Gingival Fibroblasts as a Promising Source of Induced
Pluripotent Stem Cells
Hiroshi Egusa1*, Keisuke Okita2, Hiroki Kayashima1, Guannan Yu1, Sho Fukuyasu1, Makio Saeki3, Takuya
Matsumoto4, Shinya Yamanaka2, Hirofumi Yatani1
1Department of Fixed Prosthodontics, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan, 2Center for iPS Cell Research and Application (CiRA), Kyoto
University, Kyoto, Japan, 3Department of Pharmacology, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan, 4Department of Oro-Maxillofacial
Regeneration, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan
Background: Induced pluripotent stem (iPS) cells efficiently generated from accessible tissues have the potential for clinical
applications. Oral gingiva, which is often resected during general dental treatments and treated as biomedical waste, is an
easily obtainable tissue, and cells can be isolated from patients with minimal discomfort.
Methodology/Principal Findings: We herein demonstrate iPS cell generation from adult wild-type mouse gingival fibroblasts
(GFs) via introduction of four factors (Oct3/4, Sox2, Klf4 and c-Myc; GF-iPS-4F cells) or three factors (the same as GF-iPS-4F cells,
but without the c-Myc oncogene; GF-iPS-3F cells) without drug selection. iPS cells were also generated from primary human
gingival fibroblasts via four-factor transduction. These cells exhibited the morphology and growth properties of embryonic
stem (ES) cells and expressed ES cell marker genes, with a decreased CpG methylation ratio in promoter regions of Nanog and
Oct3/4. Additionally, teratoma formation assays showed ES cell-like derivation of cells and tissues representative of all three
germ layers. In comparison to mouse GF-iPS-4F cells, GF-iPS-3F cells showed consistently more ES cell-like characteristics in
terms of DNA methylation status and gene expression, although the reprogramming process was substantially delayed and
the overall efficiency was also reduced. When transplanted into blastocysts, GF-iPS-3F cells gave rise to chimeras and
contributed to the development of the germline. Notably, the four-factor reprogramming efficiency of mouse GFs was more
than 7-fold higher than that of fibroblasts from tail-tips, possibly because of their high proliferative capacity.
Conclusions/Significance: These results suggest that GFs from the easily obtainable gingival tissues can be readily
reprogrammed into iPS cells, thus making them a promising cell source for investigating the basis of cellular
reprogramming and pluripotency for future clinical applications. In addition, high-quality iPS cells were generated from
mouse GFs without Myc transduction or a specific system for reprogrammed cell selection.
Citation: Egusa H, Okita K, Kayashima H, Yu G, Fukuyasu S, et al. (2010) Gingival Fibroblasts as a Promising Source of Induced Pluripotent Stem Cells. PLoS
ONE 5(9): e12743. doi:10.1371/journal.pone.0012743
Editor: Mike Klymkowsky, University of Colorado, Boulder, United States of America
Received May 25, 2010; Accepted August 23, 2010; Published September 14, 2010
Copyright: ? 2010 Egusa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This investigation was supported by a Grant-in-Aid for Young Scientists (A: 22689049, H.E.) from the Ministry of Education, Culture, Sports, Science and
Technology, Japan (http://www.mext.go.jp/english/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Direct reprogramming of somatic cells into induced pluripotent
stem (iPS) cells by forced expression of a small number of defined
factors (e.g., Oct3/4, Sox2, Klf4 and c-Myc) has great potential for
tissue-specific regenerative therapies, avoiding ethical issues sur-
rounding the use of embryonic stem (ES) cells and problems with
have been generated from a variety of mammalian species including
mice , monkeys , dogs , pigs  and humans [5–8]. Mouse
iPS cells have been generated from cells of all three embryonic germ
layers, including mesodermal fibroblasts  and B lymphocytes ,
endodermal hepatocytes , gastric epithelial cells  and
pancreatic cells , and ectodermal keratinocytes .
The reprogramming process appears to be highly inefficient and
is likely affected by many factors, including the age, type and
origin of the cells used. Recently, a ‘‘stochastic model’’ predicted
that most or all cells are competent for reprogramming .
However, the kinetics of reprogramming appear to vary when
target populations from different tissues are used. Mouse
hepatocytes and gastric epithelial cells appear to be more easily
reprogrammed and require less retroviral integration than
fibroblasts . Dermal papilla cells, which endogenously express
high levels of Sox2 and c-Myc, have been reported to be
reprogrammed more efficiently than skin and embryonic fibro-
blasts . Although the mechanisms underlying differences in
reprogramming efficiency are not yet clear, some cell types might
be more easily reprogrammed using specific exogenous factors
than others. Importantly, the use of cell types with a high
reprogramming efficiency could reduce the number of transduced
factors needed, decreasing the chance of retroviral insertional
mutagenesis and increasing the likelihood of ultimately replacing
the remaining factors with small molecules . For future clinical
application, it is therefore crucial to identify cell types that can be
PLoS ONE | www.plosone.org1 September 2010 | Volume 5 | Issue 9 | e12743
more easily reprogrammed; ideally, these cells should also be
derived from a feasible and accessible source tissue to permit
From the standpoint of accessibility, the oral mucosa is one of the
most convenient tissues for biopsy. Indeed, gingival tissues are
routinely resected during general dental treatments, such as tooth
extraction, periodontal surgery and dental implantation, and are
generally treated as biomedical waste. Interestingly, clinical
observations and experimental animal studies consistently indicate
that wound healing in the oral mucosa has better outcomes than in
the skin [16,17], although the healing process and sequence are
similar. Therefore, it has been postulated that oral mucosal cells
possess distinctive characteristics promoting accelerated wound
closure [18,19]. The oral mucosa is composed of a thin keratinocyte
layer with underlying connective tissue. Gingival fibroblasts (GFs),
which are the major constituents of the gingival connective tissue,
play an important role in oral wound healing, and are phenotyp-
ically and functionally different from skin fibroblasts [18–20]. The
establishment of primary GF cultures is relatively simple because
GFs adhere and spread well on culture plates, and proliferate well
without requiring specific culture conditions .
Stem cell-based therapies using bone marrow aspirates have
been successfully used in dentistry to regenerate maxillary/
mandibular bones and periodontal tissue [22–25]; however, bone
marrow aspiration from iliac crest is not an easy operation for
dentists because of limitations of the dental license and specialty.
Efficient reprogramming of GFs could make the gingiva an ideal
source for iPS cells that could be used for autologous cell therapy
and drug screening applications, especially in dentistry. In
addition, gingiva normally discarded as biomedical waste would
be an ideal source of donor cells from healthy volunteers to
establish an iPS cell bank for a wide range of medical applications.
We hypothesized that iPS cells could be produced from fibroblasts
derived from gingival tissue. Such cells could be used as a valuable
experimental tool for investigating the basis of cellular reprogram-
ming and pluripotency, with possible future clinical applications.
Generation of Mouse GF-Derived iPS Cells
Mouse GF cultures were established from either palatal mucosal
tissues(pGFs) ormandibulartissues (mGFs)obtained from adultmale
mice (Fig. 1A, 1B). After four-factor transduction, several small-cell
colonies were detected in pGFs and mGFs cultures (5 passages) under
phase contrast microscopy within 14 days (10 days on feeders). More
than 100 colonies were obtained ineach 60-mm dish of pGF (day 17)
and mGF (day 21) cultures (Fig. 1C). GFP expression in the cultures
was monitored under fluorescence microscopy because the correct
generation of iPS cells requires the silencing of the retroviral
transgenes . Ten colonies from each dish (total 20 colonies)
showing lower GFP expression were picked up for further expansion
in ES medium. After expansion, 5 clones (3 from pGF cultures and 2
from mGF cultures: Fig. 1E) displaying proliferation and morphol-
ogy characteristic of ES cells (Fig. 1G) were selected.
