84 | VOL.10 NO.1 | JANUARY 2013 | nAture methods
human neural stem cells hold great promise for research
and therapy in neural disease. We describe the generation
of integration-free and expandable human neural progenitor
cells (nPcs). We combined an episomal system to deliver
reprogramming factors with a chemically defined culture
medium to reprogram epithelial-like cells from human urine
into nPcs (huinPcs). these transgene-free huinPcs can self-
renew and can differentiate into multiple functional neuronal
subtypes and glial cells in vitro. Although functional in vivo
analysis is still needed, we report that the cells survive and
differentiate upon transplant into newborn rat brain.
Several neural disorders have no effective drug treatment at
present, and stem cells offer hope for those suffering from these
debilitating diseases. There has been intense interest in obtain-
ing human neural stem cells (NSCs) that may be used to treat
neural disorders or to study them in the laboratory. The isolation
and use of NSCs from either fetal or adult human tissue remain
challenging because of ethical concerns and immune rejection1,2.
Induced pluripotent stem cell (iPSC) technology provides a prom-
ising solution to this problem because it may be used to generate
patient-specific cells for autologous engraftment3–6. Indeed, iPSCs
from patients with neural diseases have been established success-
fully, such as with Parkinson’s disease7,8, Huntington’s disease7
and Alzheimer’s disease9. Differentiation of such iPSCs along the
neural lineage and modeling of disease in vitro could increase
our mechanistic understanding of these diseases, enable drug
screening and eventually provide functional cells for autologous
transplantation. However, the differentiation of human iPSCs into
NSCs is inefficient, time consuming and variable among different
iPSC lines6. Moreover, pluripotent cells such as iPSCs pose the
risk of teratomas when transplanted in vivo. To attempt to circum-
vent these problems, new approaches have been devised to con-
vert one somatic cell type to another without full reprogramming
Generation of integration-free neural progenitor
cells from cells in human urine
Lihui Wang1–3, Linli Wang1,2, Wenhao Huang1,2, Huanxing Su1,2,7, Yanting Xue1,2,4, Zhenghui Su1,2,4,
Baojian Liao1,2, Haitao Wang1,2, Xichen Bao1,2, Dajiang Qin1,2, Jufang He5, Wutian Wu6, Kwok Fai So6,
Guangjin Pan1,2 & Duanqing Pei1,2
to the pluripotent state. Notably, functional neurons, termed
induced neurons, have been generated directly from fibroblasts
by retroviral delivery of neural-specific transcription factors
or microRNAs10–15. The direct conversion approach has also
been successfully applied to reprogram mouse fibroblasts into
hepatocyte-like cells16,17, cardiomyocytes17 and pancreatic
β cells18, using virally delivered reprogramming factors.
An attractive alternative to using induced neurons or iPSC-
derived neurons is to generate self-renewable NPCs from a
patient’s own somatic cells, and to do so avoiding viral integra-
tion of the genes encoding reprogramming factors. In this report,
we describe the integration-free generation of hUiNPCs, human
NPCs from epithelial-like cells in human urine. The cells could
be expanded in vitro and differentiate into neuronal subtypes
Generation of integration-free nPcs from human urine cells
We have recently shown that human urine contains live cells that
can be efficiently reprogrammed into iPSCs19. In an effort to fur-
ther improve this approach to derive naive iPSCs, we adopted
integration-free and feeder-free methods to reprogram human
urine cells (HUCs). We purified viable HUCs from the urine
(Fig. 1a) of a healthy 37-year-old male donor and transfected
them with oriP/EBNA episomal vectors carrying a combination
of reprogramming factors encoded by OCT4 (POU5F1), SOX2,
SV40LT, KLF4 and microRNA cluster MIR302–367 through
electroporation20,21 and cultured the transfected cells in defined
basal medium containing FGF2 (ref. 22) and a cocktail of small
molecules (5i) known to promote reprogramming: CHIR99021,
PD0325901, A83-01, thiazovivin and DMH1 (refs. 23–29). We
observed that the transfected HUCs showed rapid morphologi-
cal changes in these cultures. Notably, as early as day 12 post-
electroporation, we began to observe the formation of compact
1Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health,
Chinese Academy of Sciences, Guangzhou, China. 2Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell
Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China. 3Department of Pathology,
Dalian Medical University, Dalian, China. 4School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China. 5Department of Rehabilitation
Science, Hong Kong Polytechnic University, Hong Kong, China. 6Department of Anatomy, Hong Kong University, Hong Kong, China. 7Present address: State Key
Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macao, China. Correspondence should be addressed
to G.P. (email@example.com) or D.P. (firstname.lastname@example.org).
