Direct conversion of mouse fibroblasts to self-renewing,
tripotent neural precursor cells
Ernesto Lujana,b, Soham Chandaa,c, Henrik Ahleniusa,d, Thomas C. Südhofc,e,1, and Marius Werniga,d,1
aInstitute for Stem Cell Biology and Regenerative Medicine, Departments ofdPathology,bGenetics, andcMolecular and Cellular Physiology, andeHoward
Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305
Contributed by Thomas C. Südhof, December 21, 2011 (sent for review August 4, 2011)
We recently showed that defined sets of transcription factors are
sufficient to convert mouse and human fibroblasts directly into
cells resembling functional neurons, referred to as “induced neu-
ronal” (iN) cells. For some applications however, it would be de-
sirable to convert fibroblasts into proliferative neural precursor
cells (NPCs) instead of neurons. We hypothesized that NPC-like
cells may be induced using the same principal approach used for
generating iN cells. Toward this goal, we infected mouse embry-
onic fibroblasts derived from Sox2-EGFP mice with a set of 11
transcription factors highly expressed in NPCs. Twenty-four days
after transgene induction, Sox2-EGFP+colonies emerged that
expressed NPC-specific genes and differentiated into neuronal
and astrocytic cells. Using stepwise elimination, we found that
Sox2 and FoxG1 are capable of generating clonal self-renewing,
bipotent induced NPCs that gave rise to astrocytes and functional
neurons. When we added the Pou and Homeobox domain-contain-
ing transcription factor Brn2 to Sox2 and FoxG1, we were able to
induce tripotent NPCs that could be differentiated not only into
neurons and astrocytes but also into oligodendrocytes. The tran-
scription factors FoxG1 and Brn2 alone also were capable of in-
ducing NPC-like cells; however, these cells generated less mature
neurons, although they did produce astrocytes and even oligoden-
drocytes capable of integration into dysmyelinated Shiverer brain.
Our data demonstrate that direct lineage reprogramming using
target cell-type–specific transcription factors can be used to induce
NPC-like cells that potentially could be used for autologous cell
transplantation-based therapies in the brain or spinal cord.
induced neural precursor cells
lineage-specific transcription factors. A key question is whether
cells retain their competence to respond to such transcription
factors even after differentiation and after their cell-type–specific
phenotype has been stabilized by epigenetic mechanisms (1). A
number of classic and recent studies have provided powerful
evidence that the differentiated state of at least some somatic
cells is more flexible than assumed. For instance, transfer of
somatic nuclei into oocytes has been shown to impose an early
embryonic program on somatic cells (2, 3). Similarly, aberrant
cell-type–specific genes could be induced following cell fusion
(4), and misexpression of defined transcription factors has been
shown to induce conversion of cells in closely related cell types
(5). For instance, the basic helix–loop–helix (bHLH) transcrip-
tion factor MyoD has been shown to induce muscle-specific
properties in fibroblasts but not in hepatocytes (6, 7); expression
of Cebpα in B cells induces features of macrophages (8); loss of
Pax5 in B cells induces dedifferentiation to a common lymphoid
progenitor (9); and the bHLH transcription factor Ngn3 or
NeuroD1, in combination with Pdx1 and MafA, efficiently con-
verts pancreatic exocrine cells or hepatic cells into functional B
cells in vivo (10, 11).
More recently, we demonstrated that mouse and human
fibroblasts can be converted into functional neurons by a defined
set of transcription factors (12, 13). Moreover, we showed that
defined factor-mediated reprogramming can be applied to dis-
tantly related lineages such as endodermal to ectodermal cells
uring development, the creation of distinct cell types de-
pends upon tightly regulated spatiotemporal expression of
(14), raising the possibility that perhaps any cell might be con-
verted into any other desired cell type if the right combination of
subsequently have shown the induction of cardiomyocytes, he-
matopoietic cells, and hepatocytes from fibroblasts (15–18).
Another, conceptually different approach to direct lineage
reprogramming is to dedifferentiate cells transiently into a plu-
ripotent state and then allow spontaneous differentiation of the
cells into the desired phenotype. Following the seminal work of
induced pluripotent stem (iPS) cell reprogramming (19), it was
shown that transient expression of the four iPS cell reprogram-
ming factors Oct4, Sox2, Klf4, and c-Myc followed by treatment
with specific media succeeded in generating both cardiomyocytes
and neural precursor cell (NPC) populations from fibroblasts (20,
low efficiency (presumably because of inefficient induction of
pluripotency and the stochastic nature of differentiation), the
described cells could not self-renew, and the NPCs apparently
lacked the ability to differentiate into oligodendrocytes.
Here we investigated whether NPCs could be induced directly
from fibroblasts using neural progenitor-specific transcription
factors as reprogramming factors and thus bypassing a partial or
complete pluripotent state. We show that specific combinations
of factors can induce bi- or tripotent NPCs efficiently. These cells
express an array of neural progenitor-specific genes and retain
their potential for differentiation after prolonged clonal expan-
sion, demonstrating a capacity for self-renewal.
