MAP2-CD4 reporter vector and magnetic
beads conjugated to anti-CD4. RNA was isolated from sorted cells
and assayed via RT-PCR for expression of a panel genes shown in
Figure 3. Gene expression in sorted neurons was compared to
astrocytes that did not receive ALN as well as mouse embryonic
stem cells that had been differentiated to dopaminergic neurons
via co-culture with PA6 stromal cells . Sorted neurons
displayed robust upregulation of genes expressed in midbrain
dopaminergic neurons such as Pitx3, Lmx1a, Engrailed-1,
aldehyde dehydrogenase, Foxa2, the vesicular monoamine
transporter Vmat2, Msx1, and the dopamine transporter.
Immunocytochemistry revealed expression of Girk2, a potassium
channel widely expressed in A9/substantia nigra dopaminergic
neurons , in virtually all (.99%) of the Th-immunoreactive
cells (Figure 2B). Otx2, a protein expressed in mature A10/ventral
tegmental area dopaminergic neurons [28,29], was not detected.
Induced dopaminergic neurons exhibited robust synaptophysin
expression (.99% of Th-immunoreactive cells, Figure 2C),
suggesting the capacity to form synaptic connections.
Functional characterization of induced dopaminergic
To evaluate the electrophysiological phenotype of induced
dopaminergic neurons, patch clamping was performed on induced
neurons between days 9 and 26 of initial ALN induction. Neurons
were identified by screening for GFP driven by the MAP2
promoter. Current clamp recordings in Day 9 cells showed an
immature spiking pattern – generally a single action potential per
step current injection. By Day 14–21, neurons demonstrated
repetitive firing of action potentials in individual step current
injections (Figure 4A). Of 30 patched cells, 24 fired action
potentials (80%). Recorded cells had an average resting membrane
potential of 255.4 mV. In voltage clamp, large sodium and
potassium currents were seen (Figure 4B), with an average
maximum INa of 1546 pA. Electrophysiological properties of
induced dopaminergic neurons are summarized in Table 1.
Recordings were made on neurons generated in four independent
It has been well established that midbrain dopaminergic
neurons are pacemaker neurons, spontaneously firing action
potentials at a rate between 1 and 9 Hz with an average of
4.561.7 Hz [30–33]. We observed spontaneous firing of action
potentials in 43% of recorded MAP2-GFP+ induced neurons, a
fraction that is consistent with the 50.963.3% of neurons that
were immunoreactive for tyrosine hydroxylase (Figure 4C). The
frequency of spontaneous firing ranged from 0.9 to 8.6 Hz, with
an average of 5.661.2 Hz. The pacemaker activity of dopami-
nergic neurons is accompanied by rhythmic fluctuations in
intracellular calcium ion concentration [34–37]. To determine
whether induced dopaminergic neurons exhibit calcium oscilla-
Figure 2. Immunocytochemical characterization of astrocyte-derived dopaminergic neurons. Astrocyte-derived dopaminergic neurons,
17 (A) to 19 (B,C) days post-induction. (A) Efficient conversion of astrocytes to dopaminergic neurons demonstrated by immunocytochemistry for the
neuronal marker TUJ1 (type III beta tubulin) and the dopaminergic marker tyrosine hydroxylase (Th); the pan-nuclear marker DAPI is also shown. (B)
Th positive neurons express the potassium channel Girk2, a marker of A9 (substantia nigra) dopaminergic neurons. (C) Punctate synaptophysin (Syp)
expression in tyrosine hydroxylase (Th) positive neurons, indicating the potential to form synaptic connections. Scale bars 50 mm.
Reprogramming Astrocytes to Dopaminergic Neurons
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tions, we transduced astrocytes (prior to reprogramming) with a
lentiviral vector containing the genetically-encoded calcium
indicator GCaMP3  driven by the MAP2 promoter. We
detected robust calcium oscillations with an average frequency of
0.4960.11 Hz (Figure 4E). A typical GCaMP3-expressing in-
duced dopaminergic neuron is shown in Movie S1.
To confirm that induced dopaminergic neurons produce and
release dopamine, we stimulated cells with 56 mM KCl and
measured dopamine release via HPLC. Cells assayed at 17 days
post-transduction with ALN released dopamine in response to
membrane depolarization while un-transduced cells (0F) did not,
as shown in Figure 4D. No significant levels of epinephrine,
norepinephrine, or serotonin were detected.
