Cell Stem Cell
Reprogramming of Pericyte-Derived Cells
of the Adult Human Brain into Induced Neuronal Cells
Marisa Karow,1,10Rodrigo Sa ´nchez,1,10Christian Schichor,2Giacomo Masserdotti,1,3Felipe Ortega,1
Christophe Heinrich,1Sergio Gasco ´n,1,3Muhammad A. Khan,4D. Chichung Lie,4,5Arianna Dellavalle,6Giulio Cossu,6,7
Roland Goldbrunner,2,8Magdalena Go ¨tz,1,3,11and Benedikt Berninger1,3,9,11,*
1Department of Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, Schillerstrasse 46,
D-80336 Munich, Germany
2TumorBiologyLab,NeurosurgicalClinic,KlinikumderUniversita ¨tMu ¨nchen,Großhadern,Marchioninistrasse15,D-81377Munich,Germany
3Institute of Stem Cell Research, National Research Center for Environment and Health, Ingolsta ¨dter Landstrasse 1,
D-85764 Neuherberg, Germany
and Health, Ingolsta ¨dter Landstrasse 1, D-85764 Neuherberg, Germany
5Institute for Biochemistry, Emil Fischer Centre University of Erlangen, Fahrstrasse 17, D-91054 Erlangen, Germany
6Division of Regenerative Medicine, San Raffaele Scientific Institute, 58 via Olgettina, Milan 20132, Italy
8Center for Neurosurgery, University Hospital of Cologne, Kerpener Strasse 62, D-50937 Cologne, Germany
9Institute of Physiological Chemistry and Focus Program Translational Neuroscience of the Johannes Gutenberg University Mainz,
Hanns-Dieter-Hu ¨sch-Weg 19, D-55128 Mainz, Germany
10These authors contributed equally to this work
11These authors contributed equally to this work
Reprogramming of somatic cells into neurons
provides a new approach toward cell-based therapy
the translation of neuronal reprogramming into
therapy is whether the adult human brain contains
cell populations amenable to direct somatic cell
conversion. Here we show that cells from the adult
human cerebral cortex expressing pericyte hall-
marks can be reprogrammed into neuronal cells by
retrovirus-mediated coexpression of the transcrip-
tion factors Sox2 and Mash1. These induced neu-
ronal cells acquire the ability of repetitive action
neurons, indicating their capability of integrating into
neural networks. Genetic fate-mapping in mice ex-
pressing an inducible Cre recombinase under the
tissue-nonspecific alkaline phosphatase promoter
corroborated the pericytic origin of the reprog-
rammed cells. Our results raise the possibility of
functional conversion of endogenous cells in the
adult human brain to induced neuronal fates.
Reprogramming of somatic cells into neurons provides a new
approach toward cell-based therapy of neurodegenerative
diseases (Vierbuchen and Wernig, 2011). Previous studies
have shown that postnatal astroglia from the mouse cerebral
cortex can be directly converted into functional neuronal cells
rich et al., 2010, 2011; Heins et al., 2002) and that the synergistic
action ofthree orfour transcription factorscan induce neurogen-
esis from rodent and human fibroblasts (Caiazzo et al., 2011;
Pang et al., 2011; Qiang et al., 2011; Son et al., 2011; Vierbuchen
et al., 2010; Yoo et al., 2011). However, a major challenge for the
translation of neuronal reprogramming into therapy is whether
direct conversion of somatic cells into neuronal cells can be
achieved from cells residing within the adult human brain. To
address this question, we prepared adherent cultures from 30
human specimens that were derived from surgical approaches
through the cerebral cortex to deep-seated nontraumatic non-
malignant lesions, i.e., epileptic foci and nonruptured vascular
lesions. In order to characterize the cellular composition of
the cultures obtained from these specimens, we performed
immunocytochemistry and fluorescence-activated cell sorting
(FACS) analyses at different stages of culturing. Intriguingly,
the majority of cells expressed platelet-derived growth factor
receptor-b (PDGFRb) (Daneman et al., 2010) (Figures 1C and
1D and Figure S1A available online), which is detected within
the human brain tissue exclusively on microvessel-associated
pericytes (Figure 1A), a cell type involved in the establishment
and maintenance of the blood-brain barrier and regulation of
local blood flow (Armulik et al., 2011). Consistent with a pericyte
identity, we also observed expression of NG2 (Karram et al.,
2005) (Figure 1B and S1B), smooth muscle actin (SMA) (Figures