Figure 1. Generation of mouse GF-derived iPS cells. (A) Palatal (upper inset) and mandibular (lower inset) gingival tissues of adult mice were
extracted for establishment of primary GFs. (B) Fibroblasts (arrow) and epithelial cells (arrowhead) migrated out of the palatal gingival tissue (asterisk).
Scale bar; 60 mm. (C) The morphology of a colony derived from mGFs 19 days after transduction of the four factors. Scale bar; 200 mm. Inset: Colonies
in a 60-mm dish after staining with crystal violet (CV) on day 21. (D) The morphology of a colony derived from mGFs 49 days after transduction of the
three factors. Scale bar; 300 mm. Inset: Colonies in a 100-mm dish after staining with CV on day 50. (E–G) Morphology of (E) mGF-iPS-4F-1 cells (Scale
bar; 200 mm), (F) mGF-iPS-3F-1 cells (Scale bar; 60 mm) and (G) mouse ES cell line (Scale bar; 60 mm). (H) Morphology of a pGF-iPS-4F-1 colony stained
with methylene blue. Scale bar; 50 mm (I) TEM photograph of a pGF-iPS-4F-1 cell colony showing tight and continual cell membrane contacts
(arrows), large nucleoi (asterisks) and scant cytoplasm. Scale bar; 5 mm.
iPS Cells from Gingival Tissue
PLoS ONE | www.plosone.org2 September 2010 | Volume 5 | Issue 9 | e12743
For three-factor transduction, a small number (approximately
50 in a 100-mm dish) of ES cell-like colonies emerged in both the
pGF and mGF cultures with few background cells within 50 days
after transduction (Fig. 1D). Twenty colonies in the mGF culture
were then mechanically picked for expansion. Most colonies were
expandable, and ten colonies were finally selected for clonal iPS
cell cultures (Fig. 1F).
The colonies in the selected clone cultures grew in a tight and
round shape (Fig. 1H). Transmission electron microscopy (TEM)
revealed tight and continual cell membrane contacts, and showed
a large nuclear to cytoplasmic ratio and prominent nucleoli
(Fig. 1I), representing the typical ultrastructure of mouse ES and
iPS cells . We refer to these ES-like cells as pGF-iPS-4F-1, -2, -
3 and mGF-iPS-4F-1, -2 cells (four-factor transduction), and mGF-
iPS-3F-1 to -10 cells (three-factor transduction).
Characteristics of Mouse GF-iPS Cells
All generated GF-iPS-3F and -4F cell colonies showed robust
staining for alkaline phosphatase (ALP) (Fig. 2A). To confirm ES
cell-like characteristics, the expression of undifferentiated ES
marker genes was determined by reverse transcription-polymerase
chain reaction (RT-PCR). All GF-iPS-4F cell clones expressed
various markers for undifferentiated ES cells, including Nanog,
ERas, Rex1 (Zfp42), and Oct3/4 (endogenous), to various extents
but at lower levels than in mouse ES cells (Fig. 2C). All GF-iPS-3F
cell clones expressed these ES cell marker genes at levels
comparable to those in ES cells (Fig. 2D). In contrast, these
genes were not expressed in parental GFs and SNLP feeder cells.
Bisulfite genomic sequencing was performed to evaluate the
methylation status of cytosine guanine dinucleotides (CpGs) in the
promoter regions of the pluripotency-associated genes, Nanog and
Oct3/4. The methylation analysis revealed the percentage
methylation of CpGs in the Nanog promoter regions of mGF,
GF-iPS-4F (average of 2 clones), GF-iPS-3F (average of 2 clones)
and ES cells to be 73.3%, 30%, 0% and 3.3%, respectively. The
respective percentages for Oct3/4 in mGF, GF-iPS-4F (average of
2 clones), GF-iPS-3F (average of 2 clones) and ES cells were
85.3%, 30.7%, 4.7% and 1.3% (Fig. 2B). These results suggest
that the highly methylated CpGs in Nanog and Oct3/4 promoters
of parental GF cells were demethylated, and that these promoters
became active during iPS cell induction.
Differentiation of Mouse GF-iPS Cells
After 3 days of floating cultivation, the GF-iPS cells and mouse
ES cells formed ball-shaped structures and embryoid bodies (EBs)
(Fig. 3A–C). Ten days after the expansion of EB-like structures
Figure 2. Characteristics of the mouse GF-iPS cells. (A) GF-iPS cell colonies (upper panels: pGF-iPS-4F-1, lower panels: mGF-iPS-3F-4) positively
stained for ALP. Scale bar; 30 mm. (B) Bisulfite sequencing of the Nanog and Oct3/4 promoters revealed that CpGs in the parental mGFs were
converted to a demethylated state in the GF-iPS cells induced by the three or four factors, resulting in a methylation pattern similar to that of mouse
ES cells. The numbers in the panel indicate CpG loci respective to the transcription start site (TSS) of the genes (blue: untranslated region, yellow:
translated region). (C and D) RT-PCR analysis of ES cell marker genes (Nanog, ERas, Rex1) and endogenous Oct3/4, Sox2, Klf4 and c-Myc genes in GF-
iPS-4F clones (C) or GF-iPS-3F clones (D), mouse ES cells, parental mGFs and SNL feeder cells. GAPDH was used as a loading control.
iPS Cells from Gingival Tissue
PLoS ONE | www.plosone.org3 September 2010 | Volume 5 | Issue 9 | e12743
from pGF-iPS-4F cells on gelatin-coated plates, the attached cells
showed various morphologies (Fig. 3D), resembling neuronal
cells, cobblestone-like cells, and epithelial cells (Fig. 3E–G). By
twenty days after expansion, some clumps of cells had started
pulsating, suggesting that they had differentiated into cardiomy-
ocytes (Fig. 3H and Movie S1). Thirty days after expansion, von
Kossa staining revealed osteogenic cells with mineralized nodule
formation (Fig. 3I). Immunocytochemistry revealed positive
staining for b-III tubulin (a marker of ectoderm), a1-fetoprotein
(AFP) (endoderm) and a-smooth muscle actin (a-SMA) (meso-
derm) in pGF-iPS-4F-1 (Fig. 3J–L) and mGF-iPS-3F-2 cell
cultures (Fig. 3M–O). Other clones of GF-iPS-4F and -3F cells
also showed positive staining for these proteins. These data
demonstrate that GF-iPS-4F and GF-iPS-3F cells could differen-
tiate into cells from all three germ layers in vitro.
Teratoma Formation by Mouse GF-Derived iPS Cells
Apparent tumor formation was observed in mGF-iPS-3F- and
pGF-iPS-4F-injected mice at weeks seven and ten after injection,
respectively (Fig. 4A). Extracted tumors containing GF-iPS-3F
Figure 3. In vitro differentiation of mouse GF-iPS cells. (A–C) In vitro EB formation of (A) mouse ES cells, (B) pGF-iPS-4F-2 cells and (C) mGF-iPS-
3F-9 cells after 3 days in floating culture. Scale bars; 60 mm. (D) Morphology of pGF-iPS-4F-3 cells cultured on gelatin-coated plates without feeder
cells for 10 days. Scale bar; 200 mm. Attached cells showed various morphologies, such as those resembling (E) neural cells (arrowheads: neurite-like
outgrowth), (F) cobblestone-like cells and (G) epithelial cells. Scale bars; 100 mm. (H) Beating myocardial cells (arrow) in the pGF-iPS-4F-3 culture after
20 days of expansion. Scale bar; 100 mm. (I) Osteogenic cells with mineralized nodule formation (asterisks) in the pGF-iPS-4F-1 cell culture detected by
von Kossa staining after 30 days of expansion. Scale bar; 30 mm. (J–L) pGF-iPS-4F-1 cells and (M–O) mGF-iPS-3F-2 cells were specifically directed to
differentiate into cells from all three germ layers at days three and ten after expansion, respectively. (J and M) b-III tubulin-positive ectodermal neural
cells (arrowheads: neurite-like outgrowth). (K and N) AFP-positive endodermal hepatic cells. (L and O) a-SMA-positive mesodermal smooth muscle
cells. Nuclei are stained with DAPI. Scale bars; 200 mm.
iPS Cells from Gingival Tissue
PLoS ONE | www.plosone.org4 September 2010 | Volume 5 | Issue 9 | e12743
cells were larger than those containing GF-iPS-4F cells (Fig. 4A).