Direct reprogramming of fibroblasts to dopaminergic
Given the widespread use of fibroblasts as a starting cell
population in reprogramming, we tested whether our polycistronic
vector was effective on mouse embryonic fibroblasts. The ALN
vector was able to reprogram mouse embryonic fibroblasts to
Th+/TUJ1+ neurons, as shown in Figure S1, with an overall
efficiency of 9.160.9%. Fibroblast-derived dopaminergic neurons
exhibited large sodium currents and spontaneous action potential
firing (Figure S1B–D). Dopaminergic neurons generated from
fibroblasts using a nearly identical set of transcription factors
(ASCL1, LMX1A, and NURR1) were recently described and
characterized in detail .
Our study is the first to demonstrate direct reprogramming of
astrocytes to dopaminergic neurons. These neurons exhibit gene
and protein expression patterns that are consistent with A9
midbrain dopaminergic neurons. Given the mounting evidence
that cells retain some epigenetic memory when driven to
pluripotency and subsequent differentiation, such memory is
almost certain to be maintained in direct reprogramming. Global
epigeneticprofiling that compares authentic
neurons to those derived from astrocytes and fibroblasts will be
informative in determining the relative completeness of these
reprogramming processes. It will also be important to compare the
ability of astrocyte- and fibroblast-derived dopaminergic neurons
to engraft and function in animal models of PD.
In addition to providing a novel source of dopaminergic
neurons for use in cell-based therapies for PD, our use of astrocytes
as the starting population allows an approach that may obviate the
need for grafting altogether – direct in vivo reprogramming to
replace lost neurons. Such an approach is facilitated by the
development of a polycistronic vector, especially since one of the
three factors, ASCL1, has been shown to reprogram astrocytes to
non-dopaminergic neurons in the absence of additional factors
. The fact that the polycistronic ALN vector reprograms
astrocytes at such a high efficiency is also noteworthy, given the
history of polycistronic vector use in generating iPS cells. A single
vector delivering transcription factors that induce pluripotency was
shown to be effective, but at a significantly lower rate than that
which could be achieved when the factors were delivered
individually . This was presumed to indicate that a particular
ratio of reprogramming factors is ideal for inducing pluripotency.
In the case of reprogramming to dopaminergic neurons, we can
conclude that either the ratio of factors is not as important or, less
likely, that our polycistronic vector fortuitously delivers the factors
in the appropriate proportions. The present study takes a crucial
step toward the ultimate goal of in vivo reprogramming of
Figure 3. Transcriptional profile of astrocyte-derived dopami-
nergic neurons. Heat map of quantitative RT-PCR results comparing
astrocyte-derived neurons magnetically sorted for a MAP2-CD4 reporter.
Ast, uninfected astrocytes. ESN, mouse embryonic stem cell-derived
neurons, generated via co-culture with PA6 stromal cells. Induced
dopaminergic neurons express markers consistent with midbrain
dopaminergic neurons. Color scale indicates change in Ct (threshold
cycle) relative to the normalizing actin control. Higher delta Ct values
correspond to lower relative gene expression, with every Ct decrease of
3.3 representing a ten-fold increase in relative expression.
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Figure 4. Functional characterization of astrocyte-derived dopaminergic neurons. (A) Action potential firing characteristics of induced
dopaminergic neurons. Five overlapping traces are depicted derived from whole-cell current clamp recording of a representative induced
dopaminergic neuron, elicited in response to hyperpolarizing and depolarizing current injection, increased from 210 to +40 pA in 10 pA increments.
(B) Voltage-dependent sodium currents in induced dopaminergic neurons. Membrane potential was initially held at 290 mV and incrementally
increased from 260 to +20 mV in 5 mV depolarizing steps. (C) Spontaneous action potential firing, consistent with a dopaminergic neuron
pacemaker phenotype. The recording was conducted at resting membrane potential (251 mV). (D) Dopamine release quantified by HPLC.
Membrane depolarization was induced with 56 mM KCl at 17 days post-infection. 0F: uninfected astrocytes; ALN: induced dopaminergic neurons. (E)
MAP2-GCaMP3 reveals rhythmic oscillations of intracellular calcium. Left panel displays a single frame of Movie S1, a recording of oscillating levels of
intracellular calcium, which is presented as a histogram of GCaMP3 fluorescence intensity in the adjacent panel.
Table 1. Electrophysiological properties of astrocyte-derived dopaminergic neurons.
