Generation of stratified squamous epithelial progenitor cells from mouse induced pluripotent stem cells.
ABSTRACT Application of induced pluripotent stem (iPS) cells in regenerative medicine will bypass ethical issues associated with use of embryonic stem cells. In addition, patient-specific IPS cells can be useful to elucidate the pathophysiology of genetic disorders, drug screening, and tailor-made medicine. However, in order to apply iPS cells to mitotic tissue, induction of tissue stem cells that give rise to progeny of the target organ is required.
We induced stratified epithelial cells from mouse iPS cells by co-culture with PA6 feeder cells (SDIA-method) with use of BMP4. Clusters of cells positive for the differentiation markers KRT1 or KRT12 were observed in KRT14-positive colonies. We successfully cloned KRT14 and p63 double-positive stratified epithelial progenitor cells from iPS-derived epithelial cells, which formed stratified epithelial sheets consisting of five- to six-polarized epithelial cells in vitro. When these clonal cells were cultured on denuded mouse corneas, a robust stratified epithelial layer was observed with physiological cell polarity including high levels of E-cadherin, p63 and K15 expression in the basal layer and ZO-1 in the superficial layer, recapitulating the apico-basal polarity of the epithelium in vivo.
These results suggest that KRT14 and p63 double-positive epithelial progenitor cells can be cloned from iPS cells in order to produce polarized multilayer epithelial cell sheets.
- [Show abstract] [Hide abstract]
ABSTRACT: The cornea is covered by a stratified epithelium that is renewed by stem cells located in the peripheral region of the cornea known as the limbus. This stroma of the limbus contains stromal keratocytes that, when expanded in culture, are termed limbal fibroblasts (LFs). It is thought that LFs exhibit similar characteristics to bone marrow mesenchymal stem cells (BM MSCs) and help maintain the epithelial stem cell phenotype in the limbal region. In this study, we aimed at reprogramming SSEA4+ LFs and BM MSCs into corneal epithelial lineage by using a 3-dimensional culture system and embryonic stem cell medium. After enrichment, SSEA4+ cells showed a higher level of stem cell marker expression such as Sox2, Oct4, Nanog, Rex1, ABCG2, and TRA-1-60, and colony forming efficiency than did SSEA4- cells. SSEA4+, as compared to SSEA4- cells, had a greater propensity to form spheres that, in turn, were induced into ectodermal lineage and further differentiated into functional corneal epithelium. Results show that LFs were similar to BM MSCs in marker profiles, and together with the differences noted between SSEA4+ and SSEA4- cells, point to LFsd' being tissue-specific MSCs. However, LFs showed a greater potential for differentiation into corneal epithelium, indicating the potential importance of tissue-specific adult progenitors in their reprogramming capacity into cells of interest. This study opens a new avenue for investigating the molecular mechanism involved in maintaining a limbal stem cell niche and thus a potentially important clinical application to treat corneal epithelial stem cell loss. Stem Cells 2013.Stem Cells 09/2013; · 7.70 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The ability to direct differentiation of mouse embryonic stem (ES) cells into specific lineages not only provides new insights into the pathways that regulate lineage selection but also has translational applications, for example in drug discovery. We set out to develop a method of differentiating ES cells into mesodermal cells at high efficiency without first having to induce embryoid body formation. ES cells were plated on a feeder layer of PA6 cells, which have membrane-associated stromal-derived inducing activity (SDIA), the molecular basis of which is currently unknown. Stimulation of ES/PA6 co-cultures with Bone Morphogenetic Protein 4 (BMP4) both favoured self-renewal of ES cells and induced differentiation into a Desmin and Nestin double positive cell population. Combined stimulation with BMP4 and all-trans Retinoic Acid (RA) inhibited self-renewal and resulted in 90% of cells expressing Desmin and Nestin. Quantitative reverse transcription-polymerase chain reaction (qPCR) analysis confirmed that the cells were of mesodermal origin and expressed markers of mesenchymal and smooth muscle cells. BMP4 activation of a MAD-homolog (Smad)-dependent reporter in undifferentiated ES cells was attenuated by co-stimulation with RA and co-culture with PA6 cells. The Notch ligand Jag1 was expressed in PA6 cells and inhibition of Notch signalling blocked the differentiation inducing activity of PA6 cells. Our data suggest that mesodermal differentiation is regulated by the level of Smad activity as a result of inputs from BMP4, RA and the Notch pathway.PLoS ONE 01/2012; 7(4):e36405. · 3.73 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: In 2006, we demonstrated that mature somatic cells can be reprogrammed to a pluripotent state by gene transfer, generating induced pluripotent stem (iPS) cells. Since that time, there has been an enormous increase in interest regarding the application of iPS cell technologies to medical science, in particular for regenerative medicine and human disease modeling. In this review article, we outline the current status of applications of iPS technology to cell therapies (particularly for spinal cord injury), as well as neurological disease-specific iPS cell research (particularly for Parkinson's disease and Alzheimer's disease). Finally, future directions of iPS cell research are discussed including a) development of an accurate assay system for disease-associated phenotypes, b) demonstration of causative relationships between genotypes and phenotypes by genome editing, c) application to sporadic and common diseases, and d) application to preemptive medicine.Molecular Brain 03/2014; 7(1):22. · 4.20 Impact Factor
Generation of Stratified Squamous Epithelial Progenitor
Cells from Mouse Induced Pluripotent Stem Cells
Satoru Yoshida1, Miyuki Yasuda1, Hideyuki Miyashita1, Yoko Ogawa1, Tetsu Yoshida1, Yumi Matsuzaki2,
Kazuo Tsubota1, Hideyuki Okano2, Shigeto Shimmura1*
1Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan, 2Department of Physiology, Keio University School of Medicine, Tokyo, Japan
Background: Application of induced pluripotent stem (iPS) cells in regenerative medicine will bypass ethical issues
associated with use of embryonic stem cells. In addition, patient-specific IPS cells can be useful to elucidate the
pathophysiology of genetic disorders, drug screening, and tailor-made medicine. However, in order to apply iPS cells to
mitotic tissue, induction of tissue stem cells that give rise to progeny of the target organ is required.