Induction of NPC-Like Cells from Mouse Embryonic Fibroblasts with
a Pool of 11 Transcription Factors. We recently showed that three
transcription factors directly and efficiently convert mouse fi-
broblasts into functional induced neuronal (iN) cells (12). Al-
though this conversion has been demonstrated to be direct, with
few or no cell divisions, we hypothesized that an intermediate
NPC population also may be produced directly from mouse
embryonic fibroblasts (MEFs) under appropriate conditions. In
an attempt to achieve this goal, MEFs were derived from Sox2-
internal ribosome entry site (IRES)-EGFP knockin mice ex-
pressing the reverse tetracycline transactivator (rtTA) under
control of the Rosa26 locus. These MEFs were infected with a
pool of 11 lineage-specific transcription factors (11F) under a
tetO promoter (12, 22, 23). The 11 factors were chosen because
of their demonstrated functions in neural development and their
high expression levels in NPCs. After infection, cells were grown
in EGF- and FGF2-containing media in the presence of doxy-
cycline (24). Twenty-four days after transgene induction, Sox2-
EGFP+cells were observed when MEFs were infected with the
11F pool. Some EGFP+cells formed colonies. Overall, 12.3% of
Author contributions: E.L., S.C., H.A., T.C.S., and M.W. designed research; E.L., S.C., H.A.,
and M.W. performed research; E.L., S.C., H.A., T.C.S., and M.W. analyzed data; and E.L.,
S.C., H.A., T.C.S., and M.W. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence may be addressed. E-mail: email@example.com or wernig@
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| February 14, 2012
| vol. 109
| no. 7
the total cell population expressed EGFP at day 25 (Fig. 1 A and
B and Table S1). To determine whether this Sox2-EGFP+cell
population exhibited a transcriptional profile similar to ES cell-
derived NPCs, Sox2-EGFP+cells were purified by FACS 25 d
after transgene induction (Fig. 1B). Endogenous expression
levels of the NPC-associated genes Sox1, Sox3, Olig2, Brn2, Ncan,
Pax6, Nkx2.2, Gpm6a, and Tox3 were quantified by quantitative
RT-PCR (qRT-PCR) (Fig. 1C). The Sox2-EGFP+population
showed induction of these genes at levels similar to those seen in
ES cell-derived NPCs, except for the region-specific gene Nkx2.2.
A small number of cells with similar Sox2-EGFP expression
levels also were detectable in control-treated MEFs after 25 d in
culture (Fig. 1B). Expression analysis of this purified population
revealed that Sox1, Sox3, Olig2, and Pax6 levels were below de-
tection limits, demonstrating that these cells were similar to
uninfected MEFs rather than NPCs (Fig. 1C). Given the ap-
pearance of multiple independent NPC markers, we termed the
converted MEFs “induced NPCs” (iNPCs).
We next sought to determine whether 11F iNPCs also dis-
played functional neural precursor properties, such as the ca-
pacity to differentiate into neurons and glia under defined
conditions. Indeed, upon withdrawal of growth factors and
doxycycline from cultured iNPCs, Tuj1+cells with typical neu-
ronal morphologies were detected (Fig. 1D). However, it is im-
portant to note that the 11 factors used also contained Ascl1,
Brn2, and Zic1, which can induce neuronal cells directly from
MEFs, and Tuj1+cells with neuronal morphologies were de-
tectable before growth factor withdrawal (12). We next attemp-
ted to differentiate 11F iNPCs into astrocytes. The NPC growth
medium was replaced 25 d after infection with medium con-
taining 5% serum, and cells were cultured for another 8 d,
a condition known to induce astrocyte differentiation (25, 26).
Subsequent immunofluorescence detection of GFAP revealed
distinct groups of GFAP+cells in this condition, but no such
cells were seen in NPC growth medium (Fig. 1D). Thus, 11F
iNPCs demonstrated the potential to differentiate into cells with
neuronal and glial morphologies and marker expression under
differentiation cues similar to those of regular NPCs.
Systematic Identification of the Critical Reprogramming Factors. To
determine which transcription factors are necessary and suffi-
cient to induce NPC-like cells, Sox2-EGFP MEFs were infected
with nine- or 10-factor pools in which one or two genes were
excluded at a time (11F − 2 pools). We ordered the 11 genes
according to expression levels in NPCs to build these groups
(27). Twenty-four days after transgene induction, a significant
decrease in Sox2-EGFP+colony numbers was observed upon
removal of (i) FoxG1 and Lhx2 or (ii) Sox2. This result suggested
that Sox2 and either FoxG1 or Lhx2 may be critical for the for-
mation of iNPCs (Fig. 2A and Table S1). We therefore tested
a pool of only the five most highly expressed transcription factors
(5F pool) in NPCs that also contained these apparently critical
three genes (27). Rewardingly, expression of the five factors
Rfx4, ID4, FoxG1, Lhx2, and Sox2 in MEFs was sufficient to
induce Sox2-EGFP+colonies as assessed 24 d after infection
(Fig. 2B and Table S2). Again, cells were tested for differentia-
tion into neuronal and glial fates without further expansion.