S1A and S1B) (Hellstro ¨m et al., 1999), CD146 (Crisan et al.,
2008), and CD13 (Crisan et al., 2008) (Figure 1E), though with
some heterogeneity with regard to coexpression of these
markers (Figures 1E, S1A, and SB). In contrast, the number of
glial acidic fibrillary protein (GFAP)-positive cells was extremely
low in these cultures (<1%), although astrocytes were readily
detected within the human tissue (data not shown). Quantitative
RT-PCR experiments confirmed the enriched expression of peri-
cytic marker genes and the virtual absence of astroglial (gfap)
Cell Stem Cell 11, 471–476, October 5, 2012 ª2012 Elsevier Inc. 471
Figure 1. Characterization and In Vitro Conversion into Induced Neuronal Cells of Human and Mouse Adult Brain Pericyte-like Cells
(A) PDGFRb expression in microvessel-associated cells in the adult human cerebral cortex. Scale bar: 100 mm.
(B)NG2expressioninmicrovessel-associated cells intheadulthuman cerebral cortex. Microvessels werevisualizedbyCD31 (green) immunoreactivityand DAPI
(blue). Scale bar: 100 mm.
(C) Immunocytochemical analysis for pericyte marker PDGFRb (red) in cell cultures obtained from human cerebral tissue; DAPI is in blue. Scale bar: 100 mm. See
also Figures S1A and S1D. Scale bar: 100 mm.
(D) Example of FACS analysis from an adult human brain culture. Depicted are the isotype controls (ctrl, left and middle panel) for establishing the gating
conditions for sorting the PDGFRb- and CD34-positive populations. See also Figure S1I.
Cell Stem Cell
Two-Factor Pericyte-to-Neuron Conversion
472 Cell Stem Cell 11, 471–476, October 5, 2012 ª2012 Elsevier Inc.
and oligodendroglial cells (olig2) in these cultures compared to
human brain tissue from which the cells had been isolated (Fig-
ure S1C). Importantly, bIII-tubulin could not be detected at any
stage of culturing (assessed from 2 days to 8 weeks after
blasts or surviving neurons (data not shown). Furthermore, these
cultures were completely devoid of expression of neural stem
cell markers such as sox2 or prom1 or neurogenic fate determi-
nants such as ascl1 or pax6 (Figure S1C). Moreover, Sox2,
Mash1, Olig2, and Pax6 were also not detected on the protein
level by immunocytochemistry (data not shown). The few
CD34-positive cells (Figures 1D and S1C) of hematopoietic or
are enriched for cells exhibiting pericyte characteristics.
Previous work has identified Mash1 (mammalian achaete-
scute homolog 1, encoded by the gene ascl1) as a powerful
reprogramming factor for direct conversion of somatic cells
into neuronal cells (Berninger et al., 2007; Caiazzo et al., 2011;
Vierbuchen et al., 2010). When we assessed the response of
our cultures to retrovirus-mediated expression of Mash1 (CAG-
ascl1-IRES-dsred), we observed the reduction of PDGFRb
expression to 23% (n[cells] = 219), indicating a loss of peri-
cyte-specific protein expression (Figure S1D). Moreover,
a subset of Mash1-transduced cells responded with the induc-
tion of bIII-tubulin, suggesting some degree of neuronal respeci-
fication (Figure 1F). Previous work has suggested that Sox2
expression may facilitate neuronal reprogramming of postnatal
astrocytes by neurogenic fate determinants (Heinrich et al.,
2010). As there was no endogenous Sox2 expression in these
cultures (Figure S1C), we hypothesized that forced expression
of sox2 may enhance the efficiency of neuronal reprogramming
by Mash1. Expression of Sox2 (CAG-sox2-IRES-gfp) alone
had no overt effect on bIII-tubulin expression (Figure 1F) or
morphology of pericyte-like cells (Figure S1F). In contrast, coex-
pression of Sox2 and Mash1 significantly increased the propor-
tion of bIII-tubulin-expressing cells to 48% ± 9% SEM (n[cells] =
1,500, analyzed after 4–5 weeks following transduction, cultures
from six different patients; compared to 10% ± 4% SEM after
Mash1 transduction alone, p = 0.0038, Figure 1F). Most strik-
ingly, many of the double-transduced cells (28% ± 5% SEM)
exhibited neuronal morphology (Figure S1F) and induced
expression of MAP2 (46% ± 11% SEM, n[cells] = 296 from three
different patients, analyzed after 5–6 weeks; Figures 1H and
S1G) and NeuN (Figure S1H), indicating a high degree of re-
programming efficiency of cells from adult human tissue.