Methodology/Principal Findings: We induced stratified epithelial cells from mouse iPS cells by co-culture with PA6 feeder
cells (SDIA-method) with use of BMP4. Clusters of cells positive for the differentiation markers KRT1 or KRT12 were observed
in KRT14-positive colonies. We successfully cloned KRT14 and p63 double-positive stratified epithelial progenitor cells from
iPS-derived epithelial cells, which formed stratified epithelial sheets consisting of five- to six-polarized epithelial cells in vitro.
When these clonal cells were cultured on denuded mouse corneas, a robust stratified epithelial layer was observed with
physiological cell polarity including high levels of E-cadherin, p63 and K15 expression in the basal layer and ZO-1 in the
superficial layer, recapitulating the apico-basal polarity of the epithelium in vivo.
Conclusions/Significance: These results suggest that KRT14 and p63 double-positive epithelial progenitor cells can be
cloned from iPS cells in order to produce polarized multilayer epithelial cell sheets.
Citation: Yoshida S, Yasuda M, Miyashita H, Ogawa Y, Yoshida T, et al. (2011) Generation of Stratified Squamous Epithelial Progenitor Cells from Mouse Induced
Pluripotent Stem Cells. PLoS ONE 6(12): e28856. doi:10.1371/journal.pone.0028856
Editor: Qiang Wu, National University of Singapore, Singapore
Received July 12, 2011; Accepted November 16, 2011; Published December 9, 2011
Copyright: ? 2011 Yoshida 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 work was supported by grants from Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science (JSPS) and the Ministry of
Education, Culture, Sports, Science and Technology of Japan (MEXT), the project for realization of regenerative medicine and support for the core institutes for iPS
cell research from Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) to S.S. and H.O. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Transplantation of cultivated epithelial sheets is an established
method for regenerating damaged skin epithelium and corneal
epithelium [1,2,3,4]. Both allogeneic donor-derived cells and
autologous cells have been used to produce the transplantable
epithelial cell sheets. ES (embryonic stem) cells, which are also
pluripotent and can differentiate into all three embryonic germ
layers, are also possible as a source of epithelial cells sheet but the
use of ES cells involves ethical issues. Recently, Takahashi and
Yamanaka have successfully developed induced pluripotent stem
(iPS) cells from somatic cells by forced reprogramming using the
transcriptional factors OCT4, SOX2, c-MYC, and KLF4 [5,6].
By applying patient-specific iPS cells to regenerative medicine,
transplantation of autologous cells will become possible. To apply
iPS cells to engineering of stratified epithelial sheets, we examined
differentiation of iPS cells into epithelial cells.
To date, several procedures to differentiate mouse ES/iPS cells
and human ES cells into epidermal keratinocytes have been
reported [7,8,9,10,11,12,13,14,15]. These procedures include the
methods using feeder cells [7,12], embryoid bodies (EBs) [8,13], and
direct differentiation of ES/iPS cells as monolayers on extra-cellular
matrix (ECM) [13,14,15]. In the case of mouse ES cells, BMP-4 has
been identified as a key factor for epidermal differentiation.
Kawasaki et al. reported that stromal cell–derived inducing activity
(SDIA) culture method using PA6 feeder cells promote neural
differentiation of mouse ES cells, and that BMP-treatment in SDIA
culture suppress the neural differentiation while promoting
epidermal differentiation [12,16] as in the embryo. For human ES
cells, Metallo et al. have developed the method using retinoic acid
(RA) and BMP-4 for EBs or mono-layer culture on collagen IV-
coating without feeder cells. Sakurai et al. also applied this
methodinmouseiPScells.However,none ofthese reportshave
developed a stratified epithelial cell sheet with physiological polarity.
In this study, we applied the SDIA method with BMP fibroblast-
derived mouse iPS cells and examined its differentiation into stratified
epithelial cells. We further optimized the timing of adding BMP in
order to produce a pure population of epithelial cells which can be
be engineered from cloned mouse iPS cells-derived epithelial cells.
Inductionofsquamous epithelial cells from mouse iPScells
To ascertain the undifferentiated state before differentiation
culture, we used Nanog-iPS cells, which express GFP (Figure 1B)
PLoS ONE | www.plosone.org1 December 2011 | Volume 6 | Issue 12 | e28856
Epithelial Progenitor Cells Derived from iPS Cells
PLoS ONE | www.plosone.org2December 2011 | Volume 6 | Issue 12 | e28856
and puromycin resistance gene under the control of Nanog
regulatory region . For differentiation into Cytokeratin 14
(KRT14)-positive squamous epithelial cells, we applied SDIA
(stromal cell-derived inducing activity) method with BMP4 for
the mouse iPS cells (Figure 1A). The iPS cells formed flattened
colonies on mitomycin C-treated PA6 feeder cells (Figure 1C) and
the expression of KRT14 and KRT18, an early ectodermal
marker, was observed in these colonies (Figure 1D). iPS-derived
KRT14-positive cells expressed the stratified squamous epithelium
marker p63 as well (Figure 1, E–G). Theses cells formed stratified
layers with higher levels of KRT14 expression in the upper layers.
On the other hand, the expression of p63 was higher in lower
layers (Figure 1G). In addition, most KRT14-positive colonies
included cells positive for KRT1, a marker for epidermal
keratinocytes (Figure 1H). KRT14-positive colonies including cells
positive for KRT12, a corneal epithelial marker, were also found
(Figure 1, I and J). Up-regulation of Krt1 expression after
stimulation with FBS was observed by RT-PCR analysis
(Figure 1K). While a certain level of Krt14 expression was found
by RT-PCR before FBS stimulation at day 9, only few KRT14-
positive cells were observed by immunocytochemistry when cells
were not stimulated with FBS. As is the case of mouse ES cells
, induction of KRT18-positive cells was observed without FBS
stimulation (data not shown) and further differentiation into
KRT14-positive cells was promoted only when cells were
stimulated with FBS.