Distinct patches of Tuj1+and MAP2+neuronal cells and
GFAP+astrocytic cells were readily identified 12 d after removal
of growth factors (Fig. 2 B and D). Furthermore, qRT-PCR
analysis of sorted Sox2-EGFP+cells 25 d after doxycycline ad-
dition showed induction of endogenous Sox1, Sox3, Olig2, Ncan,
and Pax6. Of note, unlike the 11F pool, induction of Brn2 was
not detected (Fig. 2C).
Next, we asked whether any of the five factors was dispensable
for NPC induction. We infected MEFs with pools of four fac-
tors, systematically omitting one factor at a time, and quantified
Sox2-EGFP+colonies 24 d after transgene induction (Table S2).
Consistent with the results of the 11F − 2 pools, a decrease in
Sox2-EGFP+colonies was observed when FoxG1 and Sox2 were
removed, suggesting that both factors are important for forma-
tion of Sox2-EGFP+colonies. When we tested the resulting
putative NPCs for neuronal differentiation potential, we ob-
served many Tuj1+neuronal cells with complex morphologies
under all conditions (including omission of Sox2) except when
FoxG1 was removed, after which only a few cells with fibroblastic
morphologies were labeled with Tuj1 antibodies (Fig. 2D). This
observation highlighted FoxG1 as perhaps the most critical fac-
tor for inducing NPC-like cells with neuronal differentiation
potential (Fig. 2D). Similarly, Sox1, Sox3, Olig2, Ncan, and Pax6
were induced in all conditions except when FoxG1 or Sox2 were
Sox2-EGFP+colonies 25 d after transgene induction by doxycycline. (B) Sox2-
EGFP+cells from control (infected only with rtTA) and 11F infections were
analyzed by FACS 25 d after transgene induction. (C) qPCR analysis of NPC-
specific genes from passage 9 ES cell-derived NPCs (black), passage 4 MEFs
(blue), 11F Sox2-EGFP+FACS-sorted cells (red), and negative control rtTA
infections (green) 25 d after transgene induction (n = 2). (D) Differentiation
of 11F pooled infections 25 d after induction into GFAP+astrocytes (Left) and
Tuj1+neurons after 8 d (Right). (Scale bars: 50 mM.)
Induction of a neural precursor-like population with 11 factors. (A)
| www.pnas.org/cgi/doi/10.1073/pnas.1121003109Lujan et al.
omitted, suggesting that both genes are important for inducing
an NPC profile (Fig. 2C).
FoxG1 and Sox2 Can Induce a Bipotent NPC Population. Because
FoxG1 and Sox2 were necessary for Sox2-EGFP+colony for-
mation and NPC-specific gene induction, we investigated the
possibility that these two factors alone may induce NPC-like
cells. Sox2-EGFP MEFs infected with the two factors displayed
morphological changes as early as 4 d after doxycycline addition
(Fig. S1), and distinct elongated cell morphologies were detect-
able by 7 d. Colony formation was first observed 13 d after in-
duction, and 5.0% of cells were EGFP+at day 25 (Fig. 3B). This
cell population had the capacity to differentiate into Tuj1+,
total colonies (black) and Sox2-EGFP+colonies (green) from 11F, two pools
24 d after transgene induction. Control represents infection with rtTA virus
alone. Error bars represent SDs of three experiments. (B) Sox2-EGFP+colony
25 d after 5F transgene induction (Left). MAP2+neurons and GFAP+astro-
cytes were differentiated from the 5F population by removal of mitogens
and doxycycline and culturing for 12 d (Right). (C) qPCR analysis of NPC-
specific gene induction for Sox2-EGFP+FACS-sorted populations from 5F and
5F−1 pools 25 d after transgene induction (n = 2). (D) When FoxG1 was re-
moved from the 5F pool, no Tuj1+cells with neuronal morphology could be
derived. Neuronal cells were observed in all other conditions. Culture con-
ditions are described in B. (Scale bars: 50 μm.)
Identification of critical NPC-inducing factors. (A) Quantification of
fibroblasts. (A) Sox2-EGFP MEF-derived iNPCs give rise to Tuj1+and MAP2+
neurons (Upper) and GFAP+astrocytes (Lower Right). EGFP+neuronal cells
(Lower Left) can be detected readily from Tau-EGFP MEF-derived iNPCs un-
der the same conditions. Differentiation conditions are described in the text.
(B) Sox2-EGFP+cells from FoxG1+Sox2 infections were analyzed by FACS 25 d
after infection. (C) Representative current-clamp traces in response to cur-
rent pulses, in control condition (infection with rtTA virus only; Left) and
after infection with FoxG1+ Sox2 (Right). Neurons were differentiated 25 d
after transgene induction. Asterisks indicate action potentials. (D) qPCR
analysis reveals that FoxG1 and Sox2 can induce a subset of NPC-specific
genes but not Brn2 or the region-specific gene Nkx2.2. In addition, the fi-
broblast-specific gene Col1a1 is not repressed (n = 2). (E) A clonal population
derived from the FoxG1+Sox2 condition can differentiate into Tuj1+and
MAP2+neurons (Upper) and GFAP+astrocytes (Lower) at multiple passages.