Capacitance (pF)RMP (mV) Spontaneous firing (%)
(Hz) Threshold (mV)INa-max (pA)IK-max (pA)IR (GV)
227.73 1546.00 1381.91 1.48
SEM 0.761.46 11.00 1.172.27 175.21151.510.23
N8 16 217 1111 11 16
RMP: resting membrane potential; AP freq: frequency of spontaneous action potentials; INaand IK: Maximum sodium and potassium currents; IR: input resistance.
Reprogramming Astrocytes to Dopaminergic Neurons
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astrocytes to dopaminergic neurons. Future work will determine
the feasibility of this approach in animal models of PD.
Materials and Methods
Lentiviral expression vector construction
To make a drug-inducible lentiviral vector that is compatible
with Gateway recombination (Invitrogen), we removed the OCT4
open reading frame from FU-tetO-hOCT4 (Addgene plasmid
19778, ) and replaced it with a Gateway cassette cloned from
pEF-DEST51 (Invitrogen) to generate FU-tetO-Gateway. ORFs
that lack stop codons can be cloned into this plasmid to express
proteins with a C-terminal V5 epitope tag. ORFs and cDNAs
were purchased from Open Biosystems (Table 2).
Reporter vector construction
The MAP2-GFP lentiviral reporter plasmid pGZ-hMAP2 was
purchased from System Biosciences (SR10047PA-1). The MAP2
promoter sequence from this plasmid was then cloned along with
the Gateway cassette of pEF-DEST51 into the backbone of
FUdeltaGW-rtTA (Addgene plasmid 19780) following removal of
the ubiquitin promoter-rtTA sequence to produce FU-MAP2-
Gateway. This destination vector was subsequently used to
generate MAP2-CD4 (human CD4 sequence cloned from
pMACS4-IRESII, Miltenyi Biotec) and to generate MAP2-
GCaMP3 (GCaMP3 cloned from G-CaMP3, Addgene plasmid
22692). All lentiviruses were packaged in HEK-293T cells using
psPAX2 (Addgene plasmid 12260) and pMD2.G (Addgene
plasmid 12259). Calcium imaging of neurons containing the
MAP2-GCaMP3 reporter was performed on an Olympus iX-81
with a 606 oil objective lens using Metamorph and HyperCam
software, with neurons grown on poly-D-lysine coated Fluor-
oDishes (World Precision Instruments FD35PDL-100). GCaMP3
movies were analyzed in ImageJ, corrected to account for
photobleaching, and intensities were plotted using SigmaPlot.
Polycistronic vector construction
Open reading frames of human ASCL1, LMX1B, and NURR1
were cloned from the plasmids listed above using the following
primers: B1-ASCL1-FWD: GGGGacaagtttgtacaaaaaagcaggctAC-
CATGGAAAGCTCTGCCAAG; ASCL1-REV: CATCTCCT-
CCGAACCAGTTGGTGAAGTC; LMX1B-FWD: CTCTCT-
GCCTATGTTGGACGGCATCAAG; LMX1B-REV: ACGT-
ATCCGGAGGCGAAGTAGGAACT; NURR1-FWD: GAAG-
CCCTATGCCTTGTGTTCAGGCG; B2-NURR1-REV: GG-
(extra A added to maintain reading frame with C-terminal V5-
tag). The 3 ORFs were then joined via recombinant PCR using
ONR221, and subsequently cloned into FU-tetO-Gateway via
Gateway recombination. Cleavage at 2A sites was verified by
performing in vitro transcription and translation using the TnT T7
Coupled Reticulocyte Lysate System (Promega L4610) in the
presence of biotinylated lysine (Promega L5061). 5 mL of each
TnT-generated protein sample was resolved via PAGE, trans-
ferred to PVDF membrane, incubated with streptavidin-conju-
gated horseradish peroxidase (Cell Signaling Technology 3999,
1:2000), and detected with ECL Plus (GE Healthcare RPN2132).
Primary mouse embryonic fibroblasts (strain CF-1) were
purchased from Millipore (PMEF-CFL) and cultured in DMEM
(Gibco) containing 10% fetal bovine serum (Hyclone) and 16non-
essential amino acids (Gibco). Primary postnatal mouse astrocytes
(strain CD1) isolated from cerebral cortex were purchased from
ScienCell (MA1800) and cultured in Astrocyte Growth Medium
(AGM, Lonza). For reprogramming, astrocytes or fibroblasts were
plated onto poly-L-lysine-coated glass coverslips or 6-well plates
(BD Biocoat). Expression of transcription factors was induced by
the addition of doxycycline (2 mg/mL). On Day 4, medium was
switched to NB27G neuronal medium containing Neurobasal
medium and 16B27 supplement (Gibco), with 20 ng/mL GDNF
(R&D Systems) and doxycycline. Doxycycline was removed on
Day 10. Mouse embryonic stem cells (line ESD3) were
differentiated to dopaminergic neurons by co-culturing with PA6
stromal cells according to a published protocol .