Histological examinations showed that the tumors contained
various tissues (Fig. 4B–G), including keratin-containing epider-
mal tissues (ectoderm), neural tissues (ectoderm), striated muscle
(mesoderm), cartilage (mesoderm) and gut-like epithelial tissues
(endoderm). These data demonstrate that the GF-iPS-3F and -4F
cells generated in our study were capable of differentiating into
tissues representative of the three germ layers in vivo.
Germline Chimeras from GF-iPS-3F Cells
GF-iPS-3F cells (C57BL/6J black mouse-derived) were micro-
injected into Jcl:MCH white mouse-derived blastocysts, which
were then transplanted into the uteri of pseudo-pregnant Jcl:ICR
white mice. This yielded 13 out of 52 (25%), 9 out of 22 (40.9%),
21 out of 34 (61.8%), 19 out of 22 (86.4%) and 16 out of 27
(59.3%) adult chimeric mice from mGF-iPS-3F-1, -2 -3, -4 and -6
cells, respectively, as determined by the coat color (Fig. 4H).
Chimeric mice from the mGF-iPS-3F-3 clone were then mated
with Jcl:MCH white females to verify germline transmission, and
one pup obtained from the mating was derived from mGF-iPS-3F
cells, as revealed by coat color in black (Fig. 4I). Taken together,
these data demonstrate that GF-iPS-3F cells possess in vivo
developmental potential comparable to that of ES cells.
Reprogramming Efficiency of Mouse GF-Derived iPS Cells
To compare the reprogramming efficiency between mouse GFs
and tail-tip fibroblasts (TTFs), pGF, mGF and TTF cultures were
established from the same individual mouse. The reprogramming
efficiency of the pGFs, mGFs and TTFs at passage 4 was 1.2%,
0.6% and 0.1%, respectively (Fig. 5A). During the experimental
period (4–10 passages), the reprogramming efficiency was the
highest in the pGFs, followed by the mGFs, and then the TTFs. No
EScell-likecolonieshademerged from the TTFculturestransduced
after 7 passages, whereas pGFs transduced after 10 passages were
still amendable to reprogramming, at a rate of 0.6%.
Cell proliferation assays performed on cells at passage 5 showed
that the number of pGFs and mGFs on day 8 was significantly
higher than that of TTFs (P,0.01) (Fig. 5B). The proliferative
capacity of pGFs was maintained for at least 20 passages, while
that of TTFs decreased significantly after 10 passages (data not
shown). Real-time RT-PCR showed that the expression level of
telomerase reverse transcriptase (Tert) mRNA in pGFs and mGFs
was maintained for 6 passages, while that in TTFs decreased as the
passage number increased (Fig. 5C). Similar expression levels
were detected for c-Myc, Klf4, Sox2, p53 and p21 genes among
the pGFs, mGFs and TTFs, although the expression levels of klf4,
p53 and p21 in GFs were slightly higher than in TTFs at passage 6
(Fig. 5C). Expression of Oct3/4 mRNA was not detected in the
primary GFs and TTFs.
Induction of Human Gingival Fibroblast (hGF)-Derived
When gingival tissues from the patient (Fig. 6A) were cultured
on a gelatin-coated dish, fibroblasts and epithelial cells proliferated
Figure 4. Teratoma formation and germline chimeras from mouse GF-iPS cells. (A) Transplantation of mGF-iPS-3F-1 and pGF-iPS-4F-1 cells into
mouse testes resulted in apparent teratoma formation (dotted circle: tumor formation by pGF-iPS-4F-1 cell transplantation at week 10). Insets: Extracted
teratomas from mice transplanted with pGF-iPS-4F-1 (upper) or mGF-iPS-3F-1 (lower) cells. Scale bars; 1 cm. (B–G) H&E staining of teratoma sections
showed differentiation of mGF-iPS-3F-1 cells into various tissues from all three germ layers, including keratin-containing epidermal tissues (B: ectoderm),
neural tissues (C: ectoderm), striated muscle (D: mesoderm), cartilage (E: mesoderm), adipose tissues (F: mesoderm) and gut-like epithelial tissues (G:
endoderm). Scale bars; 100 mm. (H) Chimeric micegenerated by injecting the black mouse-derived mGF-iPS-3F-1 cells into white mouse-derived blastocyst
embryos. (I) An adult old chimeric male mouse generated from mGF-iPS-3F-3 cells (arrow head) was mated with Jcl:MCH white female mice and achieved
germline transmission, as indicated by coat color in black (arrow). Inset: A newborn mouse generated via germline transmission (arrow).
iPS Cells from Gingival Tissue
PLoS ONE | www.plosone.org5 September 2010 | Volume 5 | Issue 9 | e12743
out of the tissues (Fig. 6B). Homogeneous fibroblast culture was
established in serum- and calcium- containing media (Fig. 6C).
(Fig. 6D); these colonies were picked mechanically and five clone
cultures (hGF-iPS-547A-1 to -5) were established (Fig. 6E, 6F).
The colonies could be expanded and displayed the same
morphology and growth characteristics as colonies of iPS cells
obtained from human dermal fibroblasts (Fig. 6G) and human ES
cells (Fig. 6H, 6I).
Characteristics and Differentiation of the hGF-iPS Cells
The colonies of all generated hGF-iPS cell clones stained
positively for ALP activity (Fig. 7A). RT-PCR analysis showed
that all hGF-iPS cell clones expressed ES cell specific genes,
including NANOG, REX1, TERT, endogenous OCT3/4 and
SOX2, at levels comparable to those in H9 and KhES3 human ES
cell lines (Fig. 7B). In contrast, these genes were not expressed in
parental hGFs or SNLP feeder cells. Bisulfite genomic sequencing
revealed the percentage methylation of CpGs in the NANOG
promoter regions of parental hGF, hGF-iPS (average of 2 clones)
and H9 human ES cells to be 32.8%, 3.2% and 6.3%, respectively.
The respective percentages for OCT3/4 in hGF, hGF-iPS
(average of 2 clones) and H9 human ES cells were 64.6%, 5.2%
and 7.3% (Fig. 7C).
Cloned hGF-iPS-547A-2 and -3 cells formed teratomas after
injection into SCID mouse testes. Histological examination of the
teratomas at week nine after injection revealed representative tissues
originating from the three embryonic germ layers, including
ectodermal neural tissues, mesodermal cartilage and endodermal
the hGF-iPS cells generated in this study had the capacity to
differentiate into tissues representative of the three germ layers in vivo.
Tissue engineering become a new frontier in dentistry for,
among other applications, regeneration of missing oral tissues .