To reveal the most effective time course of BMP treatment for
promoting epithelial differentiation of mouse iPS cells, we
examined the temporal effect of BMP treatment on induction of
KRT14-positive cells. After FBS-stimulation, immunostaining of
KRT14 was performed (Figure 2A) and total colony number
(Figure 2B) and the number of KRT14-positive colonies
(Figure 2C) were counted. The proportion of KRT14-positive
colonies was also calculated (Figure 2D). Although total colony
number decreased when cells were treated with BMP after culture
day 3 (Figure 2B), there was no remarkable difference in the
number of KRT14-positive colonies among the conditions tested
(Figure 2C). As a result, in terms of KRT14-positive colony
formation, epithelial induction was most effective when cells were
treated with BMP during culture days 3–5 (Figure 2D). When
colony size was used as a parameter, we found that KRT14-
positive colony area was also largest with BMP-treatment between
days 3–5 (Figure 2E). These results were consistent with
suppression of neural differentiation by BMP, and suggested that
KRT14-positive epithelial cells induced by BMP-treatment in
culture days 3–5 were the most proliferative. Without BMP
treatment, no KRT14-positive cells were found (data not shown).
Expansion and purification of iPS cells-derived epithelial
To expand and enrich epithelial cells generated from mouse iPS
cells, cells were subcultured on gelatin-coated culture dish in
media suitable for culture of stratified epithelial progenitor cells,
CnT20, supplemented with B-27 (Figure 1A). During subculture
in the media, epithelial cells proliferated preferentially and cells
other than epithelial cells such as fibroblasts decreased. However,
since proliferation of GFP-positive undifferentiated cells was found
in the media in addition to epithelial cells, GFP-negative cells were
sorted to exclude GFP-positive cells. Serial subcultures and GFP-
negative sorting were repeated to enriched KRT18- and/or
KRT14-positive epithelial cells (Figure 3A). Finally, to obtain
KRT14-positive clones, cells were seeded at a density of ,10 cells
per cm2and single colonies were selected using cloning discs.
Among selected clones, a single clone (1204SE1) was used in all
subsequent experiments. To characterize these cells, we first
examined the expression of differentiation markers. During
subculture prior to cloning, cells expressing tissue specific epithelial
keratins such as KRT1 or KRT12 disappeared (Figure 3B). Prior
to cloning, epithelial cells that expressed KRT18, but not KRT14
were observed (Figure 3A). After cloning, only KRT14-positive
cells were found although expression levels were not uniform
(Figure 3C). In addition, some cells were positive for KRT18 as
well (Figure 3D and 3E), and all of the cells were positive for p63
(Figure 3F). Clusters of cells positive for KRT15, which is
expressed in basal cells of stratified epithelia, were found
(Figure 3G). Expression of KRT19, a non-cornified squamous
epithelial cell marker, was not detected by immunocytochemistry.
We found that the cells expressed KRT16 as well, which is
expressed in proliferative epithelial cells (Figure 3H). The
expression of epidermal keratinocyte markers KRT10 (Figure 3I)
and KRT1, and the corneal epithelial marker KRT12 were not
Engineering of 3D-cultured epithelial sheets
Using the iPS-derived epithelial clone, we next engineered 3D-
culutred stratified epithelial sheets by air-lifting culture on culture
inserts without feeder cells in SHEM medium (see Materials and
Methods). Five- to six-layered epithelial sheets were formed
(Figure 4L, H&E-staining image) by this 3D-culture protocol.
For characterization, we first examined the expression of
cytokeratins in the cultivated epithelial sheet by RT-PCR analysis
(Figure 4M). The expression of Krt1 and Krt10, which was not
detected in the 2D-cultured epithelial clone 1204SE, was detected
in 3D-cultured sheet even though the expression level was low.
Up-regulation of Krt12 was not detected. Various levels of Krt14,
Krt15, Krt18, and Krt19 expression was found. The expression of
differentiation markers was examined by immunohistochemistry
as well. Expression of KRT14 was found in all layers of the
cultivated epithelial sheet (Figure 4, A, D, and G). The expression
of KRT18 was down-regulated (Figure 4, B and C). The
expression of p63 was also found in all layers, but higher levels
were observed in the basal layer while expression decreased
towards the supra-basal layers (Figure 4, G and I). The stratified
epithelial stem cell marker KRT15, which is also found in basal
layer of stratified epithelium, was also observed in the basal layer
(Figure 4H). The Expression of epidermal keratinocyte markers
Figure 1. Epidermal and corneal epithelial cells are induced from mouse iPS cells by SDIA method. A schematic diagram of the culture
method is represented in (A). (B) Nanog-iPS cell line 38C2. Undifferentiated state was monitored by the expression of GFP. (C) Mouse iPS cells formed
epithelial cell colonies on MMC-treated MPA6 cells by SDIA method with use of BMP-4 and FBS. Cell culture schedule is represented in panel K. (D)
After culture days 14, the epithelial cell colonies induced by SDIA method were positive for KRT18 and KRT14. (E, F, and G) In KRT14-positive colonies,
cells formed multilayer and the expression of p63 was observed especially at a high level in basal layer. The image in (G) represents a merged image
of (E) and (F). Cells expressing KRT1 (H) and KRT12 (I and J) were found in KRT14-positive colonies. An image at high magnification of H is shown in I.
(K) Expression of these cytokeratins were also confirmed by RT-PCR. Scale bars in B-H, 200 mM; I, 100 mM; J, 400 mM. KRT14 and p63, both stratified
squamous epithelium markers; KRT18, a marker for non squamous epithelia and early surface ectoderm; KRT1, as an epidermal marker; and KRT12, as
a corneal epithelial marker.