Received 10 August; Accepted 16 NovembeR; published oNliNe 9 decembeR 2012; doi:10.1038/Nmeth.2283
© 2013 Nature America, Inc. All rights reserved.
nAture methods | VOL.10 NO.1 | JANUARY 2013 | 85
colonies (Fig. 1b). The average colony-forming efficiency was
around 0.2% as determined from the initial number of HUCs
used in each reprogramming experiment and was consistent for
three different donors (n = 3, Supplementary Fig. 1). This is in
sharp contrast to HUCs electroporated with the same episomal
vectors but cultured in TeSR22, in which iPSC colonies appeared
at around 25 d after electroporation (Supplementary Fig. 2). We
were intrigued by the appearance of these early colonies and char-
acterized them further.
We picked colonies from both TeSR and 5i cultures and cultured
them further on Matrigel, in their original media. Surprisingly,
the domed colonies that had emerged in the 5i medium assumed
a rosette-like morphology typical of NPCs
(Fig. 1c). In contrast, colonies grown in
TeSR medium grew as typical human
iPSC colonies (Supplementary Fig. 2).
We hypothesized that cells undergoing
reprogramming in the 5i medium might
preferentially commit to the NPC fate at
an early stage (around day 12) before the
establishment of a full pluripotent state
(around day 25). To test this hypothesis,
we randomly picked 20 individual colonies
from the reprogramming culture in 5i at day 14 or 15 and exam-
ined the expression of NSC and pluripotency markers without
further re-plating or culturing. By quantitative real-time PCR
(qRT-PCR) analysis, we observed robust expression of typical
NSC genes such as SOX2 and NES (encoding nestin) in all 20
colonies. We did not observe substantial activation of genes
encoding pluripotency markers such as OCT4 and NANOG or
other lineage markers such as T (brachyury) and SOX17 (Fig. 1d
and Supplementary Fig. 2). We did detect low expression of
SOX17 in HUCs and some picked colonies, which is consistent
with the endodermal origin of HUCs (Supplementary Fig. 2).
PAX6, another NSC gene, was activated at day 14, but more
Defined medium with
Number of cells (× 106)
1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10iPS UC
Colonies picked at day 14
Colonies picked at day 15
Relative expression level
P6 P7 P8 P9 P10 P11
Figure 1 | Generation and expansion of
integration-free NPCs from human urine cells.
(a–d) Generation of NPCs from human urine
cells. Epithelial-like cells isolated from human
urine (a) were transfected with episomal vectors
encoding reprogramming factors and miR302–367
and cultured in defined medium (5i) on Matrigel.
Colonies arising at day 12 (b) were picked and
re-plated onto Matrigel (c) or directly analyzed for
marker expression by qRT-PCR (d). The plot shows
relative expression levels of the indicated genes
in colonies picked at days 14 and 15 compared
to levels in HUCs and iPSCs (abbreviated as
UC and iPS, respectively). Note that levels of
NANOG in iPSCs are invisible on the plot at this
scale. The value of NANOG in iPSCs was 0.0061,
compared with 0.0000036 in HUCs and 0.00165
in colonies at day 14 and 0.000899 in colonies
at day 15. (e–i) Micrographs show immunostains
of colonies for the indicated markers (day 15).
Lower panel of g shows a positive control for the
antibody to NANOG. (j–q) Expansion of hUiNPCs.
Representative morphology of hUiNPCs cultured on
Matrigel (j) or in suspension as neural spheres (k)
is shown. (k) Left, growth curve of hUiNPCs
cultured as neural spheres. Right, neural spheres
before and after passage (P). (l) hUiNPCs stained
for Ki67 and nestin (P6). (m–p) Immunostains of
expanded hUiNPCs (P5) for the indicated markers.
(q) qRT-PCR analysis of markers expressed by
expanded hUiNPCs (P5). (r) PCR detection of
integrated transgenes in expanded hUiNPCs
(iNPC, P6). Primers were designed to detect
exogenous reprogramming factors. Untransfected
urine cells served as the negative control.
HUCs transiently transfected with reprogramming
factors served as the positive control.
(s) Karyotype of expanded hUiNPCs (P5). Scale
bars, 50 µm. Error bars, s.d., based on three
replicates (n = 3) for d, k and q.