(Scale bars: 50 μm.)
FoxG1+Sox2 can induce a self-renewing bipotent population from
Lujan et al. PNAS
| February 14, 2012
| vol. 109
| no. 7
MAP2+neurons, demonstrating that the two factors are suffi-
cient to create a population with neurogenic potential (Fig. 3A).
Patch-clamp recordings confirmed several functional neuronal
properties with an average resting membrane potential of −57.0 ±
1.0 mV, an input resistance 1.2 ± 0.5 GΩ, and the capacity to
generate action potentials (n = 4 of 7 cells; Fig. 3C).
FACS-purified Sox2-EGFP+cells displayed a morphologically
homogeneous population and could be expanded in adherent
conditions for many passages (up to 12 passages were tested),
whereas Sox2-EGFP+cells sorted from MEFs infected with
rtTA alone resembled fibroblasts with little proliferative capacity.
After extensive expansion, these iNPCs maintained the ability
to differentiate into both neuronal and astroglial cells (up to nine
passages were tested) (Fig. 3A). Notably, when placed under
astroglial differentiation conditions, GFAP+cells differentiated as
patches, suggesting that only a subset of the cells had astroglial
potential. Despite several attempts, no oligodendrocytes could be
results were reproduced with MEFs derived from the Tau-EGFP
knockin mice, which could also produce a proliferative population
that could differentiate into Tau-EGFP+, Tuj1+, and MAP2+
neurons and GFAP+astrocytes under differentiation conditions
(Fig. 3A). Expressionanalysisof FoxG1/Sox2-infected Sox2-EGFP–
and Pax6, but, similar to results in 5F cells, Brn2 was not induced
(Fig. 3D and Fig. S2). Additionally, transcript levels of the fibro-
blast-specific genes Col1a1, Col3a1, Twist2, Snail1, and Dkk3 were
similar to those in fibroblasts, suggesting that this population had
not silenced the fibroblast-specific transcriptional program (Fig. 3D
and Fig. S3). In summary, these data suggest that FoxG1 and Sox2
are capable of inducing NPCs with bilineage-restricted differenti-
ation potential but fail to induce the NPC marker Brn2.
We next explored the self-renewal potential of FoxG1/Sox2
iNPCs. To this end, clonal analysis was performed. Three Sox2-
EGFP+colonies and six colonies from Tau-EGFP knockin
MEFs were picked manually and expanded. Differentiation was
induced at multiple passages, and Tuj1+and MAP2+neurons
were detected in all clones (Fig. 3E). In contrast, only one clone
was able to differentiate also into GFAP+astrocytes (Fig. 3E).
Efficient differentiation into GFAP+astrocytes was observed
from this clone at passages 4, 11, and 17 when exposed to 5%
serum (Fig. 3E). Thus, the FoxG1/Sox2 iNPCs have self-renewal
potential and consist mostly of neuron-restricted progenitor cells
and some neuron/astroglial-restricted bipotent precursor cells,
confirming our observations with the nonclonal iNPC cultures.
No astroglial-restricted progenitors and no cells with oligoden-
droglial differentiation potential were observed.
Expression analysis of the bipotent and two unipotent clones
showed that NPC markers were expressed at levels similar to
those in the nonclonal iNPC population (Fig. S2).
Addition of Brn2 Induces NPCs with Trilineage Differentiation
Potential. As described above, FoxG1/Sox2 iNPCs lacked oligo-
dendrocyte differentiation potential and failed to activate the
Brn2 locus. We hypothesized that an additional factor might be
able to induce a tripotent NPC population with a more complete
NPC expression profile. To this end, we screened the remaining
nine of our 11 candidate factors in combination with FoxG1
and Sox2 for their potential to induce NPC-like cells that could
be differentiated into oligodendrocytes. Twenty-five days after
transgene induction, Sox2-EGFP+cells were sorted by FACS,
passaged twice, and exposed to oligodendrocyte differentiation
media (Materials and Methods). Strikingly, O4+cells with a typi-
cal oligodendrocytic morphology could be produced only from
the EGFP+population generated with the addition of Brn2 (Fig.
4A). The addition of Ascl1 also induced O4+cells, but these cells
did not acquire a mature oligodendrocytic morphology. Char-
acterization of the FoxG1/Sox2/Brn2 Sox2-EGFP+–sorted pop-
ulation showed that this population also had the potential to
differentiate into GFAP+astrocytes and Tuj1+/MAP2+neurons,
suggesting that all three cell types of the central nervous system
could be derived from this three-factor population (Fig. 4A).
