Direct generation of functional dopaminergic
neurons from mouse and human fibroblasts
Massimiliano Caiazzo1, Maria Teresa Dell’Anno1*, Elena Dvoretskova2*, Dejan Lazarevic3,4, Stefano Taverna2, Damiana Leo2,
Tatyana D. Sotnikova2, Andrea Menegon5, Paola Roncaglia4, Giorgia Colciago1, Giovanni Russo2, Piero Carninci6, Gianni Pezzoli7,
Raul R. Gainetdinov2, Stefano Gustincich4,8, Alexander Dityatev2& Vania Broccoli1
resulting from degeneration of mesencephalic dopaminergic neu-
rons1,2. In particular, transplantation of embryonic-stem-cell-
derived dopaminergic neurons has been shown to be efficient in
restoring motor symptoms in conditions of dopamine defi-
ciency3,4. However, the use of pluripotent-derived cells might lead
identified a minimal set of three transcription factors—Mash1
(also known as Ascl1), Nurr1 (also known as Nr4a2) and
neurons from mouseand humanfibroblasts without reverting toa
progenitor cell stage. Induced dopaminergic (iDA) cells release
dopamine and show spontaneous electrical activity organized in
regular spikes consistent with the pacemaker activity featured by
brain dopaminergic neurons. The three factors were able to elicit
dopaminergic neuronal conversion in prenatal and adult fibro-
blasts from healthy donors and Parkinson’s disease patients.
Direct generation of iDA cells from somatic cells might have sig-
onal development, in vitro disease modelling and cell replacement
Seminal studies have demonstrated that functional neurons can be
genetics-based approaches6. More recently, in a set of elegant experi-
ments, fibroblasts have been directly converted into neuronal cells
Mash1, Brn2 (also known as Pou3f2) and Myt1l7. However, iNs rep-
resent a heterogeneous population of glutamatergic and GABAergic
neurons and their degree of global reprogramming remains to be
properly characterized. It is thus unclear whether a specific neuronal
subtype can be preferentially induced from direct reprogramming of
rons through the direct conversion of somatic cells by forced expres-
sion of lineage-specific factors that act during brain development8,9.
Initially, we transduced mouse embryonic fibroblasts (MEFs) from
TH-GFP transgenic mice10with a mixture of doxycycline (dox)-
inducible lentiviruses expressing all selected factors (11 dopaminergic
and 3iN; Supplementary Table 1) or with DsRed retrovirus (negative
control) (Fig. 1a–d). We did not observe any GFP1cells in MEFs 10
days after Ds-Red retrovirus infection or in culture without any viral
in the generation of a small number of bright GFP1cells (1.860.8%)
(Supplementary Fig. 1d–f). We next sought to determine the minimal
set of genes required for dopaminergic neuronal induction. Given its
essential role as a proneural gene during neurogenesis, Mash1 was
introduced into MEFs together with each other single dopaminergic
factor. Reporter gene expression was elicited only when Mash1 was
specification and survival during development and in adulthood11.
a third molecule of the 12 remaining and scored for the rate and mor-
phology of GFP1cells in each combination. Surprisingly, only Lmx1a
and in part Lmx1b (1863% versus 1363% of GFP1cells, respec-
tively) were able to synergize with Mash1/Nurr1, robustly increasing
phology (Fig. 1h and Supplementary Fig. 1s–y). Using the Mash1/
*These authors contributed equally to this work.
1Stem Cells and Neurogenesis Unit, Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy.2Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, via
34136 Trieste, Italy.5Advanced Light and Electron Microscopy Bio-Imaging Centre, Experimental Imaging Centre, San Raffaele Scientific Institute, 20132 Milan, Italy.6Omics Science Center, RIKEN
Foundation Laboratory, 34136 Trieste, Italy.