© 2013 Nature America, Inc. All rights reserved.
86 | VOL.10 NO.1 | JANUARY 2013 | nAture methods
substantially so at day 15, which suggests a dynamic process of
reprogramming toward a neural fate (Fig. 1d).
We further immunostained the 5i colonies at day 15 for known
embryonic stem cell (ESC) or NSC markers. The colonies were neg-
ative for pluripotency markers such as TRA-1-60, TRA-1-81 and
NANOG but stained positively for PAX6 and nestin (Fig. 1e–i).
We also failed to detect substantial activation of OCT4 and
NANOG at earlier stages of reprogramming
(days 6–8) (Supplementary Fig. 2). It has
been reported that mouse embryonic fibroblasts reprogrammed
with standard pluripotency factors (Oct4, Sox2, Klf4 and c-Myc)
can be switched to a neural identity by transferring the virally
infected cells to neural medium containing fibroblast growth
factors and epidermal growth factor30. However, we did not
observe NPC-like colonies among the transfected HUCs grown
in the identical neural medium reported earlier (data not shown).
UriC1 UriC2 UiNPC1 UiNPC2 Uri_iPSC1 Uri_iPSC2
hUiNPC vs. Uri_iPSC
5 1015 20
Uri_iPSC expression (log2)
Selected neural genes
Selected pluripotency genes
R = 0.95
Log fold change (log2)
NSC marker genes
ESC marker genes
Generation of neurons
Transmission of nerve
Regulation of neuron
Regulation of cell migration
Regulation of cell motility
Regulation of cellular-component movement
Cardiovascular system development
Circulatory system development
Positive regulation of cell migration
Positive regulation of cell motility
Figure 2 | Global gene expression analysis of hUiNPCs. (a) Pearson correlation analyses of global gene expression in HUCs (UriC1 and UriC2) and in neural
progenitor cells (UiNPC1 and UiNPC2) and iPSCs (Uri_iPSC1 and Uri_iPSC2) derived from them. (b) Comparison of global gene expression profiles of a
Uri_iPSC and UiNPC line derived from the same starting cell sample. R, Pearson correlation coefficient. Selected neural-specific (purple) and embryonic
stem cell (ESC)-specific (red) genes are highlighted. (c) Differential expression profile between parental HUCs (above) and hUiNPCs (below). Yellow
dashed lines correspond to a twofold change. The differentially expressed genes (red) are those with an adjusted P value 0.05 and fold change 3. Known
NPC-specific (green) and ESC-specific (purple) genes are depicted. (d) Functional annotations of genes differentially expressed between HUCs (top)
and hUiNPCs (bottom). Gene ontology (GO) statistics for these genes were computed using the hypergeometric test, and enriched GO terms (biological
processes) for each cell type are plotted with −log10 of the adjusted P values (adjpvalue).
MAP2- or TUJ1-positive neurons
Percentage of total
Figure 3 | Differentiation of hUiNPCs in vitro.
(a) Bright-field image of spontaneously
differentiated cells from hUiNPCs.
(b–j) Immunostains of spontaneously
differentiated hUiNPCs with antibodies
against the indicated markers: GFAP, astrocyte
marker; TUJ1, pan-neuronal marker; NeuN
and MAP2, mature neuronal markers; DCX,
immature neuronal marker; glutamate (Glu),
GABA and TH, subtype-specific neuronal
markers; O4, oligodendrocyte marker; PDGFR-α,
oligodendrocyte progenitor marker; SYN (and
arrow in j), synapsin I. (k) Percentage of each
neuronal subtype (left) and of neurons with
synapsin staining (right) out of total neurons.
Total neurons were determined by staining with
pan-neuronal marker MAP2 or TUJ1. At least 500
DAPI-positive cells from five randomly selected
fields were counted to calculate the fraction of
each lineage (Online Methods). (l) Percentage
of neurons (TUJ1-positive cells) and astrocytes
(GFAP-positive cells) in spontaneously
differentiated hUiNPCs at different passages
(P). Total cells were determined by DAPI stain.
Scale bars: 50 µm (a–g), 20 µm (h–j).
Error bars, s.e.m.; n = 3 experiments.
© 2013 Nature America, Inc. All rights reserved.
nAture methods | VOL.10 NO.1 | JANUARY 2013 | 87
Furthermore, when we replaced HUCs with human dermal
fibroblasts, we observed no NPC-like cells under the conditions
that we used for HUC reprogramming with 5i (data not shown).