Epithelial Progenitor Cells Derived from iPS Cells
PLoS ONE | www.plosone.org3December 2011 | Volume 6 | Issue 12 | e28856
Epithelial Progenitor Cells Derived from iPS Cells
PLoS ONE | www.plosone.org4December 2011 | Volume 6 | Issue 12 | e28856
KRT1/KRT10, were slightly up-regulated in the stratified sheet
(Figure 4, E and F), as well as shown by RT-PCR, while the
corneal epithelial cells marker KRT12 was not detected. A
terminal differentiation marker involucrin (IVL) was detected in
suprabasal layers (Figure 4I). The expression of an adherence
marker E-cadherin through out the sheet, especially at a high level
in basal layers, was found (Figure 4J). In addition, the tight-
junction marker, ZO-1, was found in the superficial layer,
however, Claudin1 was not observed (Figure 4K). Notably, the
dense expression pattern of E-cadherin in the basal side and ZO-1
in the apical side reflects well the apico-basal polarity of the
epithelium in vivo. We further examined the epithelial sheets by
electron microscopy. In addition to tight-junctions, desmosomes
were formed in the cell junctions as shown by transmission
electron microscopy (TEM, Figure 4N). Microvilli can also be
observed on the surface of epithelial sheets by scanning electron
microscopy (SEM, Figure 4O). These results suggested that the
iPS-derived epithelial clone maintained an undifferentiated state in
2D culture and can be induced to differentiate in 3D culture and
suggested that cultivated epithelial sheets, which reproduce the
structure and polarity of stratified epithelium in vivo such as the
expression patterns of p63, KRT15, E-cadherin, and ZO-1, can
be developed with the epithelial clone.
Differentiation of the iPS-derived epithelial clone on
To further examine the ability of the iPS-derived epithelial
clones to proliferate and differentiate on corneal stroma, the
epithelial clone was labeled with mRFP and seeded and cultured
ex-vivo on corneal epithelium-denuded mouse eyes (Figure 5A). As
in the case of cultivated sheet on culture inserts, we examined the
expression of differentiation markers in the epithelial cells cultured
on mouse denuded-cornea by immunohistochemistry of tissue
sections (Figure 5 B–O). To confirm that the markers were
expressed in the iPS cells-derived epithelial clone, sections were
counter stained for mRFP as well (B, E, F, G, K and L). Residual
mRFP fluorescence was detected in Figures 5 D, H, I, J, M, N
and O. We found that the cornea was completely covered with
Figure 3. Characterization of mouse iPS cell-derived epithelial clone 1204SE1. Phase contrast image (A left) and immunofluorescent image
of mouse iPS cell-derived epithelial cells before cloning (A right). (B) RT-PCR analysis of the Krt1, Krt12, Krt14 and Krt18 in the epithelial cells before
cloning revealed the expression of Krt14 and Krt18. (C–H) Immunohistochemical analysis of cloned iPS cell-derived epithelial cells, 1204SE1. Merged
image of (C) KRT14, and (D) KRT18 is represented in (E). After cloning, all cells were positive for KRT14 and p63 (F) but negative for the epidermal
marker KRT10 (I). Some cells were KRT18-positive as well. Clusters of KRT15-positive cells, a marker for basal cells of stratified epithelia, were found in
2D-cultures but cells were negative for the non-cornified squamous epithelial cell marker KRT19 (G). Cells were positive for KRT16, a marker for hyper
proliferative epithelial cells (H). Scale bars, 100 mm in panel A left and C-H; 50 mm in panel A right.
Figure 2. Temporal effect of BMP-treatment to promote KRT 14-positive stratified epithelial cells. Mouse iPS cells cultured by SDIA-
method were treated with BMP during different culture days as indicated in the figure. Images of the whole culture dish immunostained for KRT14
(red) were obtained using BIOREVO (A). Colonies negative for KRT14 are also visualized by auto-fluorescence. The number of total colonies (B) and
KRT14-positive colonies (C) were used to calculate the percentage of KRT14-positive colonies (D). (E) KRT14-positive area was calculated as pixel
numbers using ImageJ. Scale bar in A=2 mm.
Epithelial Progenitor Cells Derived from iPS Cells
PLoS ONE | www.plosone.org5 December 2011 | Volume 6 | Issue 12 | e28856
mRFP-labeled cells (Figure 5B), all of which were positive for
KRT14 (Figure 5C). The cells formed a stratified, four- to six-layer
epithelium that was KRT14-positive in all layers (Figure 5D).
Polarized expression of p63 was observed in the basal layers
(Figure 5E) as well as in the cultivated sheet on culture insert. High
expression levels of KRT15 was also found in basal most layer
(Figure 5F). The expression of KRT18 remained in some
(Figure 5H) and involucrin (Figure 5I) was found in suprabasal
layers. Low levels of epidermal keratinocytes markers KRT1 and
KRT10 was observed (Figure 5, J and K), but corneal epithelial
cells specific markers such as KRT12 was not observed under
these conditions (Figure 5L). E-cadherin was expressed in all cell
layers, although more prominently in basal cells (Figure 5M). Low
cells (Figure5G). The expression ofKRT19
Figure 4. Characterization of cultivated epithelial sheets developed from clone 1204SE1. Stratified epithelial cell sheets were developed
from clone 1204SE1. An H&E-stained image of the five- to six-layered 3D sheet is shown in (L). The expression of cytokeratins and other epithelial
markers in the 3D-cultured cell sheet were examined by immunohistochemical (A-K) and RT-PCR (M) analysis. Images are shown with the basal side
down. Merged images of (A) KRT14 and (B) KRT18, and (D) KRT14 and (E) KRT10 were represented in (C) and (F), respectively. Cells were KRT14-
positive throughout the epithelial sheet. Remaining KRT18-positive cells were found in the superficial layer and the expression of epidermal
keratinocyte markers KRT1/KRT10 was slightly up-regulated in the stratified sheet (E and M). High expression of p63 was found in basal layers of the
stratified epithelial sheet and the expression decreased in suprabasal layers (H). KRT15, which is found in basal layer of stratified epithelium, was also
found in the most basal layer of the cultivated sheet (G). The cells were positive for KRT19 but negative for KRT12 (G and M). The terminally-
differentiated marker Involucrin (IVL) was positive (I) and high levels of E-cadherin (ECAD) expression were found throughout the sheet (J). Tight-
junction marker ZO-1 was found in the superficial layer, however, Claudin1 (CLDN1) was not observed (K). Formation of tight-junctions (arrow head in
N), and abundant desmosomes (arrows in N) were observed by TEM. SEM (O) revealed formation of microvilli on the surface of epithelial sheet. Scale
bars, 50 mm in A-L, 0.2 mm in N, 2 mm in O.