9 kb 9 kb
TH promoterGFP GFP TH promoter
NI AN ANLa
Figure 1 | Mash1,Nurr1andLmx1areprogrammousefibroblastsintoiDA
cells. a, b, TH and GFP detection in TH-GFP adult brain (a) and ventral
midbrain primary cell culture (b). DIV, days in vitro. SN, substantia nigra;
VTA, ventral tegmental area. P112, postnatal day 112. c, Scheme of
and in uninfected MEFs (inset) after 16 days in vitro. e–g, i–l, iDA cells are
positive for the dopaminergic markers TH (e–g), VMAT2, ALDH1A1,
calbindin and DAT (i–l). h, Quantification of TuJ11and TH1cells. AN,
Mash1, Nurr1; ANLa, Mash1, Nurr1, Lmx1a; NI, non-induced. Data are
presented as mean6s.e.m. Scale bars: 500mm (a), 20mm (b, j), 50mm (e–
g, k, l) and 100mm (d, i).
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further increase in GFP1cells, with Brn2 and Myt1l, the other two iN
factors, even reducing the overall reprogramming efficiency (data not
shown). For these reasons we focused on cells reprogrammed exclu-
sively with the Mash1/Nurr1/Lmx1a factor combination. The same
genecocktail was also proficient in reprogramming adult mouse fibro-
blasts with high efficiency (Supplementary Fig. 2).
Sixteen days after reprogramming, a large number of GFP1cells
expressed many of the distinctive components of the dopaminergic
also known as SLC18A2), dopamine transporter (DAT; also known
as SLC6A3), as well as aldehyde dehydrogenase 1a1 (ALDH1A1) and
calbindin (Fig. 1e–l). Conversely, markers associated with adrener-
gic (dopamine-b-hydroxylase (DBH)) or serotonergic (tryptophan
hydroxylase 1 or 2 (TPH1/2)) serotonin transporter (SERT; also
known as SLC6A4) neurons were not induced (data not shown).
Transcriptional analysis by reverse transcription–polymerase chain
gene network including the endogenous expression of Nurr1 and
by hierarchical clustering (Fig. 2a, b) and the general degree of gene
expression overlap (Fig. 2c). Of note, many representative genes of the
Ret, Gfra1, Foxa1, Gdnf and Drd2 were highly enriched (Fig. 2d).
Conversely, genes coding for adrenergic and serotonergic biosynthetic
enzymes were found to be not upregulated in the reprogrammed cells
(Fig. 2e). Moreover, the fibroblast markers Twist2, Zeb2, Tgfb1i1 and
Chd2 (ref.12)weredownregulated iniDA cells(Fig.2f).Thesefindings
indicate that the genetic reprogramming erased the majority of the
evident expression hallmarks of the cell of origin, while specifically
inducing the dopaminergic neuronal phenotype and not those of other
160 genes differently expressed with a$5-fold change (Supplementary
gene expression in iDA cells remains to be addressed.
lated in dopaminergic neuronal cells whereas they were fully methy-
lated in parental fibroblasts, indicating their epigenetic reactivation
during dopaminergic neuronal conversion (Supplementary Fig. 4).
morphology with multiple and long processes (Fig. 1d–l). Hence, we
asked whether induced neuronal cells establish synaptic contacts in
culture. Notably, synaptic resident proteins such as synaptotagmin I
(SYT1) andsynapsin (SYN)werelocalized in discrete punctaand colo-
calized with TH immunolabelling, suggesting the establishment of
dopaminergic synaptic terminals (Supplementary Fig. 5). Moreover,
synaptic processes (Supplementary Fig. 5).
Next, we performed patch-clamp recordings of GFP1iDA cells
(n516) as well as primary mDA neurons (n512) to compare their
respective physiological properties13,14. iDA cells had higher cell res-
istance and lower capacitance than primary dopaminergic neurons,
(Fig. 3a), overshooting action potentials (Fig. 3c), and even more
prominent K1currents (Fig. 3b) and afterspike hyperpolarizations
(Supplementary Table 4). More than 80% of iDA cells showed rhyth-
mic discharges (Fig. 3d, e) at an average frequency of 2.6Hz. The
identity of voltage-gated inward Na1and outward K1currents in
iDA cells has been verified pharmacologically (Supplementary Fig. 6).
the D2 receptor (Fig. 3f). To verify whether the dopamine receptors
were functional, we applied the specific D2/D3 receptor agonist quin-
pirole (1mM), which markedly suppressed neuronal firing in 6 of 10
metry for real-time electrochemical detection of monoamine secretion
from iDA cells15,16. When carbon fibre electrodes were placed adjacent
of monoamines. Furthermore, high-performance liquid chromato-
graphy (HPLC) measurements revealed that iDA cells contain high
KCl (Fig. 3j). Thus, reprogrammed cells show several major properties
of dopaminergic neurons in terms of spontaneous spiking activity,
temporal parameters of action potentials, inhibition of cell firing
through D2 autoreceptors and controlled dopamine release.