Taken together, our results suggest that the compact, dome-shaped
colonies that appeared early during reprogramming in 5i medium
may have committed to an NPC fate rather than a pluripotent
one. We observed the same outcome for HUCs donated by two
additional individuals ages 10 and 25 at an average reprogram-
ming efficiency of around 0.2% based on the starting number of
HUCs (Supplementary Fig. 1).
To characterize the proliferative potential of hUiNPCs, we dis-
sociated the re-plated rosette colonies and seeded them as single
cells on Matrigel (Fig. 1j). We observed that these cells grew well
and exhibited typical NPC morphology. When cultured in sus-
pension, the hUiNPCs grew as neural spheres, a typical property
of NSCs31, and could be expanded over multiple passages with
a high proliferation rate (Fig. 1k). The cells expressed high lev-
els of the proliferation marker Ki67 (Fig. 1l). hUiNPC spheres
expanded for 11 passages also showed homogenous expression
(>90% of cells) of the neural ectoderm transcription factors PAX6
(ref. 32) and SOX1 as well as the neural stem cell markers SOX2
and nestin (Fig. 1m–o and Supplementary Fig. 2). In contrast,
we did not detect the expression of pluripotency markers such as
OCT4 or markers for other germ layer lineages such as SOX17
and T (Fig. 1p,q and Supplementary Fig. 2). The expanded
hUiNPCs at passage 6 no longer harbored the reprogramming
factors encoded by OCT4, SOX2, KLF4, SV40LT or MIR302–367,
nor did they carry the genes from the episomal vector back-
bone (Fig. 1r), yet they possessed normal karyotypes (Fig. 1s).
We also performed whole-genome sequencing of select hUiNPCs
and confirmed that they are of human origin (data not shown).
huinPcs show distinct gene expression profiles
We profiled the global gene expression patterns of hUiNPCs, the
parental HUCs and the urine cell–derived iPSCs (Uri_iPSCs).
The hUiNPCs clustered independently from the starting HUCs
(UriC1 and UriC2) and the urine iPSCs (Uri_iPSC1, Uri_iPSC2)
(Fig. 2a). Further paired-comparison analysis revealed distinct
expression profiles between hUiNPCs and Uri_iPSCs derived
from the same starting cells: neural genes were more highly
expressed in hUiNPCs, and pluripotency genes were more highly
expressed in Uri_iPSCs (Fig. 2b). By comparing the expression
profiles between HUCs and hUiNPCs, we identified 720 genes
related to neuronal function that were upregulated in hUiNPCs
(Fig. 2c,d and Supplementary Table 1). In contrast, the HUC
expression profiles were enriched for genes related to endothelial,
angiogenic or epithelial functions (Fig. 2c,d and Supplementary
Table 2), indicating that the HUCs might be of endothelial or
huinPcs differentiate into cell subtypes of neural lineage
The promise of NPCs relies on their capacity to differentiate into
functional subtypes of neural cells (Fig. 3). We observed that
hUiNPCs (at passage 5) could efficiently give rise to β III tubulin
(TUJ1)-positive neurons (at an average efficiency of 76.7% of total
cells in three experiments) and glial fibrillary acid protein (GFAP)-
positive astrocytes (at an average efficiency of 7.8% of total cells
in three experiments) upon in vitro spontaneous differentiation
(Fig. 3a,b,l). Moreover, this differentiation potential was well main-
tained throughout prolonged in vitro expansion (passage 8) (Fig. 3l).
Unlike mouse NPCs, human NPCs do not give rise to oligodendro-
cytes during spontaneous in vitro differentiation33,34. However,
in the presence (for 3 weeks) of factors (PDGF-AA, NT3, IGF1)
–80 mV –80 mV
+10 µm TEA
+10 µm TTX
Figure 4 | hUiNPC-derived neurons are functional in vitro. (a) Current-clamp recording showing a
representative train of action potentials in a neuron differentiated from hUiNPCs. Intracellular injected
currents were step currents of −20, 30, 50, 60, 70, 80, 90 and 100 pA. Right, current trace at 90-pA injected
current. (b,c) Representative recordings of voltage-gated ion channels from a neuron differentiated from
hUiNPCs. An inward current was observed and could be blocked by tetrodotoxin (TTX) (b), and an outward
current could be blocked by tetraethylammonium (TEA) (c). (d–f) Analysis of postsynaptic currents in
hUiNPC-derived neurons. Shown are representative traces of spontaneous postsynaptic currents (PSCs) (d),
of excitatory postsynaptic currents in cells clamped at −80 mV in response to l-glutamate puffs (e), and of
inhibitory postsynaptic currents in cells clamped at 0 mV in response to GABA puffs (f).