Cells on glass coverslips were fixed in 4% paraformaldehyde
with 0.15% picric acid, blocked in 10% chicken serum, 1% bovine
serum albumin (w/v), 0.3% Triton X-100 in PBS and stained with
antibodies to tyrosine hydroxylase (Millipore MAB318 or Santa
Cruz sc-14007, both 1:200), type III beta-tubulin (clone TUJ1,
Covance MMS-435P, 1:400), synaptophysin (Santa Cruz sc-9116,
1:200), Girk2 (Santa Cruz sc-16135, 1:50), Otx2 (Abcam ab21990,
1:100), and V5 (Invitrogen R96025, 1:400). Detection was
performed with alexa-fluor labeled secondary antibodies (Molec-
ular Probes) and coverslips were mounted in ProLong Gold
Antifade Reagent with DAPI (Invitrogen). Imaging was performed
on an Olympus iX-81 with Metamorph software. Quantification
of TUJ1+ and Th+ cells was performed by counting a total of 3357
cells in 20 fields of view for astrocytes and 885 cells in 4 fields of
view for fibroblasts.
Induced dopaminergic neurons containing the MAP2-CD4
reporter were grown on 10 cm dishes coated in poly-L-lysine. On
Day 14, cells were harvested with Accutase (Sigma A6964),
incubated with anti-CD4-conjugated MACSelect 4 Microbeads
(Miltenyi Biotec 130-070-101) and sorted on MS columns
(Miltenyi Biotec 130-042-201).
Table 2. Open Reading Frames and cDNAs cloned into FU-
Transcription Factor Accession Number Open Biosystems Clone ID
BRN2 (POU3F2)BC051699 4817001
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RNA was collected using the RNeasy Mini Kit (Qiagen 74104)
and first-strand cDNA synthesis was performed using Superscript
III reverse transcriptase (Invitrogen 18080051) with random
hexamer primers. Quantitative real-time PCR was performed on
an ABI 7900HT system (Applied Biosystems) using the TaqMan
gene expression assays listed in Table 3 and normalized to beta
actin (Mm00607939_s1). Analysis of gene expression data was
performed with DataAssist version 3.0 (Applied Biosystems).
Voltage- and current-clamp whole cell patch clamp recordings
were performed on MAP2-GFP+ cells with neuronal morphology,
visualized using an upright fixed stage microscope (Olympus) and
a 406water immersion objective. All recordings were performed
at room temperature (20–24uC) using glass patch pipettes,
fabricated on a Flaming-Brown micropipette puller (P-97, Sutter
Instruments). The electrodes were 6–8 MV in resistance when
filled with potassium gluconate-based intracellular solution (in
mM: 145 K-gluconate, 2 MgCl2, 2.5 KCl, 2.5 NaCl, 0.5
GTP?Tris, 0.1 BAPTA, 2 ATP?Mg and 10 HEPES). The
recording chamber was perfused with aCSF solution with the
following composition: 155 mM NaCl, 3 mM KCl, 1 mM MgCl2,
3 mM CaCl2, 25 mM glucose and 10 mM HEPES, pH, 7.35.
Neurons were recorded using an Axopatch 200B amplifier
(Molecular Devices, Sunnyvale, CA), sampled at 10 kHz and
digitized using Digitizer 1320A (Molecular Devices), with data
stored for off-line analysis (Clampfit 10; Axon Instruments, Inc.,
Foster City, CA). In voltage clamp mode, cells were held at a
membrane potential of 260 mV to record the potential
pacemaker current fluctuations. To evoke voltage-gated currents,
the command potential was held initially at 290 mV, then
stepped to potentials ranging from 260 mV to +20 mV in 5 mV
increments. In current clamp mode, cells were recorded for 2–
10 min to examine spontaneous fluctuations in membrane
potential and possible spontaneous firing. In order to calculate
input resistance, all cells were injected with at least one
hyperpolarized command current.