factors to induce a stable reprogrammed cell state, infected MEFs were
treated with dox for different time windows, after which dox was with-
drawn. Only when fibroblasts were treated with dox for 6 or more days
were numerous neuronal cells, mostly TH1, observed (Supplementary
Fig. 7). Thus, reprogramming is a relatively rapid process that requires
transgene expression and even at 18 days of dox withdrawal, iDA cells
Fig. 8 and Supplementary Table 4).
Reprogramming of fibroblasts into differentiated neuronal cells
might occur directly or by passing first through neural progenitors.
label the proliferating cells, virtually all neuronal cells were already
post-mitotic after this time (Supplementary Fig. 9b, c, h). Despite
the fact that during the first 2 days infected cells were actively prolif-
erating in serum-containing medium, none showed expression of the
neural progenitor molecular markers Sox2, Ngn2, Otx2, Lmx1b and
mDA.A9.1mDA.A9.5 mDA.A10.4mDA.A10.3 mDA.A10.6 mDA.A9.2
0 1020 30
Other monoaminergic genesFibroblast-specific genes
Figure 2 | Mouse iDA cells expression profiling. a, Heat-map of genes
differentiallyexpressed in RNA-microarray analysisperformed on MEFs (NI),
iDA cells and brain mDAs (A9–A10). b, c, Hierarchical clustering (b) and
general degree of overlapping expression (c) among the three cell populations
(DA) markers are increased, whereas other monoaminergic neuronal markers
are not activated and fibroblasts markers are silenced.
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En1 (ref. 17) (Supplementary Fig. 9i). Furthermore, we used a genetic
tracing system based on activation of the Sox2b-geoLacZ reporter18
showing that, as a proof-of-principle, LacZ activity was easily visua-
lized upon reprogramming of Sox21/b-geofibroblasts into induced
pluripotent stem cells. By contrast, the reporter was never activated
from the same cells when engaged into direct iDA reprogramming
(Supplementary Fig. 9j). Altogether, these findings are inconsistent
with the occurrence of detectable cell intermediates during the repro-
gramming of fibroblasts into iDA cells.
Next, the in vivo differentiation potential of iDA cells was assessed
by orthotopic transplantations into neonatal mouse brains. Four days
after viral transgene induction, infected cells were grafted into the
extremely elaborated morphology (Supplementary Figs 10 and 11).
Most of the GFP1grafted neuronal cells were positive for TH,
AADC, VMAT2 and DAT, indicating the acquirement of a full neur-
onal dopaminergic cell fate (Supplementary Fig. 11b–g, i–l). Injection
of brief supra-threshold current pulses evoked overshooting action
potentials, and large Na1and K1currents were activated by depol-
arizing voltage steps (Supplementary Fig. 11o, p).Therefore, iDAcells
period of time from grafting.
We then translated the same procedure to the human system by
we scored numerous TuJ11and TH1neuronal cells accounting,
respectively, for 1064% and 662% of the infected cells (Sup-
plementary Fig. 12a–c). We then reprogrammed adult human fibro-
with genetic forms of Parkinson’s disease (Supplementary Table 5).
to convert into neuronal cells, accounting for an estimated efficiency
for TuJ11and TH1cells of 561% and 361%, respectively (Fig. 4a–
g). iDA cells were positive for ALDH1A1, TH, AADC, VMAT2 and
DAT by immunocytochemistry (Fig. 4a–f) and gene expression ana-
lysis (Supplementary Fig. 13). Cell conversion was stable over time as
the number and morphology of human iDA cells was not obviously
affected up to day 24 after reprogramming even when dox was with-
drawn from day 6 onwards (Fig. 4h, i).