© 2013 Nature America, Inc. All rights reserved.
88 | VOL.10 NO.1 | JANUARY 2013 | nAture methods
known to promote oligodendrocyte differentiation, we observed
O4- and PDGFR-α–positive oligodendrocyte-like cells (Fig. 3h,i).
We observed, by staining with neuronal subtype–specific markers,
that hUiNPCs could generate various subtypes of neurons includ-
ing mature glutamatergic, GABAergic and dopaminergic neurons
at 37.6%, 15.2% and 6.5%, respectively (Fig. 3e–g, respectively, and
Fig. 3k). We also observed DCX-positive immature neurons in the
differentiation culture (Fig. 3d). Finally, 85.4% of neurons derived
from hUiNPC were positive for synapsin, indicating that most of
the neurons were likely to be excitable (Fig. 3j,k).
huinPc-derived neurons are functional in vitro
We used standard whole-cell patch-clamp recordings to examine
the function of hUiNPC-derived neurons (Fig. 4). We observed
rapidly inactivating inward currents and persistent outward
currents in response to depolarizing voltage steps that could be
blocked by tetrodotoxin and tetraethylammonium (Fig. 4b,c).
We also observed that the neurons generated repetitive trains
of action potentials (Fig. 4a). Notably, the hUiNPC-derived
neurons exhibited strong postsynaptic currents spontaneously
or in response to excitatory or inhibitory neurotransmitter
stimulation (Fig. 4d–f; 5 out of 5 neurons for each experiment).
Together, these data demonstrate that
hUiNPCs can give rise to mature, functional
neurons in vitro.
transplantation of huinPcs in vivo
To examine the potential of hUiNPCs
in vivo, we transplanted these cells into
the striatum of newborn rats (n = 12
rats) (Fig. 5a) and analyzed the brains
of the animals 4 weeks after transplanta-
tion. We observed human nuclear antigen
(hNA)-stained cells that had survived and
migrated in the host brain (Fig. 5) and did
not observe teratoma formation in any
of the transplanted rats (Fig. 5a). Some
hNA-positive cells apparently remained
as NPCs, as they stained positively for
the NSC marker nestin (Fig. 5b and
Supplementary Fig. 3). We also observed
hNA-positive cells that expressed the
astrocyte marker GFAP and the neuronal
marker TUJ1 (Fig. 5b), which indicated
that the engrafted hUiNPCs could give
rise to both neurons and astrocytes in vivo.
A full characterization of the stability, dif-
ferentiation potential and function of these
cells in vivo will require further study.
We describe here the notable finding that
functional NPCs can be generated from
somatic cells with the same factors known
to be capable of reprogramming to pluripo-
tency when cells are cultured in specially
defined conditions. Recently, several
reports have shown that mouse fibroblasts
can be reprogrammed directly into neural
stem cells using virally delivered reprogramming factors35–38. In
contrast, our method uses episomal factors for rapid and efficient
derivation of integration-free NPCs from cells in human urine.
We showed that hUiNPCs arise at day 12–15, before the emer-
gence of iPSCs at day 24–28 (Fig. 1). Further, the colonies express
NSC markers, but we did not detect the expression of pluripotency
markers (Fig. 1d–i). However, on the basis of our current data,
which were obtained from mixed populations of cells, we can-
not rule out the possibility that some cells in the reprogramming
culture pass through a fully pluripotent state. Single-cell analysis
will help clarify this question39,40 and may also yield insight at the
molecular level into how different cell fate decisions can be trig-
gered in somatic cells by the same reprogramming factors.
We have applied our approach successfully to generate hUiN-
PCs from three individuals at ages 10, 25 and 37 years, with an
average reprogramming efficiency around 0.2% (Supplementary
Fig. 1). We envision that our protocols can be further applied
to HUCs isolated from patients with neural disorders such as
Parkinson’s disease, Alzheimer’s disease or other neurodegenera-
tive diseases. These patient-specific hUiNPCs should be useful for
modeling disease and for drug screening. hUiNPCs may in the
future also prove useful for cell therapy, such as for patients with
hNA GFAP hNA/GFAP/DAPI
hNA Nestin hNA/nestin/DAPI
Figure 5 | In vivo transplant of hUiNPCs. (a) Schematic shows the transplant location (striatum) of
hUiNPCs into the brain of newborn rats. Micrographs show immunostains for human nuclear-specific
antigen (hNA) and DAPI stain on a brain section at the transplant site. (b) Immunostains for the
indicated markers of brain sections at the transplant site 4 weeks after cells were transplanted.