However, engineering applications for tooth, jawbone, temporo-
mandibular joint cartilage and periodontal tissues await the
Figure 5. Reprogramming efficiency of mouse GF- and TTF-derived iPS cells. (A) Fibroblasts established from palatal (pGFs) and mandibular
(mGFs) gingival tissues as well as tail-tips (TTFs) of the same mouse were simultaneously induced into iPS cells by four-factor transduction. Left
panels: ES cell-like colony formation from each cell type at passage 4 was determined by ALP staining (Scale bars; 200 mm). Right panel: The
reprogramming efficiency at passages 4, 7 and 10 was calculated as the number of ALP-stained iPS colonies formed per number of infected cells
seeded. The data represent the mean values 6 s.d. (n=4). Significant differences (*P,0.01: ANOVA with Dunnett’s correction for multiple
comparisons) were evaluated with respect to the values for TTF at each passage number of cultures. (B) A cell proliferation assay was performed on
the pGF, mGF and TTF cultures at passage 5. The data represent the mean values 6 s.d. (n=3). Significant differences (*P,0.01: ANOVA with
Dunnett’s correction for multiple comparisons) were evaluated with respect to the values for TTF at each time point. (C) Real-time RT-PCR analysis for
endogenous expression of c-Myc, Klf4, Sox2, p53, p21 and Tert genes in pGFs, mGFs and TTFs at passages 4 and 6 (left panel). The expression level of
Tert mRNA in pGFs was maintained for 6 passages, while that in TTFs decreased as the passage number increased (right panel). Expression of GAPDH
was used as an internal control. The data represent the relative mRNA expression levels with respect to the expression levels of each gene in TTFs at
iPS Cells from Gingival Tissue
PLoS ONE | www.plosone.org6 September 2010 | Volume 5 | Issue 9 | e12743
establishment of a stem cell source that allows easy collection by
dentists . In view of the potential clinical applications of iPS
cells, various types of discarded or easily obtainable normal human
tissues, especially those that can be obtained with minimal patient
discomfort such as peripheral blood , should be considered as
potential sources of iPS cells. From a practical standpoint, the
complicated cell isolation process, low numbers of isolated cells
and slow proliferation necessitate a long-term ex vivo expansion
step for obtaining sufficient cells for iPS induction. Such a step is
costly, time-consuming, and increases the risk of cell contamina-
tion and loss.
Several sources have shown more efficient iPS cell generation,
such as human keratinocytes from hair follicles or epidermal
biopsies  as well as mesenchymal stem cells of dental origin
from dental pulp [32,33] and impacted third molars [33,34].
However, there are several practical limitations to using these cells
for tissue engineering. For example, expansion of keratinocytes
requires serum-free low-calcium medium, which is relatively
costly, to prevent terminal differentiation. Isolation of the dental
stem cells may not be sufficiently convenient to allow easy
harvesting whenever the cells are needed because it requires tooth
or pulp extraction surgery, and the missing tissues can not
regenerate. In addition, culture of these non-terminally differen-
tiated cells requires a high level of skill to adequately maintain
cellular homogeneity. Similar limitations apply to many other
possible sources of iPS cells. An ideal autologous source should
thus allow easy collection of a large number of cells that can be
grown in a simple culture system, and that can quickly be cultured
to quantities sufficient to obviate extensive and long-term
expansion. Oral gingival tissue may represent such a source. In
this study, GFs from both mouse and human gingival tissues were
The mandibular mucosa of a 10-week-old mouse is too small to
extract only gingival tissues. Therefore, muscle and bone tissues
around the mandibular gingival tissues were carefully removed,
and mGFs were obtained from the outgrowth of fibroblastic cells
from the remaining tissues. This technical limitation may have
resulted in the possible contamination of mGFs with some
myoblasts or bone marrow stromal fibroblasts. On the other
hand, the mouse palatal mucosa was easily extracted en bloc
without any contamination by surrounding tissues (Fig. 1A).
Therefore, the pGFs used in this study may have been more
homogeneous than the mGFs. Nevertheless, both types of adult
mouse primary GFs proliferated well and could be successfully
reprogrammed into iPS cells that differentiated into cells and
tissues representing all three germ layers in vitro and in vivo.
All reprogramming approaches investigated to date seem to
involve epigenomic modification. GF-iPS-3F showed a greater
decrease in the CpG methylation ratio in the promoter regions of
Nanog and Oct3/4 in comparison to GF-iPS-4F cells that resulted
in a methylation pattern similar to that of mouse ES cells.
Nakagawa et al.  reported that the omission of c-Myc
transduction resulted in the generation of high-quality iPS cells
from mouse TTFs, in which Nanog is strongly activated and the
retroviruses are silenced. Consistently, our results showed that GF-
iPS-3F cell clones highly expressed ES cell marker genes, including
Nanog and endogenous Oct3/4. On the other hand, GF-iPS-4F
cell clones expressed these genes at lower levels than in mouse ES
Figure 6. Generation of human GF-derived iPS cells. (A) Resection of gingival tissue from the adult patient during dental implant surgery. The
resected gingival tissue (inset: scale bar; 1 mm) is generally treated as biomedical waste. (B) Fibroblasts (arrow) and epithelial cells (arrowhead)
migrated out of the human gingival tissue (asterisk). Scale bar; 100 mm. (C) The morphology of established hGFs (Passage 4). Scale bar; 60 mm. (D) The
morphology of a colony derived from hGFs 26 days after transduction of the four factors. Scale bar; 500 mm. (E–I) Morphology of (E and F) clonal hGF-
derived iPS cells (clone 547A-1), (G) human dermal fibroblast-derived iPS cells (DF-iPS cells) and (H and I) human KhES-3 ES cells. Scale bars; 500 mm
for E, G and H, and 50 mm for F and I.
iPS Cells from Gingival Tissue
PLoS ONE | www.plosone.org7 September 2010 | Volume 5 | Issue 9 | e12743
cells and showed partial DNA methylation in restricted areas of
the promoters. These results may in part be due to incomplete
reprogramming effects of four-factor transduction during GF-iPS-
4F cell induction in our system, which did not utilize a specific
system for reprogrammed cell selection (e.g., drug selection and
mice genetically modified for Nanog expression ). Because
incompletely reprogrammed GF-iPS-4F cells at least possess
multipotency, they might be used in some tissue regenerative
approaches; however, concerns remain that reactivation of the c-
Myc oncogene in iPS cells could increase tumorigenicity, thereby
hindering potential clinical applications .
c-Myc has recently been shown to be dispensable for direct
reprogramming [35,37]. It is conceivable that one major function
of c-Myc is to enhance proliferation, thereby accelerating the
reprogramming process, possibly by increasing the speed of
stochastic events that lead to the formation of iPS cells .
Consistently, the three factors without c-Myc were able to initiate
a slower reprogramming process that was sufficient to fully
reprogram mouse GFs after a longer time period. Indeed, GF-iPS-
3F cells demonstrated almost complete DNA demethylation in the
promoter regions of Nanog and Oct3/4. In addition to the specific
induction, the long reprogramming time course in GF-iPS-3F cells
may be responsible for the low level of methylation in the
promoters. All five tested GF-iPS-3F clones readily produced
viable chimeric newborn and adult mice. Moreover, mGF-iPS-3F-
3 cells contributed to germline transmission, which definitively
demonstrates that they were pluripotent and functionally indistin-
guishable from ES cells. So far, 4 out of the 78 mGF-iPS-3F cell
chimeras (5%) produced in the study died within six months;
however, apparent tumors were not observed in the dead mice.
These results suggest that high-quality iPS cells can be generated
from adult mouse GFs by transduction of the three factors (Oct3/
4, Sox2 and Klf4) without any specific system for the selection of
reprogrammed cells. However, because Klf4, the remaining
oncogenic factor, or insertional mutagenesis due to retroviral
transduction itself might also cause tumor formation, it will be
important to investigate the possibility of using recombinant
proteins (and small molecules) to reduce the number of genetically
transduced factors required for iPS cell induction, or even to
entirely obviate the need for viral gene delivery.