Consistent with the acquisition of a neuronal phenotype and
a loss of pericyte identity, Sox2- and Mash1-coexpressing cells
downregulated PDGFRb (Figure S1E). Of note, some cultures
of which 46% of the Mash1 and Sox2 cotransduced cells differ-
entiated into bIII-tubulin-positive cells, with 26% exhibiting
neuronal morphology (n[cells] = 203). In the following we refer
to these neuronal cells derived from human pericyte-like cells
as human pericyte-derived induced neuronal cells (hPdiNs).
Despite the high frequency of PDGFRb-positive cells infected
by the retroviral vectors, the remainder of PDGFRb-negative
cells may still act as the main source of induced neuronal cells
upon Mash1 and Sox2 transduction. Thus, we proceeded to
follow the fate conversion of pericytes by live imaging (Rieger
et al., 2009). Cultured cells were FACS-sorted for surface
expression of PDGFRb (Figure S1I), transduced 48 hr later with
retroviral vectors encoding sox2 and ascl1, and subsequently
imaged by time-lapse video microscopy (Movie S1). Figure 1G
shows an example of an anti-PDGFRb FACS-sorted cell
undergoing Sox2- and Mash1-induced neurogenesis. The cell
acquired a polarized morphology within 12 days following trans-
duction and could be shown to express bIII-tubulin at the end of
the live imaging (Figure 1G0). Intriguingly, following the onset of
reporter expression, this PDGFRb-sorted cell did not undergo
any cell division, providing evidence for direct conversion from
an adult human nonneuronal somatic cell into an hPdiN. Like-
wise, only 1 of 36 (3%) Sox2- and Mash1-coexpressing cells
that we followed over time underwent cell division, in sharp
contrast to untransduced (n[cells] = 11/30; 36%], Mash1-only
(n[cells] = 8/30; 26%), and Sox2-only transduced cells (n[cells] =
13/30; 46%), indicating that Sox2- and Mash1-induced re-
programming does not only not require cell division, but is
accompanied by immediate cell cycle exit. Of all the tracked
cells coexpressing Sox2 and Mash1, 36% endured cell death.
This percentage was considerably higher than that of untrans-
duced cells (3%) and Sox2-only transduced cells (7%). Of
note, Mash1-only transduced cells also exhibited a higher rate
of cell death (33%), suggesting that Mash1 or Sox2 and Mash1
coexpression can induce a catastrophic conflict of cell fates in
(E) Relative coexpression of pericyte markers as analyzed by FACS analysis. Each data point represents the relative coexpression of PDGFRb and CD146 (mean
40.7% ± 28.1%) or CD13 (mean 46.4% ± 29.1%).
(F) Quantification of the effect on bIII-tubulin expression and morphology following DsRed only for control, Sox2, Mash1, and combined Sox2 and Mash1
expression. Cells were categorized for exhibiting a flat polygonal morphology, round morphology without processes, or neuronal morphology with processes.
analyzed. For each condition >1,000 cells were analyzed. Error bars are SEM.