HPLC for dopamine quantification
Membrane depolarization was evoked by incubating cells in a
well of a 6-well dish with 1 mL aCSF containing 56 mM KCl for
15 minutes at 37uC. The aCSF solution was then collected and
catecholamines were extracted onto 30 mg activated alumina
(Wako 018-09561). The alumina was washed with ultrapure water
and dried on Ultrafree-MC GV filters (Millipore UFC30GV00).
Catecholamines were eluted into 100 mL 2% acetic acid (v/v) with
100 mM EDTA. 20 mL samples were analyzed on an HTEC-500
HPLC system with electrochemical detection (Eicom) using a
reversed phase C18 separation column (Eicompak CA-5ODS).
Mobile phase consisted of 88% 0.1 M phosphate buffer pH 6.0,
12% methanol, 600 mg/L sodium octanesulfonate, and 50 mg/L
EDTA?2Na. Analysis was performed at 25uC with a flow rate of
230 mL/min and dopamine quantities were calculated by
comparison of area under curve measurements to known standard
dilutions of dopamine hydrochloride (Sigma H8502).
Table 3. TaqMan assays for RT-PCR.
Gene Symbol Gene Product TaqMan Assay
Aldh1a7 aldehyde dehydrogenaseMm00496380_m1
Cacna1g Cav3.1 calcium channel Mm00486572_m1
Calb1 Calbindin-1 Mm00486645_m1
Ddc DOPA decarboxylaseMm00516688_m1
Foxa2 forkhead box A2Mm01976556_s1
Gabra1 GABA A receptor subunit alpha-1 Mm00439046_m1
Gad2glutamic acid decarboxylase 2 Mm00484623_m1
Grin1 NMDA receptor 1Mm00433800_m1
Kcnn3 KCa2.3 potassium channelMm00446516_m1
Lmx1aLIM homeobox transcription factor 1 alphaMm00473947_m1
Maptmicrotubule-associated protein tauMm00521988_m1
Msx1msh-like homeobox 1Mm00440330_m1
Pax2paired box gene 2Mm01217939_m1
Pax5paired box gene 5Mm00435501_m1
Pitx3 paired-like homeodomain transcription factor 3Mm01194166_g1
Pou3f2POU domain, class 3, transcription factor 2 (Brn2)Mm00843777_s1
Scn3aNav1.3 sodium channel, alpha subunitMm00658167_m1
Slc17a7vesicular glutamate transporter 1 (vGlut1)Mm00812886_m1
Slc18a2vesicular monoamine transporter 2 (Vmat2)Mm00553058_m1
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Supporting Information Download full-text
The ALN polycistronic vector is effective in converting fibroblasts to
dopaminergic neurons, demonstrated by immunocytochemistry for
the neuronal marker TUJ1 (14.962.3% positive) and the dopami-
nergic marker tyrosine hydroxylase (9.160.9% positive); the pan-
nuclear marker DAPI is also shown. Scale bars 50 mm. (B) Action
potential firing characteristics of fibroblast-derived dopaminergic
neurons. Four overlapping traces are depicted derived from whole-
neuron, elicited in response to hyperpolarizing and depolarizing
current injection, increased from 220 to +10 pA in 10 pA
increments. (C) Voltage-dependent sodium currents in fibroblast-
derived dopaminergic neurons. Membrane potential was initially held
at 290 mV and incrementally increased from 260 to +40 mV in
5 mV depolarizing steps. (D) Spontaneous action potential firing,
consistent with a dopaminergic neuron pacemaker phenotype. The
recording was conducted at resting membrane potential (257 mV).
Fibroblast-derived dopaminergic neurons. (A)
indicator GCaMP3 is expressed under the control of the
neuron-specific MAP2 promoter. Fluorescence level increases with
increased levels of intracellular calcium. Rhythmic oscillations of
calcium levels are seen, consistent with the midbrain dopaminergic
Calcium oscillations in an astrocyte-derived
The authors thank Hajime Takano for microscopy assistance.
Conceived and designed the experiments: RCA MAD DAC JDG.
Performed the experiments: RCA F-CH RLW. Analyzed the data: RCA
F-CH MAD DAC JDG. Contributed reagents/materials/analysis tools:
RCA F-CH RLW. Wrote the paper: RCA.
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Reprogramming Astrocytes to Dopaminergic Neurons
PLoS ONE | www.plosone.org8 December 2011 | Volume 6 | Issue 12 | e28719