Epithelial Progenitor Cells Derived from iPS Cells
PLoS ONE | www.plosone.org6December 2011 | Volume 6 | Issue 12 | e28856
levels of Claudin1 (Figure 5N), and another tight-junction marker
ZO-1 (Figure 5O) was observed in suprabasal layers, especially in
the superficial layer. Thus, the expression pattern of epithelial
markers in cells cultured on denuded corneas were similar to those
observed in cultivated epithelial sheets showing that a polarized
stratified epithelial layer can be engineered.
There is a high expectancy for the use of iPS cells in medicine.
Original iPS cell lines, including those used in this study, were
originally generated by forced expression of reprogramming
transcription factors which were delivered into host cells by
retroviral vectors. However, use of the retroviral vectors involves
increasing the risk of tumor formation since it causes random
integration of the transgenes into host genome DNA .
Therefore, there have been many attempts to generate iPS by
delivering reprogramming factors with non-integrating methods
using adenovirus vectors , RNA virus vectors , plasmid
vectors [19,20], transposons [21,22] or non-viral magnetic
nanoparticles. These achievements have lowered the hurdle
towards the clinical use of iPS cells.
However, the accessibility of target tissue and delivery of cells
are still issues that need to be addressed prior to clinical use of iPS
cells. Both the epidermis and corneal epithelium are present on the
body surface, and are therefore suitable for the clinical application
Figure 5. Mouse iPS cell-derived epithelial cells on mouse cornea. The iPS-derived epithelial clone 1204SE1, labeled with mRFP, were seeded
and cultured on enucleated mouse eyes (A). Stratification on the denuded cornea was promoted by air-exposed culture. The expression of
differentiation markers was examined by immunohistochemistry (C–L). Whole images of cultured cornea sections were stained for mRFP (B) or KRT14
(C). Since the fluorescence of mRFP was impaired by PFA-fixation, the expression of mRFP was confirmed by immunohistochemistry as well using
rabbit anti-DsRed antibody (B, E, F, G, K and L). In D, H, I, J, M, N and O, residual fluorescence of mRFP was detected, in which sections were not
immunostained for mRFP because of overlap with the primary antibody. Merged images were represented in lower half of each panel (D–O). Images
are shown with basal side down. As on the culture insert, cells were KRT14-positive in all layers (D) and high expression levels of p63 and KRT15 was
found in the basal layer (E and F, respectively). KRT18-positive cells were found in the superficial layer (G). The expression of epidermal marker KRT1 (I)
and KRT10 (J) was found, but the corneal marker KRT12 (K) was not detected. Cells in suprabasal layers were positive for IVL (L). Higher levels of E-
cadherin was found in basal layer (M). Although staining for Claudin1 was faint (N), the expression of ZO-1 was found especially in superficial layer (O).
Scale bars, in A–C, 500 mm; in D–O, 50 mm.
Epithelial Progenitor Cells Derived from iPS Cells
PLoS ONE | www.plosone.org7 December 2011 | Volume 6 | Issue 12 | e28856
of iPS cells. Derivation of ocular epithelial tissue from iPS cells was
first reported in retinal pigment epithelium (RPE)[24,25]. The RPE
is a monolayer epithelium of neuroectoderm origin, and differen-
tiates from the outer layer of the optic cup driven by a series of
transcription factors such as PAX6, Rx, SIX3, MITF, and RPE-
65[26,27]. The corneal epithelium, on the other hand, is a stratified
epithelium that forms the outermost layer of the ocular surface and
is derived from the surface ectoderm. It is distinguished from the
epidermis and other mucosal epithelia by the expression of
PAX6[28,29]. We demonstrated stratified epithelial differentiation
of mouse iPS cells by the SDIA method with BMP-4 treatment.
Mizuseki et al. have described in their working hypothesis that
epidermogenesis is strongly promoted when BMP was used in the
early phase (days 0–3) of SDIA culture . In addition, judging
from the expression of the neuronal differentiation marker
Neuronal Class III b-Tubulin, they also reported that neural
differentiation was effectively suppressed when mouse ES cells
cultured by SDIA were treated with BMP during days 3–5 .
Our results on the temporal effect of BMP-4 on epithelial
differentiation (Figure 2) were consistent with their observation.
Recently, Bilousova et al. reported the differentiation of mouse
iPS cells into a keratinocyte lineage by the method of Metallo et al.
with modifications , which was originally developed for
keratinocyte differentiation of human ES cells . They also
showed the iPS cells-derived keratinocyte lineage cells (named
iPSC-KCs) had the ability to regenerate epidermis, hair follicles,
and sebaceous glands in vivo by grafting iPSC-KCs with mouse
dermal fibroblast into the skin of athymic nude mouse using silicon
graft chambers. On the other hand, in therapeutic treatment of
epidermis and corneal epithelium, stratified epithelial cell sheets
have been used for transplantation. In our iPS cell-derived
epithelial cell sheets, we found higher levels of the tight junction
protein ZO-1 in the apical side, and the adherence junction
marker E-cadherin in basal layers. KRT15 and p63 were also
highly expressed in the basal layers. These results suggest that the
characteristics of in vivo stratified epithelium were well regenerated
in these sheets. In addition, similar stratification and differentiation
was observed ex vivo on mouse corneas as well (Figure 5). Re-
direction of corneal epithelial cells  and thymic epithelial cells
 into epidermal cells by graft on mouse skin has been reported.
In addition, Blazejewska et al. have reported the transdifferentia-
tion of hair follicle stem cells into cornea epithelial-like cells using
corneal limbal fibroblasts . These reports suggest that signals
from tissue-specific mesenchymal cells in the microenvironment
define the differentiation of epithelial cells. Therefore we
examined differentiation of iPS-derived epithelial clones into
corneal epithelial cells on denuded mouse corneas. However,
contrary to our expectations, expression of corneal epithelium
marker K12 and PAX6 was not observed.