Recordings in five infected fetal human iDA cells showed that the
electrophysiological properties of these cells resemble mouse iDA cells
0.760.1nA for Na1currents; 1.260.1nA for delayed rectifier K1
pharmacologically (Fig. 4m, n). Most importantly, depolarization of
Baseline (+750 mV)
K+ stimulation (+750 mV)
K+ stimulation (–750 mV)
0 2 4 8 12 16 24 DIV
Figure 4 | Characterization of human fibroblasts reprogrammed into iDA
g, Quantification of iDA cells obtained from fetal (IMR90), healthy and
a time-course study from 0 to 24 days in vitro (DIV). i, Quantification of TuJ11
and TH1reprogrammed cells kept with (w) orwithout (w/o) doxycycline for 6,
12, 18 or 24 DIV. Data are presented as mean6s.e.m. j, k, Whole-cell voltage-
ofsingle actionpotentialelicited by aminimaldepolarization.m,n,Suppression
4-AP. o, Amperometric recordings of release events after K1stimulation; high-
resolution pattern is shown below the image of the recorded cell. All cells are 18
Baseline (+750 mV)
K+ stimulation (+750 mV)
K+ stimulation (–750 mV)
0 200 400 600
Dopamine (ng ml–1)
Figure 3 | Functional characterization of mouse iDA cells. a, b, Whole-cell
voltage-clamp recording of Na1and K1currents. c, Current-clamp recording
of multiple action potentials evoked by current injection. d, e, Current-clamp
recording and interspike interval frequency of spontaneous action potentials.
f, D2 receptor (D2R) staining. g, h, Effect of the D2/D3 agonist quinpirole on
spiking frequency (g) and its statistical analysis (h) (*P50.005, paired t-test,
n56). i, Amperometric recordings of monoamine release after K1
stimulation; release events are shown with high-resolution below the image of
the recorded cell. j, Dopamine content measured by HPLC in uninfected (NI)
and iDA cells, both in cell pellets and in the supernatant (SN) after K1
stimulation. All cells are 16 days in vitro unless otherwise stated. Scale bars:
50mm (b) and 20mm (i). Data are presented as mean6s.e.m.
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amperometric measurements, as described earlier for mouse iDA cells
(Fig. 4o). In summary, these experiments indicate that actively spiking
dopamine-secreting cells can be induced by forced expression of the
three factors in adult human cells from both healthy donors and
Parkinson’s disease patients.
Here, we demonstrated that the combination of three transcription
dopaminergic neuronal cells from mouse and human fibroblasts.
be modulated via D2 receptors. Importantly, this cell conversion
diverges with respect to developmental neuronal lineage commitment
need to be tested further in long-term in vivo transplantation studies.
Generation of functional dopaminergic neuronal cells by direct
reprogramming opens new possibilities for regenerative therapies for
Parkinson’s disease and related disorders. However, to be clinically
and the addition of supplementary factors might be helpful19.
gic-like neurons from human fibroblasts has been identified20. This
opens the intriguing possibility that different molecular fate determi-
nants reach a similar endpoint even though acting on different tran-
to tumours in their undifferentiated state. Moreover, the process
described here does not pass through proliferativeprogenitors that also
drawback of stem cell therapies while providing a sufficient number of
wild-type or TH-GFP mouse embryos. Adult human fibroblasts isolated from
healthysubjectsandParkinson’s disease patientsaswellasfromhumanfetallung
fibroblasts (IMR90) were grown in MEF media. Cells were infected with dox-
inducible lentiviruses as previously reported7.
Electrophysiology and amperometry. Electrophysiological recordings were per-
formed in on-cell and whole-cell configurations. Carbon-fibre microelectrodes
were used for amperometric recordings15.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 3 March 2011; accepted 15 June 2011.
Published online 3 July 2011.
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Supplementary Information is linked to the online version of the paper at
Acknowledgements We are thankful to D. Bonanomi, S.-L. Ang, S. El Mestikawy,
M. P. Smidt, M. German and F. Valtorta for providing valuable antibodies. We thank
A. Sessa and V.B. laboratory members for helpful discussion. M. Wernig is
acknowledged for providing the iN-inducing lentiviral vectors. We are thankful to
S. Nicolis for sharing Sox2b-geomice. L. Muzio, C. Laterza and G. Martino are
acknowledged for the generation of Sox2b-geoinduced pluripotent stem cells.