Scale bars: 50 µm (a), 20 µm (b, left), 10 µm (b, right).
© 2013 Nature America, Inc. All rights reserved.
nAture methods | VOL.10 NO.1 | JANUARY 2013 | 89
spinal cord injury. Our feeder- and serum-free culture system
for generating these cells can enable this application. The overall
efficiency of ~0.2% for the generation of hUiNPCs from HUCs
is operationally adequate for current research, although future
improvements to efficiency could be made, as has been accom-
plished for iPSC generation41.
Methods and any associated references are available in the online
version of the paper.
Accession codes. The microarray data are available in the Gene
Expression Omnibus database under the accession number
Note: Supplementary information is available in the online version of the paper.
We thank M. Esterban for helpful suggestions, Z. Li for providing support in
the initial phase of this work and members of our labs for their kind help. This
work is supported by the Strategic Priority Research Program of the Chinese
Academy of Sciences (grant nos. XDA01020202 and XDA01020401); National
Basic Research Program of China, 973 Program of China (2012CB966503
and 2012CB966802); National S&T Major Special Project on Major New Drug
Innovation (2011ZX09102-010); and National Natural Science Foundation of
China (31200970 and 91213304). D.P. and G.P. are supported by the 100 Talents
Project of Chinese Academy of Sciences, China.
G.P., Lihui, W. and D.P. conceived hypotheses and designed the experiments.
Lihui, W. and W.H. performed the experiments and generated data in all figures.
In addition, Linli, W., D.Q., Y.X. and Z.S. performed experiments for Figure 1 and
supplementary Figure 2; H.S., W.W. and K.F.S. participated in experiments and
analysis for Figure 5; X.B. provided reagents and experimental assistance for
miR302–367; B.L. performed the experiments for Figure 1; and H.W. and J.H.
performed the experiments for Figure 4. G.P. and D.P. wrote the paper.
comPetinG FinAnciAl interests
The authors declare no competing financial interests.
Published online at http://www.nature.com/doifinder/10.1038/nmeth.2283.
reprints and permissions information is available online at http://www.nature.
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Isolation and culture of HUCs. Three donors were recruited for
urine samples with informed consent based on IRB approval (no.
GIBH-IRB02-2009002) at Guangzhou Institutes of Biomedicine
and Health (GIBH). The procedures and purposes for isolating
urine cells and generating stem cells were explained to the donors in
detail, and questions, if any, were answered in full. We then obtained
a formal signed consent form and collected a total of ~500 ml
of urine from each donor. The subsequent procedures for isolat-
ing human urine cells and generating stem cells were performed
as approved. The method for isolating urine cells was modified
from a previous established protocol19. Briefly, urine samples were
collected at the mid-stream from three individuals and centrifuged
to collect the exfoliated cells. The primary urine cells were then
processed and cultured in urine cell medium consisting of a 1:1
mixture of DMEM/F12 culture medium supplemented with 10%
of FBS (FBS, PAA), 0.1 mM non-essential amino acids (NEAA),
1 mM GlutaMAX (Life Technologies), 0.1 mM β-mercaptoethanol
and SingleQuot Kit CC-4127 REGM (Lonza).
hUiNPC generation. For reprogramming, an oriP/EBNA1-based
pCEP4 episomal vector containing the OCT4, SOX2, KLF4 and
SV40LT genes20 and a pCEP4 vector carrying the miR302–367
precursor21 were co-transfected into urine cells via nucleofection
(Amaxa Basic Nucleofector Kit for primary mammalian epithelial
cells with the T-013 program, Lonza). Transfected urine cells were
directly plated to Matrigel-coated six-well plates (1–3 × 105 cells
per well) in urine cell culture medium. On day 2 post-transfection,
the media were changed into reprogramming media mTeSR or 5i
(mTeSR supplemented with 5i 0.5 µM A83-01, 1 µM PD0325901,
3 µM CHIR99021, 0.5 µM thiazovivin and 0.2 µM DMH1).
Medium was changed every 2 d during the reprogramming.