Further investigations are required to induce fully differentiated
epithelial sheets that are specific to tissue such as the skin and
cornea. However, since highly proliferative tissues such as the
various epithelia in the body require the regeneration of tissue stem
cells, it is unclear as to the level of differentiation required when
inducing these cells from iPS cells. Clinical studies of cultivated
epithelial sheet transplantation using ectopic autologous epithelial
cells such as the oral mucosa are already underway, many of which
report clinical success [33,34,35]. Ectopic tissue-derived epithelial
sheets transplanted to the cornea also do not express cornea-specific
cytokeratins despite the improved transparency. Therefore, our
protocol for inducing epithelial cells expressing the progenitor
markers KRT 14, KRT 15 and p63 is a major step towards the
application of iPS-derived cells in translational medicine.
Materials and Methods
Undifferentiated mouse induced pluripotent stem cells (iPS
cells), clone 38C2 (courtesy of Dr. S. Yamanaka, Kyoto University,
Kyoto, Japan), were maintained as described previously . In
brief, iPS cells were maintained on mitomycin C (Nacalai tesque
Inc, Japan) -treated SNL feeder cells in DMEM, supplemented
with 10% FBS, NEAA, 2-melcapt ethanol, L-Gln, LIF, at 37C, in
5% CO2, and passaged every 3 days using TrypleExpress
(invitrogen, Life Technologies Corp., Carlsbad, CA, USA). To
exclude differentiated cells, 1 mg/ml of puromycin was added in
the media and undifferentiated state was monitored with the
expression of Nanog-GFP. PA6 cells were maintained in DMEM
with 10%FBS and passaged every 3–4 days.
Epithelial cell induction, expansion, and purification
For epithelial differentiation, iPS cells were subcultured by SDIA
(stromal differentiation inducing activity) method with the use of
BMP-4 . Briefly,dissociated iPScells wereplatedon mytomycin
C-treated PA6 cells at a density of 300 ,500 cells per dish in SDIA
medium (GMEM, supplemented with 0.1 mM NEAA, 1mM
Sodium Pyruvate, 5 mM HEPES, 0.11 mM 2-melcapt ethanol,
2mM L-Gln, and10%KSR). BMP-4 was added from culture day 3
to day 5, or as indicated in figures. To induce KRT14-poitive
epithelial cells, cells were cultured in medium supplemented with
10% FBS instead of 10% KSR after day 9. A schematic diagram of
in FBS-containing medium, cells were replated on gelatin-coated
dishes and subcultured several times in CnT20 medium, which is
optimized for epithelial progenitor cells (CELLnTEC advanced cell
systems AG, Bern, Switzerland), supplemented with B-27 supple-
ment (invitrogen). To remove undifferentiated cells, Nanog-GFP-
positive cells were excluded by Flowcytometer (MoFlo cell sorter,
DakoCytomation Co.). Finally, cells were seeded at a density of
,10 cells/cm2and resulting single colonies were picked up with
cloning discs (Sigma-Aldrich, St. Louis, MO) to clone the iPS cells-
derived KRT14-positive epithelial cells.
Epithelial cells derived from an iPS-derived KRT14-positive
clone (clone no. 1204SE1) were seeded on culture insert (Corning
Incorporated, Corning, NY) and cultured in CnT20 supplemented
with B27. After the culture became confluent, culture medium was
switched to FBS-containing differentiation medium, SHEM
(DMEM/F12 supplemented with 10% FBS, 10 ng/ml EGF,
5 mg/ml insulin, 500 ng/ml hydrocortisone, 2 nM triiodothyro-
nine, 250 ng/ml isoproterenol hydrochloride, and antibiotics), and
cultured in air-exposed conditions for a week. Frozen sections of
the resultant stratified cell sheet embedded in carboxymethylcel-
lulose (4% CMC, Section-Lab Co. Ltd., Hiroshima, Japan) were
prepared for subsequent immunohistochmical staining.
Cell culture on corneal epithelium-denuded mouse eye
To culture epithelial cells on mouse cornea, corneal epithelium
of anesthetized animal was debribed using a corneal rust ring
remover (Algerbrush II, Algerbrush Company Inc., Lago Vista,
TX). The animals were sacrificed and the whole eye balls were
excised after debridement. To label iPS cell-derived epithelial cells,
the cells were transduced with a lentiviral vector for mRFP
expression (CSII-EF-mRFP1, courtesy of Dr. H. Miyoshi). The
labeled cells were seeded on the corneal epithelium-denuded
mouse eye in CnT20 medium supplemented with B-27. After
engraftment was observed, culture medium was switched to
Epithelial Progenitor Cells Derived from iPS Cells
PLoS ONE | www.plosone.org8 December 2011 | Volume 6 | Issue 12 | e28856
SHEM and cells were cultured in air-exposed conditions for a few
weeks. The eyeballs were embedded in 4% CMC and frozen
sections were prepared for subsequent immunohistochemical
Immunohistochemistry was performed as described previously
. In brief, cultured cells or frozen-sections fixed with 4%
paraformaldehyde (PFA), were incubated in fixative (Morphosave;
Ventana Medical Systems, Tucson, AZ) for 15 minutes. Blocking
was performed with 10% donkey or goat serum in phosphate-
buffered saline (PBS) for 30 minutes. The cells and sections were
then incubated with primary antibodies for 1 hour at room
temperature. The primary antibodies used in this study are, anti-
KRT1 (Abcam Inc., Cambridge, MA.), anti-KRT10 (PROGEN
Biotechnik GmbH, Heidelberg, Deutschland), anti-KRT12 (Santa
Cruz Biotechnology, Inc., Santa Cruz, CA), anti-KRT14
(Covance Inc., Princeton, NJ), anti-KRT15 (Covance Inc. ),
anti-KRT18 (Abcam Inc. ), anti-KRT19 (Thermo Fisher Scientific
Inc., Fremont, CA), anti-p63 (Santa Cruz Biotechnology, Inc. ),
anti-Pax6 (Covance Inc. ), anti-ZO-1 (Santa Cruz Biotechnology,
Inc. ), anti-Claudin-1 (invitrogen, Life Technologies Corp. ), anti-
Involucrin (Covance Inc. ), anti-E-cadherin (Takara Bio Inc.,
Shiga, Japan), and anti-mRFP (Takara Bio Inc. ). Immunoreac-
tivity of primary antibodies was visualized with secondary
antibodies conjugated with FITC (fluorescein isothiocyanate), or
Cy3 (Jackson ImmunoResearch Laboratories, West Grove, PA).