Further experimentation showed that cells infected with
FoxG1 and Brn2 alone could induce a Sox2-EGFP+population,
suggesting that Sox2 may be dispensable for the induction of
NPC-like cells. We therefore characterized these potential
FoxG1/Brn2-iNPCs in greater detail. Just like the cells infected
with the three factors, the FoxG1/Brn2-infected cell population
gave rise to cells with typical oligodendrocytic morphologies that
could be labeled with antibodies against O4, Olig2, 2’, 3′-cyclic
nucleotide 3′-phosphodiesterase (CNP), and myelin basic pro-
tein (MBP) when exposed to oligodendrocyte differentiation
media (Fig. 4B, Top). To investigate the potential of FoxG1/Brn2
cells to differentiate into functional oligodendrocytes, passage 17
proliferating EGFP-infected iNPCs were injected into the neo-
natal brain of Shiverer mice, which are dysmyelinated and
lack the wild-type form of MBP. Therefore, the presence of
EGFP+and wild-type MBP+cells were assessed in Shiverer
mice 6 and 10 wk after transplantation. Strikingly, 10 wk after
transplantation EGFP+cells and MPB+myelin sheets were
detected in white-matter tracts of the cerebellum (Fig. 4H). This
result indicates that iNPC-derived oligodendrocytes behave
similarly to oligodendrocytes derived from endogenous neural
progenitor cells, differentiating into oligodendrocytes and ap-
propriately myelinating axons of the developing brain. No MBP
was detected at 6 wk, indicating that differentiation into fully
mature oligodendrocytes is relatively slow in vivo.
When exposed to serum-containing media, the great majority
of the FoxG1/Brn2 iNPCs assumed a flat, astrocyte-like mor-
phology very similar to the behavior of ES cell-derived NPCs.
These astrocytic cells homogenously expressed GFAP, and the
majority of these cells coexpressed S-100 protein (65.8 ± 3.5% at
day 9 after addition of serum). The astrocyte-associated genes
Aldh1l1, S100b, Aqp4, Igfppb3, and GFAP were expressed clearly,
based on qRT-PCR (Fig. 4G, but note the moderate expression
levels for both Aldh1l1 and Igfbp3 in MEFs). Moreover, char-
acterization of the astrocytic population by patch-clamp recordings
showed the development of an average resting membrane po-
tential of −80.2 ± 3.9 mV with an input resistance of 1.2 ± 0.2
GΩ (n = 15 cells) and the lack of action potential generation
upon depolarization (Fig. 4F). These properties were highly
similar to those of astrocytes differentiated from ES cell-derived
NPCs, which had an average resting membrane potential of
−77.7 ± 4.4 mV and an input resistance of 1.3 ± 0.2 GΩ (n = 12
cells) (Fig. 4F).
Furthermore, upon withdrawal of growth factors and doxycy-
cline, cells with typical neuronal morphologies and expression of
Tuj1 and MAP2 could be identified readily (Fig. 4B). We per-
formed patch-clamp recordings from neurons differentiated at
passages 2, 4, and 20. These cells showed an average resting
membrane potential of −47.9 ± 4.1 mV and an input resistance of
1.1 ± 0.8 GΩ (n = 7 of 39 cells) and had the capacity to generate
action potential-like events, demonstrating the expression of
functional voltage-gated ion channels (Fig. 4E). However, ste-
reotypic action potentials could not be identified in this cell
population, suggesting that FoxG1/Brn2 iNPCs do not differen-
tiate into mature neurons under standard conditions. On the
other hand, when FoxG1/Brn2/Sox2 iNPCs were differentiated,
cells capable of action potential formation were readily detectable
(n = 1 of 3 cells). This finding suggests that Sox2 is a critical factor
in conferring a full neuronal differentiation potential to iNPCs.
After examining their capacity for differentiation, we further
explored the characteristics of undifferentiated iNPCs. Both
FoxG1/Brn2 and FoxG1/Brn2/Sox2 iNPC populations could be
expanded easily under standard NPC growth conditions, and
FoxG1/Brn2 iNPCs exhibited a homogeneous population of
Nestin+, brain lipid-binding protein–positive (BLBP+), Pax6+,
Olig1−cells (Fig. S4). Similar to Sox2/FoxG1 iNPCs, FoxG1/
Sox2/Brn2 and FoxG1/Brn2 iNPCs expressed Sox1, Sox3, Olig2,
Ncan, and Pax6 (Fig. S4). Importantly, iNPCs generated with
pools containing Brn2 also showed activation of the endogenous
| www.pnas.org/cgi/doi/10.1073/pnas.1121003109Lujan et al.
Brn2 gene and a reduction in the fibroblast-specific genes Co-
l1a1, Col3a1, Twist2, Snail1, and Dkk3 to levels similar to those
in ES cell-derived NPCs (Figs. S3 and S4). Both findings contrast
with the previously discussed 5F iNPCs and the FoxG1/Sox2
iNPCs (compare with Figs. 2C and 3D). Thus, the addition of
Brn2 not only confers oligodendrocytic potential but also leads to
repression of fibroblast-specific transcriptional characteristics.