Fifteen days after transfection, colonies were picked up in 5i and
transferred onto a new Matrigel plate. For further passaging, the
cells were dissociated to small clusters or single cells for suspen-
sion in flasks containing neural growth medium as neural spheres
(1:1 of DMEM/F12 supplemented with 1% N2 (Invitrogen) and
Neurobasal medium supplemented with 2% B27 (Invitrogen) sup-
plemented with 20 ng/ml bFGF and 20 ng/ml EGF).
Neural differentiation in vitro. hUiNPCs were cultured in neural
medium N2B27 containing EGF and bFGF. For pan-neuronal
differentiation, hUiNPC spheres were plated on Matrigel-coated
coverslips and cultured in neural N2B27 medium with the with-
drawal of EGF and bFGF and the addition of neurotrophic factors,
BDNF, GDNF, CNTF, IGF1 (all at 10 ng/ml, Peprotech) and 1 µM
cAMP to improve neuronal survival. hUiNPCs differentiated for
2 weeks were then examined for the expression of neuronal
markers and an astrocyte marker. To derive oligodendrocytes,
hUiNPCs were plated on poly-l-ornithine/laminin substrate
and cultured in DMEM/F12 supplemented with 1% N1 (Sigma),
biotin (100 ng/ml), PDGF-AA (20 ng/ml, Peprotech), NT3
(20 ng/ml, Peprotech), 1 µM cAMP and bFGF (10 ng/ml) for
1 week. Afterward, bFGF was replaced by IGF1 (10 ng/ml) for
another 3 weeks. All media were replenished every 2 d.
Quantitative real-time PCR. Total RNAs were extracted with
Trizol (Invitrogen). qRT-PCR was performed using a Thermal
Cycler Dice Real Time System and SYBR Premix EX Taq
(Takara). β-actin was used for qRT-PCR normalization, and all
data were measured in triplicate. Primer sequences are listed in
Supplementary Table 3.
Analysis of gene integration by PCR. Genomic DNAs were
extracted using the Wizard Genomic DNA Purification Kit
(Promega) for PCR analysis using primers (Supplementary
Table 3) that specifically amplify the exogenous transgenes
Immunocytochemistry. Cells were fixed in 4% paraformalde-
hyde dissolved in 0.1 M phosphate buffer (PB) for 20 min. After
several washes with 0.01 M phosphate-buffered saline (PBS), the
cultures were incubated with the primary antibodies in PBS plus
1% BSA, 10% normal goat serum and 0.3% Triton X-100 over-
night at 4 °C. The primary antibodies are listed in Supplementary
Table 4. Primary antibodies were visualized with species-specific
secondary antibody conjugated to the fluorescent labels Alexa
568 or 488 (1:400; Invitrogen). Cells were mounted in anti-fade
medium containing 4′,6-diamidino-2-phenylindole (Sigma) to
counterstain nuclei. At least 500 DAPI-positive cells from five
randomly selected fields were counted to calculate the fraction of
each lineage. Results are mean ± s.e.m. of data from three experi-
ments unless stated otherwise in legends. Cells were imaged on
a Zeiss Axio Imager A1 microscope or a Leica TCS SP2 Spectral
Electrophysiological analysis. Whole-cell patch-clamp record-
ing techniques were used to study the physiological properties of
hUiNPC-derived neurons in culture with borosilicate glass pipettes
(resistance 5–10 MΩ) using an Axopatch 200B amplifier (Axon
Instruments for Molecular Devices). Both the spontaneous post-
synaptic current and current response to exogenous focal applica-
tion of glutamate and GABA were recorded. Pressure ejection was
used to puff 1 mM glutamate (10 p.s.i., 100 ms) and 1 mM GABA
(10 p.s.i., 100 ms), and the holding voltages were −80 mV and 0 mV,
respectively. The patch pipette internal solution contained (in mM):
136.5 K-gluconate, 0.2 EGTA, 10 HEPES, 9 NaCl, 17.5 KCl, 4 Mg-
ATP and 0.3 Na-GTP, adjusted with KOH to pH 7.2, 285 osmol/l.