Imaging of the stained samples was performed by a microscope
(Axio Imager; Carl Zeiss Inc., Thornwood, NY) equipped with a
digital camera (Axiocam; Carl Zeiss Inc.).
Transmission electron microscopy (TEM) was performed as
described previously . In brief, sections of cultivated epithelial
sheets were immediately fixed with 2.5% glutaraldehyde in 0.1 M
phosphate buffer (pH 7.4) for 1 hour. The specimen was
dehydrated in graded ethyl alcohols and embedded in Epoc 812.
An ultrathin section was cut using a RT-7000 (RMC, USA),
stained with uranyl acetate and lead citrate, and then examined
with transmission electron microscope (1230 EXII; JEOL, Tokyo,
Japan). Epithelial sheets were examined by scanning electron
microscopy (SEM) as well. The specimens were fixed in 2.5%
glutaraldehyde for 2 h, washed with cacodylate buffer, postfixed in
1.0% osmium tetroxide, and then passed through a graded ethanol
series (50%, 70%, 80%, 90%, and 100%). The specimens were
immersed twice in hexamethyldisilazane (TAAB Laboratories
Equipment Ltd., Aldermaston, UK) for 10 min, air dried,
mounted on aluminum stubs, sputter coated with gold, and
examined on the H-7000 microscope (Hitachi, Tokyo, Japan).
Imaging of immunostained culture dishes and
calculation of K14-positive clone area
To calculate KRT14-positive clonal growth induced by SDIA
method, the cultured dishes were immunostained for KRT14 and
images of the whole dish were captured and processed using
BIOREVO (Keyence Corporation, Osaka, Japan). KRT14-
poistive area was calculated as pixel numbers using ImageJ
software (NIH, Bethesda, MD).
Total RNA was prepared from the cells using RNeasyH kit
(Qiagen, Hilden, Germany) and cDNA was synthesized from the
total RNA using the Rever Tra Ace-aH first-strand cDNA
synthesis kit (TOYOBO Co., Ltd., Osaka, Japan). Primers used
for Krt1, Krt10, Krt12, Krt14, Krt15, Krt18, Krt19, and Gapdh are
shown in Table 1. Polymerase chain reaction (PCR) was
performed using GeneAmp 9700 (Applied Biosystems, Inc, Foster
We especially thank Tomomi Sekiguchi for expert assistance, Dr. Kyoko
Miura for guidance of iPS cell culture, Sadafumi Suzuki for assistance in
cell sorting, Toshihiro Nagai for expert technical assistance on transmission
electron microscopy, Dr. Hiroyuki Miyoshi for the lentiviral vector CSII-
EF-mRFP1 (RIKEN BioResource Center, Tsukuba, Japan), and Prof.
Shinya Yamanaka (Center for iPS Cell Research and Application, Kyoto
University) for providing the Nanog-GFP mouse iPS cells.
Conceived and designed the experiments: SY HO SS. Performed the
experiments: SY MY HM YO TY YM. Analyzed the data: KT HO SS.
Contributed reagents/materials/analysis tools: TY YM. Wrote the paper:
1. O’Connor N, Mulliken J, Banks-Schlegel S, Kehinde O, Green H (1981)
Grafting of burns with cultured epithelium prepared from autologous epidermal
cells. Lancet 317: 75–78.
2. Hefton JM, Madden MR, Finkelstein JL, Shires GT (1983) Grafting of burn
patients with allografts of cultured epidermal cells. Lancet 2: 428–430.
3. Pellegrini G, Traverso CE, Franzi AT, Zingirian M, Cancedda R, et al. (1997)
Long-term restoration of damaged corneal surfaces with autologous cultivated
corneal epithelium. Lancet 349: 990–993.
4. Shimazaki J, Aiba M, Goto E, Kato N, Shimmura S, et al. (2002) Transplantation
severe ocular surface disorders. Ophthalmology 109: 1285–1290.
5. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from
mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:
6. Okita K, Ichisaka T, Yamanaka S (2007) Generation of germline-competent
induced pluripotent stem cells. Nature 448: 313–317.
Table 1. Oligonucleotide sequences for primer pairs used in
Gene Primer sequence (59-39)
Krt1 (Forward) GTGCTACAAACCAAATGGGAGC
Krt10 (Forward) GGTCTGGAGATTGAACTACAGTCCC
Krt12 (Forward) TCCTCCTGCAGATTGACAACG
Krt12 (Reverse) TTCCAGGGACGACTTCATGG
Krt14 (Forward) TGCTGGATGTGAAGACAAGGC
Krt14 (Reverse) GACAAGGGTCAAGTAAAGAGTGAAGC
Krt18 (Forward) CACCACCAAGTCTGCCGAAATCAGG
Krt19 (Forward) GGACCCTCCCGAGATTACAACC
Epithelial Progenitor Cells Derived from iPS Cells
PLoS ONE | www.plosone.org9 December 2011 | Volume 6 | Issue 12 | e28856
7. Aberdam E, Barak E, Rouleau M, de LaForest S, Berrih-Aknin S, et al. (2008) A
pure population of ectodermal cells derived from human embryonic stem cells.
Stem Cells 26: 440–444.
8. Bilousova G, Chen J, Roop DR (2010) Differentiation of Mouse Induced
Pluripotent Stem Cells into a Multipotent Keratinocyte Lineage. J Invest
9. Coraux C, Hilmi C, Rouleau M, Spadafora A, Hinnrasky J, et al. (2003)
Reconstituted skin from murine embryonic stem cells. Curr Biol 13: 849–853.
10. Green H, Easley K, Iuchi S (2003) Marker succession during the development of
keratinocytes from cultured human embryonic stem cells. Proc Natl Acad
Sci U S A 100: 15625–15630.
11. Ji L, Allen-Hoffmann BL, de Pablo JJ, Palecek SP (2006) Generation and
differentiation of human embryonic stem cell-derived keratinocyte precursors.