Finally, to investigate the self-renewal potential of iNPCs,
clonal analysis was performed. Four colonies were picked 29 d
after infection of the fibroblasts with FoxG1/Brn2. After three
passages, one of the four clonal populations showed the poten-
tial to differentiate into Tuj1+, GFAP+, and CNP+cells 12 d
after removal of growth factors; the other three populations did
not show NPC-like characteristics and could not differentiate
into cells expressing any of these markers (Fig. S5). The differ-
entiation potential of this clone was similar to that of the nonclonal
iNPC population, and both clonal and nonclonal iNPCs remained
tripotent over many passages when differentiated for 9 d in N3
medium alone and 3 d in N2B27 plus 1% serum (Fig. 4C).
Here we show that specific NPC populations with defined dif-
ferentiation potentials can be induced from mouse fibroblasts
transduced with different combinations of lineage-specific tran-
scription factors. Starting with a list of 11 candidate factors, we
found that the combination of FoxG1 and Sox2 can induce NPCs
(referred to as “iNPCs”), the majority of which exhibited a neu-
ron-restricted differentiation potential and the minority a bili-
neage neuron/astroglial differentiation capacity. The addition
of Brn2 was sufficient to induce tripotent iNPCs from mouse
fibroblasts, whereas transduction of FoxG1 and Brn2 alone
produced iNPCs capable of differentiating into astro- and oli-
godendrocytes and only immature neurons. All tested bipotent
and tripotent populations had the potential to self-renew on the
single-cell level while maintaining their initial differentiation
potential. Also, the tripotent iNPCs activated many genes se-
lectively expressed in regular neural precursor populations and
silenced a set of fibroblast-specific genes, suggesting the in-
duction of NPC identity and elimination of at least some fibro-
blast-specific properties. Moreover, iNPCs responded to the same
external stimuli as regular NPCs both for induction of self-renewal
and differentiation into neurons, astrocytes, or oligodendrocytes
(21). For example, when bi- or tripotent iNPCs were exposed
to serum, astrocytic differentiation was induced very rapidly and
efficiently, a phenomenon known to occur in primary or ES
cell-derived NPCs and mediated by BMP and JAK/Stat signaling
(25, 26, 28, 29). Thyroid hormone T3 induced the generation
of oligodendrocytes, as described for other NPC populations
(26, 30). These differentiated cell states are stable in the absence
of the reprogramming factors, demonstrating that the transcrip-
tional network maintaining these cell fates has been reactivated.
Recently, Kim et al. (21) described the generation of an iNPC
population from mouse fibroblasts by transient expression of the
four iPS cell-reprogramming factors Oct4, Sox2, Klf4, and c-Myc
for 6 d, followed by exposure to neural media. Following this
protocol, NPC-like colonies appeared spontaneously at low fre-
quencies (0.5–0.7%). After manual picking, these cultures gave
rise to functional neurons and GFAP+astrocytes. However, the
cells could not be maintained for more than three to five pas-
sages and apparently lacked the potential to differentiate into
neurons, and GFAP+astrocytes can be induced from MEFs infected with FoxG1, Sox2, and Brn2. (B) FoxG1 and Brn2 alone induce a population that can give
rise to mature CNP+, Olig2+, and MBP+oligodendrocytes, GFAP+and S100+astrocytes, and TUJ1+and MAP2+neurons. (C) Quantification of TUJ1+, GFAP+,
and CNP+cells from a nonclonal (Left) and clonal (Right) FoxG1+Brn2 iNPC population at multiple passages after 9 d in N3 and 3 d in N2B27 plus 1% serum.
(D) Sox2-EGFP+cells from FoxG1+Brn2 infections were analyzed by FACS 21 d after infection. (E) Representative current-clamp traces in response to current
pulses. Neurons were differentiated from FoxG1+Sox2+Brn2 (Upper) or FoxG1+Brn2 (Lower) iNPCs 25 d after transgene induction and after 20 passages, re-
spectively. (F) Electrophysiological characterization of FoxG1/Brn2 iNPC-derived and ESC-derived astrocytes. Representative current-clamp traces recorded from
iNPC-derived (Left) and ESC-derived (Right) astrocytes in response to current-pulses as in Fig. 3C. (G) FoxG1/Brn2 iNPC-derived astrocytes express the astrocyte-
associated markers Aldh1L1, S100B, Aqp4, Igfbp3, and GFAP (n = 2). (H) Passage 17 EGFP-labeled FoxG1/Brn2 iNPCs were transplanted into P1 Shiverer mice
targeted to the cerebellum. EGFP+transplanted cells and MBP+myelin tracks were detected 10 wk after transplantation. Right panel shows boxed area in left
panel. (Scale bars: 50 μm.)