TTFs were the first type of adult cells to be reprogrammed into
iPS cells . Since then, other adult cell types with the potential
for easier reprogramming have been tested [10,14]. Our results
show that pGFs can be reprogrammed to pluripotency at least 7-
fold more efficiently than TTFs at the same passage number
derived from the same mouse. In addition, GFs, especially pGFs,
maintained their high reprogramming efficiency for at least ten
passages. On the other hand, no-ES cell-like colonies emerged
from TTFs transduced after 7 passages, possibly due to replicative
senescence . The lower reprogramming efficiency of mGFs
compared to pGFs in our system may have been due to the
establishment of a heterogeneous cell population in the mGFs.
It was initially speculated that the high efficiency of iPS cell
generation from GFs might have been due to high endogenous
expression levels of at least one of the four defined pluripotency-
inducing factors, or due to reduced activation of the p53 pathway
[39,40]. However, no significant differences in the endogenous
expression of the four factor genes or of p53 or p21 were detected.
Figure 7. Characteristics and differentiation of the hGF-iPS cells. (A) hGF-iPS cell colonies positively stained for ALP (clone 547A-1). Scale bar;
300 mm. (B) RT-PCR analysis of ES cell specific genes (NANOG, REX1, TERT, endogenous OCT3/4 and SOX2) in hGF-iPS clones, human ES cell lines (H9
and KhES-3), parental hGFs and SNL feeder cells. GAPDH was used as a loading control. (C) Bisulfite sequencing of the NANOG and OCT3/4 promoters
demonstrated that CpGs in the parental hGFs were converted to a demethylated state in the hGF-iPS cells (clones 547A-4 and -5), resulting in a
methylation pattern similar to that of human H9 ES cells. (D–F) H&E staining of teratoma sections showed differentiation of hGF-iPS-547A-3 cells into
neural tissues (D: ectoderm), cartilage (E: mesoderm), and gut-like epithelial tissues (F: endoderm). Scale bars; 50 mm.
iPS Cells from Gingival Tissue
PLoS ONE | www.plosone.org8 September 2010 | Volume 5 | Issue 9 | e12743
On the other hand, GFs showed significantly greater proliferation
in comparison to TTFs. Additionally, pGFs proliferated well for at
least 20 passages, while the proliferation of TTFs decreased after
10 passages (data not shown). Moreover, GFs consistently showed
higher expression of Tert mRNA compared to TTFs. Tert is one
of the major subunits in the telomerase complex , and the
transcription of Tert gene correlates with telomerase activity in
most cells . The high expression of the Tert gene in GFs may
therefore explain their high proliferative capacity. The high
proliferative capacity of the GFs should be advantageous for
retroviral integration due to increased likelihood of cell division
during transduction. The higher efficiency of GF reprogramming,
therefore, may at least partially be due to the high proliferation
rate of the GFs. Wounds in the oral mucosa show faster closure
with less scar formation than skin wounds [16,17], partly because
oral GFs differentially express early wound closure-related genes,
such as FGFR1OP2/wit3.0 [18,19]. It is therefore possible that
intrinsic differences in gene expression patterns between the GFs
and TTFs may also underlie differences in reprogramming
We also demonstrated that iPS cells could be generated from
human gingival tissues, which underscores the potential value of
this promising cell source for human applications. When we
transduced human dermal fibroblasts (HDF1388 ) in parallel
with human GFs under the same experimental setting and
infection protocols, fewer ES cell-like colonies emerged from the
dermal fibroblasts, suggesting that human GFs might be more
readily reprogrammed into iPS cells. Yan et al.  recently
reported that dental stem cells can be efficiently reprogrammed
into iPS cells by lentiviral transduction of LIN28/NANOG/
OCT4/SOX2 and by retroviral transduction of c-MYC/KLF4/
OCT4/SOX2. However, their protocol did not generate ES cell-
like colonies from human gingival fibroblasts and foreskin
fibroblasts. They also indicated that dental stem cells express a
number of ES cell-associated genes, thus suggesting that these stem
cells have epigenetic advantages for reprogramming. Therefore,
the reprogramming efficiency of terminally differentiated GFs may
be inferior to that of undifferentiated stem cells.
It is unknown at present whether iPS cells derived from different
types of cells behave in the same manner . Specifically, iPS
cells from different cell types may differ in their ability to undergo
guided differentiation . Therefore, GF-iPS cells should be
further characterized and compared to ES cells and iPS cells
derived from other sources. Potential differences in the repro-
gramming efficiency between cells isolated from humans and mice
also remain to be elucidated. Nonetheless, the high replication
capacity of GFs should permit not only the generation of sufficient
cells for iPS cell induction, but also the efficient generation of iPS
cells from multiple expanded cell cultures. The intrinsic features of
GFs from easily obtainable gingival tissues could be of benefit for
regenerative medicine and drug screening, especially in dentistry,
as it is easy for dental associates to establish primary cell cultures
with minimal patient discomfort. Additionally, establishment of
iPS cell banks with various human leukocyte antigen (HLA) types
should be useful for general regenerative medicine, as the
establishment of clinical-grade iPS cell lines from individual
patients would require much time and high cost . Collection of
gingiva considered until now to be biomedical waste from healthy
volunteers and efficient iPS cell generation from this tissue may
allow the development of a cell banking system for a wide range of
In conclusion, the efficient reprogramming of mouse gingival
fibroblasts to pluripotency is expected to provide a valuable
experimental model for investigating the basis of cell source-
dependent cellular reprogramming and pluripotency, which may
thus lead to a practical alternative for the generation of patient-
and disease-specific pluripotent stem cells.
Materials and Methods
All animal experiments in this study strictly followed a protocol
approved by the Institutional Animal Care and Use Committee of
Osaka University Graduate School of Dentistry (approval number:
20-009). Written approval for human gingival tissue collection and
subsequent iPS cell generation and genome/gene analyses
performed in this study was obtained from the Institutional
Review Board at Osaka University Graduate School of Dentistry
(approval number: H21-E7) and the Ethics Committee for Human
Genome/Gene Analysis Research at Osaka University (approval
number: 233), and written informed consent was obtained from
each individual participant.
Mouse GF cultures were established from either pGFs or mGFs
obtained from 10-week-old adult male C57BL/6J mice. To
establish TTF cultures, tails from mice were peeled and minced
into 1-cm pieces . The extracted palatal and molar mucosal
tissues or minced tails were placed on a 0.1% gelatin-coated 30-
mm tissue culture dish and maintained in MF-start medium
(Toyobo, Osaka, Japan) at 37uC with 5% CO2. When fibroblasts
migrated out of the tissues (Fig. 1B), the tissues were removed.
When the cells reached subconfluence, they were harvested and
transferred to a gelatin-coated 60-mm tissue culture dish (Passage
1) and cultured in ‘‘fibroblasts and Platinum-E (FP) medium’’,
which consists of Dulbecco’s Modified Eagle’s Medium (DMEM
without sodium pyruvate; Nacalai Tesque, Kyoto, Japan), 10%
fetal bovine serum (FBS; Sigma, St. Louis, MO), 50 units/ml
penicillin, and 50 mg/ml streptomycin (Nacalai Tesque). Culture
in serum- and calcium-containing media favorably selects
fibroblasts from heterogeneous populations of migrating epithelial
cells and fibroblasts in the mucosal tissues .
Primary hGF cultures were established according to the
previously described protocol  from healthy gingival tissues
discarded during surgery on a 24-year-old man.