Sox2 and Mash1. Pictures show phase contrast and fluorescence (Mash1-DsRed and Sox2-GFP) images at different time points (Days-Hours:Minutes) during
the reprogramming process. Note the change of the cotransduced cell from a protoplasmic to a neuron-like morphology. See also Movie S1.
(G0) Depicted is the last recorded time point in phase contrast (LT) and the postimmunocytochemistry (Post IC) of the reprogrammed cell for GFP (green), DsRed
(red), and bIII-tubulin (white).
(H) Example of MAP2 and bIII-tubulin coexpression after 5 weeks following transduction. See also Figure S1G.
(I) Specific b-galactosidase expression associated with CD31-positive blood vessels in the cerebral cortex of Tg:TN-AP-CreERT2:R26RNZGmice.
b-galactosidase-positive cells express the pericyte marker PDGFRb.Note the restricted expressionaround microvessels. b-galactosidase,green; PDGFRb,red;
CD31, blue. Scale bars: left panel, 50 mm; right panels, 10 mm.
(J) Reprogramming of EYFP-positive cells isolated from the cerebral cortex of adult Tg:TN-AP-CreERT2:R26REYFPmice into induced neuronal cells.
EYFP-positive cells (green) transduced with Mash1 (red) and Sox2 (without reporter) display a neuronal morphology and express bIII-tubulin; 14 days
postinfection. Scale bar: 100 mm. For the efficiency of reprogramming of mouse pericytic cells, see Figures S1J–S1K.
Cell Stem Cell
Two-Factor Pericyte-to-Neuron Conversion
Cell Stem Cell 11, 471–476, October 5, 2012 ª2012 Elsevier Inc. 473
imaging revealed thatnone of the Sox2-only cells(n[cells] >300),
7% of Mash1-only (n[cells] = 88) cells, and 25% of double-
positive cells (n[cells] = 786; two independent experiments)
expressed bIII-tubulin. Inanadditional experiment, in which cells
had been sorted simultaneously for PDGFRb and CD146 and
had been time-lapsed, a reprogramming efficiency of 37% was
observed (n[cells] = 209). Combining all time-lapse experiments,
the overall reprogramming efficiency was 19% of the coinfected
cells, taking proliferation and cell death into account.
To unequivocally determine the origin of the reprogrammed
cells from pericytes in vivo, we turned to genetic fate-mapping
in mice. We took advantage of a transgenic mouse that ex-
presses an inducible Cre recombinase (CreERT2) under control
of the tissue-nonspecific alkaline phosphatase (TN-AP) pro-
moter for genetic fate mapping of pericytes (Dellavalle et al.,
2011). These mice were crossed to reporter lines (Tg:TN-AP-
CreERT2:R26RNZGand Tg:TN-AP-CreERT2:R26REYFP) to aid
identification of cells of pericytic origin either by b-galactosidase
or yellow fluorescent protein (YFP) immunoreactivity following
tamoxifen-induced Cre-mediated excision of the stop cassette.
As expected b-galactosidase expression was confined to micro-
vessel-associated cells coexpressing PDGFRb (Figure 1I) and
NG2 (data not shown) (Dellavalle et al., 2011) in the cerebral
cortex of young adult mice following induction at postnatal
stages, indicating that the TN-AP promoter allows reliable fate-
mapping of pericyte-derived cells in the adult brain. Next we
prepared cultures from the adult cerebral cortex of Tg:TN-AP-
CreERT2:R26REYFPmice under the same culture conditions
as used for human samples. As in the adult cerebral cortex,
reporter-positive cells coexpressed the pericytic markers
PDGFRb, NG2, and CD146 and could be expanded in vitro
(data not shown). In contrast to control vector-transduced
reporter-positive pericyte-derived cells (data not shown),
Sox2- and Mash1-expressing cells gave rise to bIII-tubulin-posi-
tive PdiNs (Figure 1J). Neuronal reprogramming of wild-type
mouse pericyte-derived cells occurred at an even higher
frequency compared to adult human pericyte-derived cells: co-
expression of Sox2 and Mash1 significantly increased the
proportion of bIII-tubulin-positive cells to 92% ± 3% SEM
(compared to 41% ± 10% SEM after Mash1 transduction alone,
p = 0.0028) (Figure S1K), and most of the double-transduced
cells (73% ± 7% SEM) exhibited neuronal morphology (Fig-
ure S1J) and were capable of repetitive action potential firing
(Figure S2F and Table S1).