M. Bacigaluppi is acknowledged for advice on stereological countings. We thank the
‘‘Cell Line and DNA Biobank’’ (G. Gaslini Institute) and ‘‘Human Genetic Bank of
Patients affected by Parkinson Disease and parkinsonism’’ (Parkinson Institute of
Milan) of the Telethon Genetic Biobank Network for human fibroblast samples. This
FGBRCVNI10310-001-V.B.), Eranet Neuron (V.B.), Cariplo Foundation (V.B.), Ministry
S.G., T.S., R.G.).
Author Contributions M.C. and V.B. designed and conceived the experiments. M.C.,
M.T.D. and G.C. performed the lentiviral infections, characterized reprogrammed cells
and analysed their fate after in vivo transplantation. E.D. and A.D. designed,performed
and analysed all electrophysiological experiments. P.R., D.L., P.C. and S.G. performed
the microarray gene expression profiling and analysed the data. D.L., A.D. and R.R.G.
designedtheprotocoland performedtheassessmentofdopaminelevels.S.Tand G.R.
performed patch-clamp recording on brain slices. A.M. performed the functional
analysis of synaptic activity. G.P. supervised the selection of the Parkinson’s disease
as co-senior authors and wrote the manuscript.
Author Information Data have been deposited in NCBI’s Gene Expression Omnibus
and are accessible through GEO series accession number GSE27174 (http://
information is available at www.nature.com/reprints. The authors declare competing
financial interests: details accompany the full-text HTML version of the paper at
www.nature.com/nature. Readers are welcome to comment on the online version of
this article at www.nature.com/nature. Correspondence and requests for materials
should be addressed to V.B. (firstname.lastname@example.org).
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embryos. Head, vertebral column, dorsalroot ganglia and allinternal organswere
removed and discarded and the remaining embryonic tissue was manually dis-
sociated and incubated in 0.25% trypsin (Sigma) for 10–15min. Cells from each
embryo were plated onto a 15-cm tissue culture dish in MEF media (Dulbecco’s
modified Eagle medium (DMEM; Invitrogen) containing 10% fetal bovine serum
(FBS; Hyclone), non-essential amino acids (Invitrogen), sodium pyruvate and
penicillin/streptomycin (Invitrogen)).In all experiments cells were not split more
than four times. Mouse adult fibroblasts were isolated from tail tip samples. Tails
were peeled, minced into 1cm pieces, placed on culture dishes, and incubated in
MEF media for 5 days. Adult human fibroblasts were isolated from skin biopsy
samples of healthy and Parkinson’s disease patients22,23provided from the ‘‘Cell
Line and DNA Biobank from Patients affected by Genetic Diseases’’ (G. Gaslini
Institute) and ‘‘Parkinson Institute Biobank’’ (Milan, http://www.parkinson.
it/dnabank.html) of the Telethon Genetic Biobank Network (http://www.
biobanknetwork.org). The informed consent as issued by the ICP Ethical com-
mittee was obtained by healthy and Parkinson’s disease patients enrolled for the
DNA and cell biobank collection.
Human skin samples were mechanically dissociated and plated on matrigel-
coated dishes. Human fibroblasts were cultured as MEFs. Mouse and adult fibro-
blasts were grown in MEF media as well as human fetal lung fibroblasts IMR90
(ATCC). Mesencephalic dopaminergic primary cell cultures from TH-GFP mice
were prepared as previously described24. Mice were maintained at San Raffaele
accordance with experimental protocols approved by local Institutional Animal
Care and Use Committees.
Molecular cloning and viral infection. Complementary DNAs for the dopami-
nergic transcription factors were cloned into lentiviral vectors under the control of
were infected in MEF media. 16–20h after infection cells were switched into fresh
MEF media containing doxycycline (2mgml21; Sigma). After 48h medium was
lin (Sigma), 50mgml21transferrin (Sigma), 30nM sodium selenite, 20nM proges-
terone (Sigma), 100nM putrescine (Sigma) and penicillin/streptomycin (Sigma))
containing doxycycline. The medium was changed every 2–3 days for a further
10–22 days. For proliferation assay, MEFs were treated with a 48h pulse of 10mM
Immunohistochemistry. For immunocytochemical analysis, 53104mouse or
human fibroblasts were plated on matrigel-coated glass coverslips the day before
the infection. 10–28 days after viral infection cells were fixed for 20min at room
in PBS containing 0.1% Triton X-100 and 10% normal goat serum (NGS), and
incubated overnight at 4uC in PBS containing 10% NGS and primary antibodies.