For the recording of voltage-gated currents and action potentials,
we used the following composition of the intracellular solution
(in mM): 140 potassium methanesulfonate, 10 HEPES, 5 NaCl,
1 CaCl2, 0.2 EGTA, 3 ATP-Na2, 0.4 GTP-Na2, pH 7.3 (adjusted
with KOH). The external solution contained (in mM): 120 NaCl,
1.2 KH2PO4, 1.9 KCl, 26 NaHCO3, 2.2 CaCl2, 1.4 MgSO4, 10 d-
glucose, 7.5 HEPES (pH with NaOH to 7.3). The bath solution
was equilibrated with 95% O2 and 5% CO2 before use. Resting
potentials were maintained at about −60 mV. Signals were sampled
at 10 kHz using a Digidata1440A analog-to-digital converter and
acquired and stored on a computer hard drive using pClamp10
software. Data were analyzed using pClamp10 (Clampfit).
Karyotype analysis. hUiNPCs were used for karyotype analysis as
described42. Cells were grown in 10-cm plates, and demecolcine
(Dahui Biotech) was added to a final concentration of 50 µg/ml
for 40 min. Cells were then trypsinized, pelleted by centrifuga-
tion at 200g for 5 min, resuspended in 8 ml of 0.075 M KCl and
incubated for 20 min at 37 °C. Fixative solution composed of one
part acetic acid and three parts methanol was added to a final
© 2013 Nature America, Inc. All rights reserved.
doi:10.1038/nmeth.2283 Download full-text
volume of 10 ml, mixed gently and incubated for 10 min at 37 °C.
After further centrifugation, the supernatant was removed, and
ice-cold fixative solution composed of one part acetic acid and
three parts methanol was added to a final volume of 10 ml. Cells
were dropped on a cold slide and incubated at 75 °C for 3 h. Belts
were treated with trypsin and colorant, and metaphase states were
analyzed on an Olympus BX51 microscope.
Whole-genome expression analysis. Total RNAs were extracted
from HUCs, hUiNPCs and Uri_iPSCs and quantified by the
NanoDrop ND-1000. RNA integrity was assessed by standard
denaturing agarose gel electrophoresis. About 5 µg total RNA was
used for the reverse transcription with Invitrogen’s Superscript
Double-Stranded cDNA Synthesis Kit. Labeled cDNA was syn-
thesized by in vitro transcription using NimbleGen one-color
DNA labeling kit. Array hybridization was performed with the
NimbleGen Hybridization System and followed by a wash with
the NimbleGen wash buffer kit. Arrays were scanned with the
Axon GenePix 4000B microarray scanner. Data were analyzed
with NimbleScan software (v.2.5).
In vivo transplantation and tissue processing. The protocol used
for newborn rat was reviewed and approved by the animal care
committee at GIBH. For hUiNPCs transplantation, 12 newborn
Sprague Dawley rats were used in the study. hUiNPC neuro-
spheres were gently dissociated into single cells with Accutase
(Millipore) and resuspended in N2 medium supplemented with
10 µg/ml BDNF, 10 µg/ml GDNF, 10 µg/ml CNTF and 10 µg/ml
IGF1 at a concentration of 1.0 × 105 cells/µl and placed on ice
for the duration of the grafting session. One microliter of cell
suspension was slowly injected into each of the striatum of cryo-
anesthetized newborn rats through a Hamilton syringe with the
sharpened tip. At 2 and 4 weeks following transplantation, rats
were anesthetized and perfused intracardially with 0.01 M PBS,
pH 7.4, followed by 100–200 ml of fixative solution containing
4% paraformaldehyde in 0.1 M PBS, pH 7.4. The brains were
harvested and post-fixed in fresh fixative solution overnight and
subsequently placed in 30% sucrose, 0.1 M PBS at 4 °C for 2–3 d.
The samples were then cut into 25-µm cross-sections on a micro-
tome. The serial sections were collected in 0.01 M PBS and kept
at 4 °C for further study.
Immunohistochemistry of brain sections. Donor cells were
identified by an antibody to a human-specific nuclear antigen
(hNA). Immunopositive cells were double labeled with antibodies
to β III tubulin (TUJ1), GFAP and nestin (Supplementary
Table 4). Species-specific fluorescence-conjugated secondary
antibodies conjugated to Alexa 488 (1:400; Molecular Probes) were
applied for 2 h at 20 °C. Sections were then counterstained with
4′,6-diamidino-2-phenylindole to stain nuclei and coverslipped
with anti-fade mounting medium (FluorSave; Calbiochem).
A Zeiss 710 NLO spectral confocal microscope was used for all
42. Esteban, M.A. et al. Generation of induced pluripotent stem cell lines
from Tibetan miniature pig. J. Biol. Chem. 284, 17634–17640 (2009).
© 2013 Nature America, Inc. All rights reserved.