We next analyzed whether the hPdiNs expressing neuron-
specific proteins also acquire the functional membrane proper-
ties of neurons. In Mash1 (n[cells] = 7) and Sox2 (n[cells] = 6)
singly transduced cells, step-current injection failed to elicit
any action potentials (Figures S2A, S2A0, S2B, and S2B0), indi-
cating that neither transcription factor alone induces neuronal
electrical properties. In sharp contrast, a substantial proportion
of cells (71% of 17 cells tested, cultures from five different
patients) coexpressing both factors responded typically with
the generation of a single action potential that could be blocked
by the sodium channel antagonist tetrodotoxin (TTX) (Figures
S2C and S2C0). Moreover, in voltage-clamp these cells exhibited
shown) currents. However, these hPdiNs exhibited immature
properties, as reflected by the relatively high input resistances,
low action potential, and peak sodium current amplitudes,
even after prolonged time in culture, consistent with the slow
maturation of human neurons (Table S1). In order to further
promote maturation and to investigate whether hPdiNs can
integrate into a neuronal network, we cocultured hPdiNs with
neurons from the mouse embryonic neocortex. Under these
conditions hPdiNs exhibited a more complex morphology
(Figures 2A, 2B, and 2E) and were capable of repetitive action
potential firing (Figure 2C), although input resistances were still
high (Table S1). Importantly, hPdiNs were found to receive func-
tional glutamatergic input from cocultured neurons (4 out of
12 cells analyzed, Figures 2D–2D00), demonstrating that they
express functional transmitter receptors, are capable of assem-
bling a postsynaptic compartment, and can be recognized by
other neurons as functional targets. Consistent with functional
glutamatergic input, dendrites of hPdiNs were decorated with
presynaptic terminals containing vesicular glutamate trans-
porters (Figure 2F). Of note, hPdiNs exhibited immunoreactivity
for the inhibitory neurotransmitter b-aminobutyric acid (GABA,
14/14 hPdiNs analyzed) (Figure S2D). Moreover, qRT-PCR
showed the expression of the interneuron calcium binding
protein pvalb (Figure S2E), pointing toward acquisition of an
interneuron-like phenotype. In contrast, none of the Sox2 and
Mash1 cotransduced cells expressed the glutamatergic lineage
marker tbr1 (T-box brain gene 1; data not shown) or slc17a7
(encoding the vesicular glutamate transporter [vGluT]-1; Fig-
ure S2E). However, a definitive proof for a GABAergic inter-
neuron-like identity awaits the demonstration of functional
Here we provide evidence for high-efficiency reprogramming
of pericyte-derived cells of the adult human cerebral cortex
into induced neuronal cells by coexpression of only two tran-
scription factors. The fact that only coexpressing cells convert
into neuronal cells provides direct evidence for a cell-autono-
mous effect. Different scenarios may account for the synergism
of these two transcription factors. Sox2 may facilitate Mash1-
induced reprogramming by rendering the somatic genome
more susceptible to the neurogenic activity exerted by Mash1.
Alternatively, Sox2 may be required to directly interact with
Mash1 on common target genes. While we can currently not
discern between these two modes of action, the fact that
cerebral cortex (data not shown) argues partially against the first
mechanism as the solely important one. Recent studies on the
role of Mash1 and Neurog2 during cortical development suggest
that these factors activate distinct programs in neural progeni-
tors (Castro et al., 2011). Mash1 also has been found as a key
transcription factor in the direct reprogramming of fibroblasts
(Pang et al., 2011; Vierbuchen et al., 2010) and hepatocytes
(Marro et al., 2011) where it synergizes with Brn2 and Myt1l.