Then cells were washed three times with PBS and incubated for 2h at room
temperature with anti-rabbit or anti-mouse secondary antibodies Alexa Fluor-
488 or Alexa Fluor-594 (1:500, Invitrogen). For immunohistochemical analysis
15- or 40-mm-thick sections with a cryostat and processed for immunostaining.
Sections were boiled for 3min in 10mM citrate buffer solution pH 6 for antigen
0.25% Triton X-100 and 10% NGS. Primary antibodies were as follows: mouse
anti-TH (1:200, Millipore), rabbit anti-TH (1:200, Immunological Sciences),
mouse anti-bIII-tubulin (1:500, Covance), rabbit anti-bIII-tubulin (TuJ1)
(1:500, Covance), rabbit anti-VMAT2 (1:200, Chemicon), rat anti-DAT (1:500,
Swant), rabbit anti-AADC (1:100, Novus Biologicals), rabbit anti-ALDH1A1
(1:200, Abcam), mouse anti-synaptotagmin I (1:200, Synaptic Systems), mouse
anti-synapsin (1:200; Synaptic Systems), chicken anti-GFP (1:2,000, Molecular
Probes), rat anti-BrdU (1:200, BD), mouse anti-MAP2 (1:500, Immunological
Sciences), rabbitanti-Otx2(1:100R&D). b-Galactosidase stainingwas performed
as previously described25.
Statistical analysis. The total numbers of Th1and Tuj11cells were quantified
12–24 days after infection. Cell counting was performed on ten fields from three
replicates for each condition and normalized with the number of cells plated
before the infection. Data were expressed as mean6s.e.m.
RT–PCR. RNA was extracted from single cultures, using Trizol isolation system
(Invitrogen) according to manufacturer’s instructions. The yield and integrity of
the RNA were determined by the spectrophotometric measurement of A260 and
by agarose-gel electrophoresis, respectively. Total RNA was treated with DNase I
(Qiagen) to prevent DNA contamination. Two micrograms of RNA were reverse
transcribed using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche).
reactionmixturecontainingTaqpolymerase buffer(Fisher BioReagents),0.2mM
dNTPs (Finnzymes OY), 0.4 micromolar of each primer, 1U Taq polymerase
(Fisher BioReagents). The primers used to amplify cDNA samples are listed in
Supplementary Table 6.
Cell sorting, laser capture dissection and microarray analysis. TH-GFP-
positive iDA cells were directly sorted in Trizol (Invitrogen) using the cell sorter
earlier and biotin-labelled cRNA was obtained using the Ovation kit (NuGEN).
Labelled cRNA was hybridized (CBM genexpression facility, SISSA) on
Affymetrix Mouse Gene 1.0 ST Arrays, containing 35,557 probe sets correspond-
ing to 28,853 genes. Hybridized arrays were stained and washed (GeneChip
Fluidics Station 450) and scanned (GeneChip Scanner 3000 7G). Cell intensity
values were computed using the Affymetrix GeneChip Operating Software
(GCOS). Further data processing was performed in the R computing environ-
ment (http://www.r-project.org/) version 2.8.0 with BioConductor packages
Statistical analysis was performed with limma27. P values were adjusted for mul-
expressed. Furthermore, a fold-change threshold cutoff was set to focus on genes
whose expression level changed at least 2 times. Data were analysed through
DAVID Bioinformatics Resources v6.7 (refs 28, 29).
Gene expression profiles of adult A9 and A10 dopaminergic neurons were
by cervical dislocation.The brainswere rapidlycutto isolate the midbrain region,
and immediately immerged in 13 zinc fixative (BD Pharmingen) for 4–6h at
(Sigma) and percooled with liquid nitrogen. 14-mm cryosections were mounted
on SuperFrost plus glass slides (Menzel–Gla ¨ser) and air dried. mDA A9 and A10
neurons (each one from three different mice) were isolated from cryosections by
using a PALM LCM microdissection system (PALM Microlaser Technology). To
the section during cell selection. The sections were air dried, neurons were dis-
sected and catapulted onto PALM adhesive caps (Zeiss). Total RNA from 2,500
pooled neurons was isolated by using the Nano RNA extraction kit (Stratagene)
of age-matched female mice. Midbrain RNA was isolated using RNeasy Mini kit
(Qiagen), followed by DNase treatment. RNA from dissected neurons and all
midbrains was amplified and labelled by Ovation Pico kit, WT exon and Encore
biotin labelling kit (Nugene), following manufacturer’s instructions. Once pre-
pared, each target was hybridizated on MoExon 1.0ST GeneChip (Affymetrix).