Addition of Brn2 induces a tripotent NPC population. (A) A Sox2-EGFP+population that gives rise to O4+oligodendrocytes, Tuj1+and MAP2+
Lujan et al.PNAS
| February 14, 2012
| vol. 109
| no. 7
oligodendrocytes. Conceptually, the approach of Kim et al. is Download full-text
fundamentally different from the approach taken in the present
study. Although our underlying hypothesis is that lineage-de-
termining transcription factors of the target cell type will directly
induce the desired cell type, cell fate conversion using iPS cell
transcription factors presumably first induces an unstable plu-
ripotent state (perhaps without induction of endogenous pluri-
potency genes) followed by spontaneous differentiation into a
neural lineage. In contrast, we believe that lineage reprogram-
ming using target cell-type–specific transcription factors repre-
involving an intermediate pluripotent state. Consistent with this
hypothesis, c-Myc was required for the dedifferentiation ap-
proach (21), presumably because without c-Myc the induction of
pluripotency is decreased by orders of magnitude (31, 32), and
cell types representing a different germ layer, such as car-
diomyocytes, can be achieved from a similar transient pluripo-
tent state (20). Future studies will be required to determine
whether specific NPC populations with defined differentiation
potential, as shown here, can be generated using the transient
The generation of iNPCs from fibroblasts with subsequent
differentiation to neurons may have several advantages over di-
rect conversion of fibroblasts into postmitotic neuronal or glial
cells. First, iNPCs can self-renew and can be expanded for many
passages. This property should facilitate applications in which
large cell numbers are needed, such as high-throughput drug
screening or cell-transplantation therapy. Second, clonal pop-
ulations can be generated that should lead to homogeneous
cell populations more amenable to characterization than the
conversion of mixed fibroblast populations. Additionally, direct
iNPC generation may be more advantageous, at least for some
applications, than NPC generation from iPS cells. For example,
one of the major complications of potential pluripotent stem
cell therapies is the risk of teratoma formation resulting from
a lack of purity of the differentiated population or reactivation
of the reprogramming factors. Unlike iPS cell or transient de-
differentiation, no overt oncogenes, such as c-Myc, or any other
specific pluripotency factors are required to produce iNPCs.
However, many questions remain: Can similar cells be gener-
ated from human fibroblasts? Can iNPCs be patterned to gener-
ate any of the many neuronal and glial subtypes in the brain?
Which regular NPC populations do iNPCs most resemble, and
how complete is the reprogrammed NPC state? One of the key
factors identified in this paper is FoxG1, which is expressed pre-
described are broadly representative of rostral forebrain NPCs
the other hand, FoxG1 expression also has been interpreted in the
context of ES cell differentiation as a primitive (anterior) neu-
roectoderm capable of being patterned to many regional identi-
ties (33). These and other questions will need to be addressed
before the therapeutic potential of iNPCs can be assessed.
Materials and Methods
Rosa-rtTA, Sox2-GFP MEFs were isolated from embryos on embryonic day 14.5.
Internal organs, eyes, and spinal cord were carefully removed. Tau-GFP MEFs
of the tetracycline promoter, and lentiviral preparations were packaged in
293T cells as previously described (34). To induce lineage conversion, 200,000
cells from passage 4 MEFs were plated onto polyornithine-coated 6-cm plates
and were infected the following day in MEF medium supplemented with
Polybrene (8 μg/mL; Sigma). One day after infection, the medium was replaced
with N3 medium (24) supplemented with FGF2 (10 ng/mL; Invitrogen), EGF (10
ng/mL; R&D Systems), and doxycycline (2 μg/mL) and was replaced every 3 d.
Differentiation, immunofluorescence, FACS analysis, qRT-PCR, electrophysi-
ology, and transplantation protocols are provided in SI Materials and
Methods. Primers used in RT-PCR analysis are given in Table S3.
ACKNOWLEDGMENTS. We thank P. Lovelace and S. Marro for help with FACS
sorting; T. Vierbuchen, Y. Kokubu, S. Mitra, and C. Khajvandi for reagents
and experimental support; and N. Uchida for instrumental advice. E.L. was
supported by California Institute for Regenerative Medicine Predoctoral
Fellowship TG2-01159, and H.A. was supported by a postdoctoral fellowship
from the Swedish Research Council. This work was enabled by a Robertson
Investigator Award from New York Stem Cell Foundation; the Ellison Medi-
cal Foundation; the Stinehart-Reed Foundation; and National Institutes of
Health Grants 1R01MH092931, AG010770-18A1, and RC4 NS073015.
1. Gurdon JB, Melton DA (2008) Nuclear reprogramming in cells. Science 322:1811–1815.
2. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH (1997) Viable offspring de-
rived from fetal and adult mammalian cells. Nature 385:810–813.
3. Gurdon JB, Byrne JA, Simonsson S (2003) Nuclear reprogramming and stem cell cre-
ation. Proc Natl Acad Sci USA 100(Suppl 1):11819–11822.
4. Blau HM (1989) How fixed is the differentiated state? Lessons from heterokaryons.
Trends Genet 5:268–272.
5. Zhou Q, Melton DA (2008) Extreme makeover: Converting one cell into another. Cell
Stem Cell 3:382–388.