SNLP76.7-4 feeder cells and the mouse ES cell line (AB2.2)
were graciously supplied by Dr. Allan Bradley of the Sanger
Institute (London, UK). Platinum-E packaging cells  for
retrovirus production were graciously supplied by Dr. Toshio
Kitamura (University of Tokyo, Japan). Human ES cell line H9
was obtained from WiCellTMResearch Institute (Wilmington,
MA), and human ES cell line KhES-3 and human dermal
fibroblast-derived iPS cells  were obtained from Institute for
Frontier Medical Sciences, Kyoto University. The human ES cells
were treated according to the guidelines for utilization of human
ES cells established by the Ministry of Education, Culture, Sports,
Science and Technology, Japan.
Moloney murine leukemia virus (MMLV)-based retroviral
vectors (pMXs-IRES-puro) containing murine and human c-
Myc, Oct3/4, Sox2 or Klf4 cDNA were obtained from Addgene
(Cambridge, MA), and the pMX-GFP retroviral vector was
purchased from Cell Biolabs (San Diego, CA). Nine micrograms of
each plasmid vector were separately added to tubes containing
Opti-MEM-I mediun (Invitrogen) and FuGENE 6 transfection
reagent (Roche, Basel, Switzerland); each plasmid was then
separately transfected into 100-mm dishes containing Platinum-E
iPS Cells from Gingival Tissue
PLoS ONE | www.plosone.org9September 2010 | Volume 5 | Issue 9 | e12743
packaging cells . The transfection efficiency was monitored by
evaluation of GFP expression under a fluorescence microscope.
The efficiency of transfection into Plat-E cells was typically .60%,
as indicated by GFP expression. The next day, the culture medium
was exchanged for fresh FP medium. After 24 hours, the virus-
containing supernatants were mixed together and used for
Induction of iPS Cells
Twenty-four hours before transduction, mouse pGFs and mGFs
(5 passages) were seeded at 56105cells per 100-mm dish in FP
medium containing 3 ng/ml bFGF (Peprotech, London, UK). For
the four-factor transduction, supernatants with retroviruses coding
c-Myc, Oct3/4, Sox2, Klf4 and GFP were mixed at a ratio of
1:1:1:1:3. When the mGFs were transduced with only three
factors, the supernatants containing retroviruses coding Oct3/4,
Sox2, Klf4 and GFP were mixed at a ratio of 1:1:1:3. The cells
were incubated overnight in the virus/polybrene (4 mg/ml)/bFGF
(10 ng/ml)-containing supernatants. On days one and three after
transduction, the culture medium was exchanged for fresh FP
medium containing 3 ng/ml bFGF.
At four days after transduction, the cells in the culture dishes
were re-seeded onto 60-mm dishes at 0.1–16103cells/cm2for the
four-factor transduction, and onto 100-mm dishes at 0.7–
16104cells/cm2for three-factor transduction; mitomycin C-
inactivated SNLP76.7-4 cells were used as a feeder layer. The
next day, the culture medium was exchanged for ‘‘ES medium’’,
which consisted of DMEM, 15% FBS, 2 mM L-glutamine,
161024M nonessential amino acids, 161024M 2-mercaptoeth-
anol, 50 U penicillin, and 50 mg/ml streptomycin. The medium
was changed every day. The three-factor-infected GFs were
harvested with deteriorated feeder cells at 30 days after
transduction and then were re-seeded onto a new feeder layer.
The colonies demonstrating minimal GFP expression were
identified and mechanically picked for expansion. After expansion,
clonal colonies showing ES cell-like proliferation and morphology,
including a round shape, large nucleoli and scant cytoplasm, were
selected for establishing clonal iPS cell cultures.
To demonstrate the expression of the ES cell marker ALP in
GF-derived iPS cell colonies, a standard ALP staining protocol was
used . TEM was used to determine the structure of individual
cells from the GF-derived iPS cell colonies. The colonies were
fixed first with 1% paraformaldehyde and 1.25% glutaraldehyde
in phosphate-buffered saline (PBS), and second with 2% osmium
tetroxide. The fixed colonies were then embedded in epoxy resin
and stained with methylene blue, followed by 1-mm sectioning of
the resin for optical microscopy. Additionally, 70-nm sections were
stained with 2% uranyl acetate and Sato’s lead stain, and
examined using a JEM 1200EX (JOEL, Tokyo, Japan) operated
at 80 kV for TEM.
Induction of iPS cells from primary hGFs (7 passages) via
introduction of four factors was performed using a previously
described protocol . According to this protocol, hGFs were
first infected with a lentivirus to express the mouse Slc7a1 gene (by
a plasmid vector from Addgene) and then infected with
retroviruses coding human c-MYC, OCT3/4, SOX2 and KLF4
genes for iPS cell induction.
Total RNA derived from mouse or human clonal GF-derived
iPS or ES cell colonies was used for RT-PCR analysis. Total RNA
was extracted with an RNeasy Mini Kit (QIAGEN, Hilden,
Germany). After DNase I treatment (Ambion, Austin, TX), cDNA
was synthesized from 1 mg of total RNA using Super Script III
reverse transcriptase (Invitogen, Carlsbad, CA). The cDNA target
was amplified by PCR using Taq DNA polymerase (Promega,
Madison, WI) following the manufacturer’s recommendations.
The primer pairs used are given in Table S1. PCR products were
subjected to 1.5% agarose gel electrophoresis with ethidium
bromide staining and visualized under ultraviolet light illumina-
tion. The expression of glyceraldehyde-3-phosphate dehydroge-
nase (GAPDH) mRNA was used as an internal control.
In vitro Differentiation of Mouse GF-Derived iPS Cells
To determine the differentiation ability of GF-derived iPS cells
in vitro, we used floating cultivation to form EBs . For EB
formation, mouse GF-derived iPS cells were harvested by
trypsinization and transferred to low-attachment bacterial culture
dishes in the ES medium. After 3 days of floating cultivation to
form EBs, aggregated cells were plated onto gelatin-coated 8-well
glass chamber slides (Nalge Nunc International, Naperville, IL) or
12-well tissue culture plates, and incubated in ES medium. The
culture medium was changed twice a week.
For immunocytochemistry, cells cultured in glass chamber slides
for 3 to 10 days after expansion were fixed in 10% buffered
formalin phosphate (Wako, Osaka, Japan) and incubated in 1%
bovine serum albumin and 0.1% Triton-X100 in PBS for 20 min.
After two washes in PBS, the cells were incubated with a mouse
anti-human a-SMA monoclonal antibody (0.05 mol/L; clone
1A4, Dako, Glostrup, Denmark) or rabbit anti-human AFP
polyclonal antibody (0.05 mol/L; Dako) for 30 min at room
temperature, or a mouse anti-human b-III tubulin monoclonal
antibody (0.5 mg/ml; clone TU-20, Millipore, Temecula, CA) or
control IgG (0.5 mg/ml; mouse IgG whole molecules: Santa Cruz
Biotechnology, Santa Cruz, CA) overnight at 4uC . The cells
were then washed and incubated for 30 min at 37uC with Alexa
488 (green dye) or 568 (red dye) conjugated to goat anti-mouse or
anti-rabbit IgG (1:500; Molecular Probes, Eugene, OR), followed
by 4,69-diamidino-2-phenylindole (DAPI: Roche) nuclear staining.
For osteogenic cell detection, EBs cultured in gelatin-coated 12-
well tissue culture plates in ES medium for 30 days were stained
using a standard von Kossa staining method  to demonstrate
the extent of nodule mineralization.