Statistical analysis was performed by oneChannelGUI R package. All hierarchical
clusters were generated by TMEV software.
Bisulphite genomic sequencing. DNA from sorted TH-GFP1reprogrammed
to the manufacturer’s recommendations. Thus Th and Vmat2 promoters CpG-
rich selected regions were amplified using the PCR primers listed in Supplemen-
tary Table 5.
genized in 100ml 0.1 N HClO4and analysed by using HPLC with electrochemical
then 0.9ml of supernatants were collected with the addition of 0.1ml of 1N
HClO4, filtered and analysed by HPLC. Dopamine was separated on a reverse-
phase column (ALB-105, 3mm, 5031mm) with a mobile phase consisting of
50mM phosphate buffer, 8mM KCl, 500mgl21octyl sodium sulphate, 0.1mM
EDTA, and 3% methanol (pH 6.0) at a flow rate of 50mlmin21. Dopamine was
detected by a Decade II electrochemical detector equipped with micro VT-03
electrochemical flow cell and a 0.7-mm-diameter glassy carbon electrode (Alexis
100, Antec Leyden). The volume of the injection was 5ml. The detection limit
established as a 3:1 signal-to-noise ratio was below 0.5nM.
Electrophysiology. Recordings were performed from reprogrammed mouse and
human fibroblasts and primary mDA neurons. The mouse TH-GFP1cells
selected for the electrophysiological analysis were not so flat as the fibrobalsts,
and had several well-developed neurites. The human cells selected for the electro-
physiological analysis also had neuron-like shapes with clearly distinguishable
neurites by phase contrast microscopy. Only cells without signs of detachment
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fromthesubstratewereusedforrecordings.Cellswereperfusedcontinuouslywith Download full-text
HEPES-buffered saline (HBS)ofthe followingcomposition (inmM): 140NaCl, 5
KCl, 2 CaCl2, 2 MgCl2, 15 HEPES, and 25 glucose, pH 7.4. The patch pipette
solution contained (in mM): 130 K-gluconate, 10 KCl, 0.5 CaCl2, 15 HEPES, 5
EGTA, 8 NaCl, 2 MgATP, 0.3 Na2GTP, and 10 glucose, pH adjusted to 7.2 with
KOH. Action potentials were recorded in the on-cell and the current-clamp
whole-cell configurations. A current was injected to have membrane potentials
around 260mV, and step currents from 250pA to 40pA were injected to elicit
action potentials. Na1currents and composite K1currents were recorded in the
120mV in cells held at 260mV. Delayed rectifier K1currents were activated by
0.5s voltage steps from 240mV to 120mV after a 0.5-s-long step to 240mV.
A-type K1currents were isolated by subtraction of delayed rectifier K1currents
from those activated by voltage steps after a 0.5-s-long step to 2100mV.
Recordings were performed using an EPC10 USB patch clamp amplifier and
PATCHMASTER software (HEKA Elektronik). Data were digitized at 10kHz
and analysed with FITMASTER Software (HEKA Elektronik). Detection and
measurements of action potentials were performed using MiniAnalysis software
day 42. The brains were quickly removed from the skull in ice-cold artificial cere-
brospinal fluid (ACSF) containing the following (in mM): 125 NaCl, 25 NaHCO3,
2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, and 25 glucose, pH 7.4 (bubbled with
95% O2and 5% CO2). Coronal slices (300-mm thick) were cut using a vibratome
(VT1000S; Leica) and stored in ACSF at 25–28uC. For recording, slices were
transferred to a recording chamber continuously superfused with ACSF (1–2ml
min21at 30–32uC). Whole-cell recordings were performed in both current- and
the following solution (in mM): 124 KH2PO4, 10 NaCl, 2 MgCl2, 0.5 EGTA, 10
HEPES, 2 Na2-ATP, 0.03 Na-GTP (pH 7.2, adjusted with KOH). Signals were
sampled at 10kHz, filtered at 2kHz, and acquired using a MultiClamp 700A
amplifier and pClamp 10 software (Molecular Devices).