This may suggest that Mash1 acts as a core factor in direct
neuronal reprogramming. Interestingly, we observed a very
slight induction of endogenous ascl1 mRNA expression (Fig-
ure S2E). It is noteworthy that, while fibroblasts coexpressing
different combinations of transcription factors have been shown
to give rise to induced neuronal cells of glutamatergic identity
(Pang et al., 2011; Vierbuchen et al., 2010), dopaminergic
(Caiazzo et al., 2011; Kim et al., 2011; Pfisterer et al., 2011)
Cell Stem Cell
Two-Factor Pericyte-to-Neuron Conversion
474 Cell Stem Cell 11, 471–476, October 5, 2012 ª2012 Elsevier Inc.
and cholinergic motor neuron identity (Son et al., 2011), the
combination of Sox2 and Mash1 appears to favor a GABAergic
phenotype in hPdiNs. It will be important to understand whether
this is largely dependent on the factor combination used or the
cellular context determined by the origin and nature of the
Local CNS pericytes have been recently recognized as
a major source of proliferating scar-forming cells following
CNS injury (Go ¨ritz et al., 2011). A key finding of the pre-
sent study is that progeny of brain pericytes represent a
potential target for direct reprogramming. While much needs
to be learned about adapting a direct neuronal reprogramming
strategy to meaningful repair in vivo, e.g., by using a noninvasive
approach to activate these transcription factors (Kormann et al.,
2011), our data provide strong support for the notion that
neuronal reprogramming of cells of pericytic origin within the
Figure 2. Neuronal Morphology and Membrane Properties of hPdiNs
(A) Bright-field micrograph depicts an hPdiN (red arrowhead) after 26 days of coculture with E14 mouse cerebral cortical neurons, 46 days following retroviral
(B) DsRed fluorescence indicating transduction with ascl1 and dsred-encoding retroviruses. Inset: GFP fluorescence indicating transduction with sox2- and
(C) Step current injection in current-clamp results in repetitive action potential firing. For comparison with cells transduced with a single transcription factor or
cotransduced, but cultured without mouse cortical neurons, see Figures S2A–S2C00.
(D) The graph depicts spontaneous synaptic events recorded from the same hPdiN as shown in (C). The enlarged trace shows individual synaptic events.
(D0) The synaptic events are blocked by the application of CNQX (10 mM).
(D00) Recovery of spontaneous synaptic input following washout of CNQX. For a summary of the electrophysiological properties, see Table S1.
(E) Micrograph depicting an hPdiN stained for DsRed and GFP, after 22 days of coculture with E14 neurons, 42 days following retroviral transduction.
(F) High-magnification view of a single dendrite (magenta, GFP) from the same hPdiN as shown in (E), illustrating the high density and the distribution of
vGluT1-immunoreactive puncta (green, Cy5).
Cell Stem Cell
Two-Factor Pericyte-to-Neuron Conversion
Cell Stem Cell 11, 471–476, October 5, 2012 ª2012 Elsevier Inc. 475
damaged brain may become a viable approach to replace Download full-text
Supplemental Information for this article includes two figures, one table,
Supplemental Experimental Procedures, and one movie and can be found
with this article online at http://dx.doi.org/10.1016/j.stem.2012.07.007.
We thank Dr. Marius Wernig (Stanford University) for generously providing us
with the sox2 coding sequence. We are also very grateful to Tatiana Simon-
Ebert and Gabi Ja ¨ger for excellent technical assistance. This work was sup-
ported by grants from the SPP1356 of the Deutsche Forschungsgemeinschaft
(DFG), the BMBF, and the Bavarian State Ministry of Sciences, Research and
the Arts to M.G. and B.B. C.S. and R.G. received funding from the binational
SYSTHER-INREMOS Virtual Institute (German and Slovenian Federal
Ministries of Education and Research) and the DFG (SFB 824). We are deeply
indebted to the Graduate School of Systemic Neurosciences (GSN-LMU) for
allowing the use of the live-imaging microscope.
Received: December 22, 2011
Revised: May 17, 2012
Accepted: July 10, 2012
Published: October 4, 2012
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