Bisulfite Genomic Sequencing
Genomic DNA was isolated from the aggregated mouse GF-
derived iPS cells and ES cells floating for 3 days or from the
attached human GF-derived iPS cells and H9 ES cells harvested
by CTK solution . Information about the promoter regions
and CpG loci of Nanog and Oct3/4 was obtained from a previous
study  and the Data Base of Transcriptional Start Sites (DBTSS
Ver. 7.0: http://dbtss.hgc.jp/). Bisulfite treatment was performed
using the EpiTect Bisulfite kit (Qiagen) according to the
manufacturer’s recommendations. Bisulfite PCR primers [1,5]
are listed in Table S1. Amplified products were cloned into the
pGEM-T Easy Vector (Promega). Five to eight randomly selected
clones were sequenced with the SP6 forward and reverse primers
for each gene.
Teratoma Formation and Histological Analysis
Eight-week-old immunodeficient mice (C.B-17 SCID; Clea
Japan, Tokyo, Japan) were anesthetized with diethyl ether and an
intraperitoneal injection (0.1 ml per 100 g body weight) of a 106
dilution of Nembutal (Dainippon Sumitomo Pharmaceutical,
Osaka, Japan). Twenty microliters of a mouse or human GF-
derived cell suspension (0.2–0.56106cells/testis) in cold Hank’s
balanced salt solution or DMEM/F12 (Invitrogen) were injected
into the medulla of mouse testes using a Hamilton syringe. The
iPS Cells from Gingival Tissue
PLoS ONE | www.plosone.org 10 September 2010 | Volume 5 | Issue 9 | e12743
mice were thereafter housed with free access to water and food
under specific pathogen-free conditions. After 7–10 weeks, the
teratomas were excised after perfusion with PBS followed by a
fixative solution containing 1% paraformaldehyde and 1.25%
glutaraldehyde, and subjected to histological analysis. Specimens
were embedded in paraffin, and sectioned at 3 mm for hematox-
ylin and eosin (H&E) staining.
Superovulation [intraperitoneal administration of 5 I.U. preg-
nant mare serum gonadotropin (PMSG) followed after 48 hr by
5 I.U. human chorionic gonadotropin (hCG)] was induced in
eight-week-old female mice [Jcl:MCH (ICR), CREA Japan],
which were then mated with Jcl:MCH (ICR) males. Embryos at
the 2-cell stage were collected at day 1.5 after vaginal plug
observation and flushed in M2 medium (Sigma). Embryos were
then cultured in KSOM culture medium (Chemicon) in the
incubator (37uC, 5% CO2in air) until they became blastocysts.
Mouse GF-derived iPS cells were harvested using 0.25% trypsin
to obtain a single cell suspension. Single cells were then transferred
into the micromanipulation chamber in a drop of DMEM medium
containing 10% fetal calf serum and 15 mM HEPES. Groups of 20
to 25 cells were injected into each single blastocyst. The injected
embryos were then transplanted into 2.5 dpc pseudopregnant
Jcl:ICR recipient females. Chimeric male mice were mated with
female mice [Jcl:MCH (ICR)] to validate germline transmission.
Determination of Reprogramming Efficiency
TTF, pGF and mGF cultures were established from the same
individual mouse (10 weeks of age). Four-factor transduction
(without GFP) was performed using cell cultures with identical
passage numbers. The transduction of each cell type was performed
simultaneously using the same virus-containing supernatants. The
cell cultures used for the comparisons were between passage
numbers four and ten. Each cell culture was then seeded in 6-well
tissue culture plates (16104cells/well) with the feeder cells. iPS cell
colonies were identified based on ES cell-like morphology, and ALP
staining was used to facilitate their identification. The reprogram-
ming efficiencywascalculatedas the number of iPScolonies formed
per number of transduced cells seeded.
A cell proliferation assay was performed on pGF, mGF and
TTF cultures with identical passage numbers. The cells were
seeded in 96-well tissue culture plates (26103cells per well) and
maintained in FP medium. The culture medium was renewed
every other day. The number of cells was evaluated using the
WST-1 cell counting assay (Dojindo Laboratories, Kumamoto,
Japan) as described previously .
The endogenous mRNA expression of Oct3/4, Sox2, Klf4, c-
Myc, p53, p21 and Tert in pGFs, mGFs and TTFs at passages 4 to
6 was determined by real-time RT-PCR analysis. TaqMan primer
and probe sets used are Mm00488369_sl (Sox2), Mm00516105_gl
Found at: doi:10.1371/journal.pone.0012743.s001 (0.07 MB
Primers used for RT-PCR and bisulfite genomic
ous differentiation of pGF-iPS-4F-3 cells.
Found at: doi:10.1371/journal.pone.0012743.s002 (1.53 MB
Beating cardiomyocytes observed following spontane-
We thank Dr. Jiro Miura and Shinya Uraguchi (Osaka University School
of Dentistry) for technical assistance and Dr. Devang Thakor (Harvard
Medical School) for scientific comments. We are also grateful to Dr.
Yasuhiko Tabata (Institute for Frontier Medical Sciences, Kyoto
University) and Dr. Tetsuya Ishii (CiRA, Kyoto University) for their
valuable support to initiate this work.
Conceived and designed the experiments: HE KO SY. Performed the
experiments: HE KO HK GY SF. Analyzed the data: HE. Contributed
reagents/materials/analysis tools: HE KO MS TM SY. Wrote the paper:
HE. Administrative support: HY.
1. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from
mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:
2. Liu H, Zhu F, Yong J, Zhang P, Hou P, et al. (2008) Generation of induced
pluripotent stem cells from adult rhesus monkey fibroblasts. Cell Stem Cell 3:
3. Shimada H, Nakada A, Hashimoto Y, Shigeno K, Shionoya Y, et al. (2010)
Generation of canine induced pluripotent stem cells by retroviral transduction
and chemical inhibitors. Mol Reprod Dev 77: 2.
4. Ezashi T, Telugu BP, Alexenko AP, Sachdev S, Sinha S, et al. (2009) Derivation
of induced pluripotent stem cells from pig somatic cells. Proc Natl Acad Sci U S A
5. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, et al. (2007)
Induction of pluripotent stem cells from adult human fibroblasts by defined
factors. Cell 131: 861–872.
6. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, et al.
(2007) Induced pluripotent stem cell lines derived from human somatic cells.
Science 318: 1917–1920.
7. Lowry WE, Richter L, Yachechko R, Pyle AD, Tchieu J, et al. (2008)
Generation of human induced pluripotent stem cells from dermal fibroblasts.
Proc Natl Acad Sci U S A 105: 2883–2888.
8. Park IH, Zhao R, West JA, Yabuuchi A, Huo H, et al. (2008) Reprogramming
of human somatic cells to pluripotency with defined factors. Nature 451:
9. Hanna J, Markoulaki S, Schorderet P, Carey BW, Beard C, et al. (2008) Direct
reprogramming of terminally differentiated mature B lymphocytes to pluripo-
tency. Cell 133: 250–264.
10. Aoi T, Yae K, Nakagawa M, Ichisaka T, Okita K, et al. (2008) Generation of
pluripotent stem cells from adult mouse liver and stomach cells. Science 321:
11. Stadtfeld M, Brennand K, Hochedlinger K (2008) Reprogramming of
pancreatic beta cells into induced pluripotent stem cells. Curr Biol 18: 890–
12. Wernig M, Lengner CJ, Hanna J, Lodato MA, Steine E, et al. (2008) A drug-
inducible transgenic system for direct reprogramming of multiple somatic cell
types. Nat Biotechnol 26: 916–924.
13. Yamanaka S (2009) Elite and stochastic models for induced pluripotent stem cell
generation. Nature 460: 49–52.
14. Tsai SY, Clavel C, Kim S, Ang YS, Grisanti L, et al. (2010) Oct4 and Klf4
Reprogram Dermal Papilla Cells into Induced Pluripotent Stem Cells. Stem
Cells 28: 221–228.