6. Davis RL, Weintraub H, Lassar AB (1987) Expression of a single transfected cDNA
converts fibroblasts to myoblasts. Cell 51:987–1000.
7. Schäfer BW, Blakely BT, Darlington GJ, Blau HM (1990) Effect of cell history
on response to helix-loop-helix family of myogenic regulators. Nature 344:454–458.
8. Graf T, McNagny K, Brady G, Frampton J (1992) Chicken “erythroid” cells transformed
by the Gag-Myb-Ets-encoding E26 leukemia virus are multipotent. Cell 70:201–213.
9. Cobaleda C, Jochum W, Busslinger M (2007) Conversion of mature B cells into T cells
by dedifferentiation to uncommitted progenitors. Nature 449:473–477.
10. Zhou Q, Brown J, Kanarek A, Rajagopal J, Melton DA (2008) In vivo reprogramming of
adult pancreatic exocrine cells to beta-cells. Nature 455:627–632.
11. Kaneto H, et al. (2005) PDX-1/VP16 fusion protein, together with NeuroD or Ngn3,
markedly induces insulin gene transcription and ameliorates glucose tolerance.
12. Vierbuchen T, et al. (2010) Direct conversion of fibroblasts to functional neurons by
defined factors. Nature 463:1035–1041.
13. Pang ZP, et al. (2011) Induction of human neuronal cells by defined transcription
factors. Nature 476:220–223.
14. Marro S, et al. (2011) Direct lineage conversion of terminally differentiated hep-
atocytes to functional neurons. Cell Stem Cell 9:374–382.
15. Ieda M, et al. (2010) Direct reprogramming of fibroblasts into functional car-
diomyocytes by defined factors. Cell 142:375–386.
16. Szabo E, et al. (2010) Direct conversion of human fibroblasts to multilineage blood
progenitors. Nature 468:521–526.
17. Sekiya S, Suzuki A. (2011) Direct conversion of mouse fibroblasts to hepatocyte-like
cells by defined factors. Nature 475:390–393.
18. Huang P, et al. (2011) Induction of functional hepatocyte-like cells from mouse
fibroblasts by defined factors. Nature 475:386–389.
19. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse
embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676.
20. Efe JA, et al. (2011) Conversion of mouse fibroblasts into cardiomyocytes using a di-
rect reprogramming strategy. Nat Cell Biol 13:215–222.
21. Kim J, et al. (2011) Direct reprogramming of mouse fibroblasts to neural progenitors.
Proc Natl Acad Sci USA 108:7838–7843.
22. Ellis P, et al. (2004) SOX2, a persistent marker for multipotential neural stem cells
derived from embryonic stem cells, the embryo or the adult. Dev Neurosci 26:148–165.
23. Beard C, Hochedlinger K, Plath K, Wutz A, Jaenisch R (2006) Efficient method to
generate single-copy transgenic mice by site-specific integration in embryonic stem
cells. Genesis 44:23–28.
24. Wernig M, et al. (2002) Tau EGFP embryonic stem cells: An efficient tool for neuronal
lineage selection and transplantation. J Neurosci Res 69:918–924.
25. Conti L, et al. (2005) Niche-independent symmetrical self-renewal of a mammalian
tissue stem cell. PLoS Biol 3:e283.
26. Johe KK, Hazel TG, Muller T, Dugich-Djordjevic MM, McKay RD (1996) Single factors
direct the differentiation of stem cells from the fetal and adult central nervous sys-
tem. Genes Dev 10:3129–3140.
27. Wu JQ, et al. (2010) Dynamic transcriptomes during neural differentiation of human
embryonic stem cells revealed by short, long, and paired-end sequencing. Proc Natl
Acad Sci USA 107:5254–5259.
28. Gross RE, et al. (1996) Bone morphogenetic proteins promote astroglial lineage
commitment by mammalian subventricular zone progenitor cells. Neuron 17:595–606.
29. Mabie PC, et al. (1997) Bone morphogenetic proteins induce astroglial differentiation
of oligodendroglial-astroglial progenitor cells. J Neurosci 17:4112–4120.
30. Glaser T, Perez-Bouza A, Klein K, Brüstle O (2005) Generation of purified oligoden-
drocyte progenitors from embryonic stem cells. FASEB J 19:112–114.
31. Nakagawa M, et al. (2008) Generation of induced pluripotent stem cells without Myc
from mouse and human fibroblasts. Nat Biotechnol 26:101–106.
32. Wernig M, Meissner A, Cassady JP, Jaenisch R (2008) c-Myc is dispensable for direct
reprogramming of mouse fibroblasts. Cell Stem Cell 2:10–12.
33. Elkabetz Y, et al. (2008) Human ES cell-derived neural rosettes reveal a functionally
distinct early neural stem cell stage. Genes Dev 22:152–165.
34. Wernig M, et al. (2008) A drug-inducible transgenic system for direct reprogramming
of multiple somatic cell types. Nat Biotechnol 26:916–924.
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