Tissue Eng 12: 665–679.
12. Kawasaki H, Mizuseki K, Nishikawa S, Kaneko S, Kuwana Y, et al. (2000)
Induction of midbrain dopaminergic neurons from ES cells by stromal cell-
derived inducing activity. Neuron 28: 31–40.
13. Metallo CM, Ji L, de Pablo JJ, Palecek SP (2008) Retinoic acid and bone
morphogenetic protein signaling synergize to efficiently direct epithelial
differentiation of human embryonic stem cells. Stem Cells 26: 372–380.
14. Metallo CM, Ji L, de Pablo JJ, Palecek SP (2010) Directed differentiation of
human embryonic stem cells to epidermal progenitors. Methods Mol Biol 585:
15. Sakurai M, Hayashi R, Kageyama T, Yamato M, Nishida K (2011) Induction of
putative stratified epithelial progenitor cells in vitro from mouse-induced
pluripotent stem cells. J Artif Organs 14: 58–66.
16. Mizuseki K, Sakamoto T, Watanabe K, Muguruma K, Ikeya M, et al. (2003)
Generation of neural crest-derived peripheral neurons and floor plate cells from
mouse and primate embryonic stem cells. Proc Natl Acad Sci U S A 100:
17. Zhou W, Freed CR (2009) Adenoviral gene delivery can reprogram human
fibroblasts to induced pluripotent stem cells. Stem Cells 27: 2667–2674.
18. Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M (2009) Efficient induction
of transgene-free human pluripotent stem cells using a vector based on Sendai
virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad
Ser B Phys Biol Sci 85: 348–362.
19. Montserrat N, Bahima EG, Batlle L, Hafner S, Rodrigues AM, et al. (2011)
Generation of pig iPS cells: a model for cell therapy. J Cardiovasc Transl Res 4:
20. Okita K, Hong H, Takahashi K, Yamanaka S (2010) Generation of mouse-
induced pluripotent stem cells with plasmid vectors. Nat Protoc 5: 418–428.
21. Kaji K, Norrby K, Paca A, Mileikovsky M, Mohseni P, et al. (2009) Virus-free
induction of pluripotency and subsequent excision of reprogramming factors.
Nature 458: 771–775.
22. Woltjen K, Michael IP, Mohseni P, Desai R, Mileikovsky M, et al. (2009)
piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells.
Nature 458: 766–770.
23. Lee CH, Kim JH, Lee HJ, Jeon K, Lim H, et al. (2011) The generation of iPS
cells using non-viral magnetic nanoparticle based transfection. Biomaterials 32:
24. Buchholz DE, Hikita ST, Rowland TJ, Friedrich AM, Hinman CR, et al. (2009)
Derivation of functional retinal pigmented epithelium from induced pluripotent
stem cells. Stem Cells 27: 2427–2434.
25. Kawasaki H, Suemori H, Mizuseki K, Watanabe K, Urano F, et al. (2002)
Generation of dopaminergic neurons and pigmented epithelia from primate ES
cells by stromal cell-derived inducing activity. Proc Natl Acad Sci U S A 99:
26. Martinez-Morales JR, Rodrigo I, Bovolenta P (2004) Eye development: a view
from the retina pigmented epithelium. Bioessays 26: 766–777.
27. Osakada F, Ikeda H, Sasai Y, Takahashi M (2009) Stepwise differentiation of
pluripotent stem cells into retinal cells. Nat Protoc 4: 811–824.
28. Davies SB, Chui J, Madigan MC, Provis JM, Wakefield D, et al. (2009) Stem cell
activity in the developing human cornea. Stem Cells 27: 2781–2792.
29. Davies SB, Di Girolamo N (2010) Corneal stem cells and their origins:
significance in developmental biology. Stem Cells Dev 19: 1651–1662.
30. Pearton DJ, Ferraris C, Dhouailly D (2004) Transdifferentiation of corneal
epithelium: evidence for a linkage between the segregation of epidermal stem
cells and the induction of hair follicles during embryogenesis. Int J Dev Biol 48:
31. Bonfanti P, Claudinot S, Amici AW, Farley A, Blackburn CC, et al. (2010)
Microenvironmental reprogramming of thymic epithelial cells to skin multip-
otent stem cells. Nature 466: 978–982.
32. Blazejewska EA, Schlotzer-Schrehardt U, Zenkel M, Bachmann B,
Chankiewitz E, et al. (2009) Corneal limbal microenvironment can induce
transdifferentiation of hair follicle stem cells into corneal epithelial-like cells.
Stem Cells 27: 642–652.
33. Nakamura T, Endo K, Cooper LJ, Fullwood NJ, Tanifuji N, et al. (2003) The
successful culture and autologous transplantation of rabbit oral mucosal
epithelial cells on amniotic membrane. Invest Ophthalmol Vis Sci 44: 106–116.
34. Nakamura T, Inatomi T, Sotozono C, Amemiya T, Kanamura N, et al. (2004)
Transplantation of cultivated autologous oral mucosal epithelial cells in patients
with severe ocular surface disorders. Br J Ophthalmol 88: 1280–1284.
35. Nishida K, Yamato M, Hayashida Y, Watanabe K, Yamamoto K, et al. (2004)
Corneal reconstruction with tissue-engineered cell sheets composed of
autologous oral mucosal epithelium. N Engl J Med 351: 1187–1196.
36. Yoshida S, Shimmura S, Nagoshi N, Fukuda K, Matsuzaki Y, et al. (2006)
Isolation of multipotent neural crest-derived stem cells from the adult mouse
cornea. Stem Cells 24: 2714–2722.
37. Ogawa Y, Yamazaki K, Kuwana M, Mashima Y, Nakamura Y, et al. (2001) A
significant role of stromal fibroblasts in rapidly progressive dry eye in patients
with chronic GVHD. Invest Ophthalmol Vis Sci 42: 111–119.
Epithelial Progenitor Cells Derived from iPS Cells
PLoS ONE | www.plosone.org 10 December 2011 | Volume 6 | Issue 12 | e28856