15. Li W, Ding S (2010) Small molecules that modulate embryonic stem cell fate and
somatic cell reprogramming. Trends Pharmacol Sci 31: 36–45.
16. Sciubba JJ, Waterhouse JP, Meyer J (1978) A fine structural comparison of the
healing of incisional wounds of mucosa and skin. J Oral Pathol 7: 214–227.
17. Walsh LJ, L’Estrange PR, Seymour GJ (1996) High magnification in situ viewing
of wound healing in oral mucosa. Aust Dent J 41: 75–79.
18. Sukotjo C, Lin A, Song K, Ogawa T, Wu B, et al. (2003) Oral fibroblast
expression of wound-inducible transcript 3.0 (wit3.0) accelerates the collagen gel
contraction in vitro. J Biol Chem 278: 51527–51534.
19. Lin A, Hokugo A, Choi J, Nishimura I (2010) Small cytoskeleton-associated
molecule, fibroblast growth factor receptor 1 oncogene partner 2/wound
inducible transcript-3.0 (FGFR1OP2/wit3.0), facilitates fibroblast-driven wound
closure. Am J Pathol 176: 108–121.
iPS Cells from Gingival Tissue
PLoS ONE | www.plosone.org11 September 2010 | Volume 5 | Issue 9 | e12743
20. Stephens P, Davies KJ, Occleston N, Pleass RD, Kon C, et al. (2001) Skin and Download full-text
oral fibroblasts exhibit phenotypic differences in extracellular matrix reorgani-
zation and matrix metalloproteinase activity. Br J Dermatol 144: 229–237.
21. Giannopoulou C, Cimasoni G (1996) Functional characteristics of gingival and
periodontal ligament fibroblasts. J Dent Res 75: 895–902.
22. Yamada Y, Ueda M, Hibi H, Baba S (2006) A novel approach to periodontal
tissue regeneration with mesenchymal stem cells and platelet-rich plasma using
tissue engineering technology: A clinical case report. Int J Periodontics
Restorative Dent 26: 363–369.
23. Meijer GJ, de Bruijn JD, Koole R, van Blitterswijk CA (2008) Cell based bone
tissue engineering in jaw defects. Biomaterials 29: 3053–3061.
24. Ueda M, Yamada Y, Kagami H, Hibi H (2008) Injectable bone applied for ridge
augmentation and dental implant placement: human progress study. Implant
Dent 17: 82–90.
25. Yamada Y, Nakamura S, Ito K, Kohgo T, Hibi H, et al. (2008) Injectable tissue-
engineered bone using autogenous bone marrow-derived stromal cells for
maxillary sinus augmentation: clinical application report from a 2-6-year follow-
up. Tissue Eng Part A 14: 1699–1707.
26. Brambrink T, Foreman R, Welstead GG, Lengner CJ, Wernig M, et al. (2008)
Sequential expression of pluripotency markers during direct reprogramming of
mouse somatic cells. Cell Stem Cell 2: 151–159.
27. Zeuschner D, Mildner K, Zaehres H, Scholer HR (2010) Induced Pluripotent
Stem Cells at Nanoscale. Stem Cells Dev 19: 615–620.
28. Egusa H, Saeki M, Doi M, Fukuyasu S, Matsumoto T, et al. (2010) A small-
molecule approach to bone regenerative medicine in dentistry. J Oral Biosci 52:
29. Zaky SH, Cancedda R (2009) Engineering craniofacial structures: facing the
challenge. J Dent Res 88: 1077–1091.
30. Seki T, Yuasa S, Oda M, Egashira T, Yae K, et al. (2010) Generation of induced
pluripotent stem cells from human terminally differentiated circulating T cells.
Cell Stem Cell 7: 11–14.
31. Aasen T, Raya A, Barrero MJ, Garreta E, Consiglio A, et al. (2008) Efficient and
rapid generation of induced pluripotent stem cells from human keratinocytes.
Nat Biotechnol 26: 1276–1284.
32. Tamaoki N, Takahashi K, Tanaka T, Ichisaka T, Aoki H, et al. (2010) Dental
pulp cells for induced pluripotent stem cell banking. J Dent Res 89: 773–778.
33. Yan X, Qin H, Qu C, Tuan RS, Shi S, et al. (2010) iPS cells reprogrammed
from mesenchymal-like stem/progenitor cells of dental tissue origin. Stem Cells
Dev 19: 469–480.
34. Oda Y, Yoshimura Y, Ohnishi H, Tadokoro M, Katsube Y, et al. Induction of
pluripotent stem cells from human third molar mesenchymal stromal cells. J Biol
Chem. In press.
35. Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, et al. (2008)
Generation of induced pluripotent stem cells without Myc from mouse and
human fibroblasts. Nat Biotechnol 26: 101–106.
36. Okita K, Ichisaka T, Yamanaka S (2007) Generation of germline-competent
induced pluripotent stem cells. Nature 448: 313–317.
37. 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.
38. Takahashi K, Okita K, Nakagawa M, Yamanaka S (2007) Induction of
pluripotent stem cells from fibroblast cultures. Nat Protoc 2: 3081–3089.
39. Hong H, Takahashi K, Ichisaka T, Aoi T, Kanagawa O, et al. (2009)
Suppression of induced pluripotent stem cell generation by the p53-p21
pathway. Nature 460: 1132–1135.
40. Kawamura T, Suzuki J, Wang YV, Menendez S, Morera LB, et al. (2009)
Linking the p53 tumour suppressor pathway to somatic cell reprogramming.
Nature 460: 1140–1144.
41. Martin-Rivera L, Herrera E, Albar JP, Blasco MA (1998) Expression of mouse
telomerase catalytic subunit in embryos and adult tissues. Proc Natl Acad
Sci U S A 95: 10471–10476.
42. Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, et al. (1998) Extension
of life-span by introduction of telomerase into normal human cells. Science 279:
43. Yamanaka S (2009) A fresh look at iPS cells. Cell 137: 13–17.
44. Nikawa H, Egusa H, Makihira S, Okamoto T, Kurihara H, et al. (2006) An in
vitro evaluation of the adhesion of Candida species to oral and lung tissue cells.
Mycoses 49: 14–17.
45. Morita S, Kojima T, Kitamura T (2000) Plat-E: an efficient and stable system for
transient packaging of retroviruses. Gene Ther 7: 1063–1066.
46. Egusa H, Schweizer FE, Wang CC, Matsuka Y, Nishimura I (2005) Neuronal
differentiation of bone marrow-derived stromal stem cells involves suppression of
discordant phenotypes through gene silencing. J Biol Chem 280: 23691–23697.
47. Ohnuki M, Takahashi K, Yamanaka S (2009) Generation and characterization
of human induced pluripotent stem cells. Curr Protoc Stem Cell Biol Chapter 4:
Unit 4A 2.
48. Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, et al. (2000)
Differentiation of human embryonic stem cells into embryoid bodies
compromising the three embryonic germ layers. Mol Med 6: 88–95.
49. Egusa H, Iida K, Kobayashi M, Lin TY, Zhu M, et al. (2007) Downregulation of
extracellular matrix-related gene clusters during osteogenic differentiation of
human bone marrow- and adipose tissue-derived stromal cells. Tissue Eng 13:
50. Egusa H, Kaneda Y, Akashi Y, Hamada Y, Matsumoto T, et al. (2009)
Enhanced bone regeneration via multimodal actions of synthetic peptide
SVVYGLR on osteoprogenitors and osteoclasts. Biomaterials 30: 4676–4686.
iPS Cells from Gingival Tissue
PLoS ONE | www.plosone.org12 September 2010 | Volume 5 | Issue 9 | e12743