amine exocytosis from single cells15,31. Carbon-fibre microelectrodes were fabri-
cated from 5mm carbon fibres (Goodfellow), inserted in a 1.230.68mm glass
capillary (A-M system, Sequim) and pulled with a PE-22 micropipette puller
(Narishige). Electrodes were sealed by dipping in Epoxy resin (Epo-Tek 301,
Epoxy Technology) and cured at 100uC for 24h. They were backfilled with 3M
KCl and trimmed to obtain a basal current between 140 and 180nA. The electro-
des’ responses were tested by cyclic voltammetry and those with unstable cyclic
voltammograms, when tested in a solution of 10mM dopamine, were rejected. A
(HEKA Elektronik). The signal was low-pass filtered at 10 kHz using a 4-pole
Bessel filter, digitalized at 50kHz and digitally refiltered at 1–1,000Hz. The latter
secretion events that was the only aim of these experiments. The electrode was
positioned adjacent to individual cells and lowered to approach the somatoden-
dritic domains of iDA cells32, using an Olympus BX51WI microscope with 340
water immersion objective. To increase the signal-to-noise ratio, cells were pre-
treated with 100mM L-DOPA (Sigma-Aldrich) for 30min although we were able
to resolve single spike-like release events in two untreated cells. The experiments
was then exchanged for a stimulation solution (25mM K1), and amperometric
signals were recorded for a further period of 7min. Catecholamine secretion was
apparent as discrete spike-like events, each corresponding to vesicular catechola-
but occasionalevents were observed also during baseline recordings. Novesicular
applied potential was 0mV or 2750mV, or at 1750mV when the electrode was
placed remotely from cells.
FM4-64 assay. FM4-64 dye uptake experiments were performed as previously
reported33. Briefly, 21 days in vitroTH-GFP1iDA cells were stimulated for 1min
with 55mM KCl, in the presence of FM4-64 (10mM). After FM4-64 loading,
neuronal cells were washed and perfused for 10min with warmed Krebs buffer
FM4-64 signals were acquired, cells were fixed and immonostained for TH and
Electron microscopy. Forultrastructuralimmunocytochemistry, 21 days in vitro
2% OsO4in PBS, and embedded in Epon. Ultrathin sections prepared from these
samples were analysed with electron microscope (H-7000; Hitachi).
and resuspended at 23105cellsml21in fresh prepared Krebs buffer containing the
following (in mM): 126 NaCl, 2.5 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.1 CaCl2, 11
glucose, 4.2 NaHCO3, 1 HEPES, and 1% vital dye Fast Green. P1 mice pups were
anaesthetized by hypothermia (4min) and fixed to a support using band-aid. The
under a Hamiltonsyringe containing 2ml ofcellsuspension. Thesyringe was placed
over the incision, positioned at the level of the skull, then lowered into the lumen of
cell solution was injected. Mice were left on a 37uC heating blanket for several
minutes after surgical manipulation to avoid fatal hypothermia.
Stereological analysis. Three mice transplanted with reprogrammed cells were
thetized and killed by transcardiac perfusion with PBS followed by 4% para-
formaldehyde. Brains were cryoprotected through incubation in an ice-cold
solution of 30% sucrose in PBS and cut in coronal 40-mm-thick cryostat sections.
so that sections were spaced at 7 section intervals (totalof16 sections permouse).
GFP immunoperoxidase staining was performed as described elsewere34. Cells
were quantified using the assistance of the Stereo Investigator v 3.0 software
(MicroBrightField) and a personal computer running the software connected to
the microscope allowed precise and well-defined movements along the x-, y- and
z-axes. Images were first acquired with a CCD-IRIS colour video camera and the
image of the section. Counting of cells was performed manually on every seventh
section using a 340 lens. To estimate the total number of GFP positive cells the
total number of neurons counted on the sections was multiplied by seven.
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