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EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS
Pluripotent Stem Cell Model Reveals Essential Roles for miR-450b-5p
and miR-184 in Embryonic Corneal Lineage Specification
RUBY SHALOM-FEUERSTEIN,a,b LAURA SERROR,b STEPHANIE DE LA FOREST DIVONNE,b ISABELLE PETIT,a
EDITH ABERDAM,b LIVIA CAMARGO,a ODILE DAMOUR,c CLOTILDE VIGOUROUX,c ABRAHAM SOLOMON,d
CÉDRIC GAGGIOLI,e JOSEPH ITSKOVITZ-ELDOR,b SAJJAD AHMAD,f DANIEL ABERDAMa,b
INSERM U898, and eINSERM U634, Nice, France; bINSERTECH, B. Rappaport Faculty of Medicine, Technion,
Haifa, Israel; cBanque de tissus et cellules, Hospices civils de Lyon et IBCP, FRE, Lyon, France; dDepartment of
Ophthalmology, Hadassah University Hospital, Jerusalem, Israel; fInstitute of Human Genetics, Newcastle
University, Newcastle upon Tyne, United Kingdom
a
Key Words. Induced pluripotent stem cells • Cell therapy • Cornea • miR-450b-5p • miR-184 • Pax6
ABSTRACT
Approximately 6 million people worldwide are suffering
from severe visual impairments or blindness due to corneal
diseases. Corneal allogeneic transplantation is often
required to restore vision; however, shortage in corneal
grafts and immunorejections remain major challenges. The
molecular basis of corneal diseases is poorly understood
largely due to lack of appropriate cellular models. Here, we
described a robust differentiation of human-induced pluripotent stem cells (hiPSCs) derived from hair follicles or
skin fibroblasts into corneal epithelial-like cells. We found
that BMP4, coupled with corneal fibroblast-derived conditioned medium and collagen IV allowed efficient corneal
epithelial commitment of hiPSCs in a manner that recapitulated corneal epithelial lineage development with high purity. Organotypic reconstitution assays suggested the ability
of these cells to stratify into a corneal-like epithelium. This
model allowed us identifying miR-450b-5p as a molecular
switch of Pax6, a major regulator of eye development. miR450b-5p and Pax6 were reciprocally distributed at the presumptive epidermis and ocular surface, respectively. miR450b-5p inhibited Pax6 expression and corneal epithelial
fate in vitro, altogether, suggesting that by repressing Pax6,
miR-450b-5p triggers epidermal specification of the ectoderm, while its absence allows ocular epithelial development. Additionally, miR-184 was detectable in early eye
development and corneal epithelial differentiation of
hiPSCs. The knockdown of miR-184 resulted in a decrease
in Pax6 and K3, in line with recent findings showing that a
point mutation in miR-184 leads to corneal dystrophy. Altogether, these data indicate that hiPSCs are valuable for
modeling corneal development and may pave the way for
future cell-based therapy. STEM CELLS 2012;30:898–909
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION
The cornea, which is the outermost tissue of the eye, plays an
essential optic role and also serves as a barrier against external insults. Like the epidermis, the corneal epithelium originates from the ectoderm, which is hallmarked by the expression of cytokeratin pair K8/K18. At day 9.5 of mouse
embryogenesis (E9.5), the expression of Pax6, a master regulator of eye development, becomes restricted to a narrow
zone at the head surface ectoderm that is committed to eye
development, while the adjacent surface ectoderm in which
Pax6 expression is switched off will give rise to the epidermis. These Pax6-positive cells thicken and form the lens placode structure that invaginates and gives rise to the lens and
corneal epithelium [1]. Further commitment of the ectoderm
into the epithelial lineages (e.g., epidermal, oral, and corneal
epithelium) is orchestrated by P63 and is hallmarked by the
substitution of K8/K18 by cytokeratins of epithelial progenitors (K5/K14), at E11-12. Corneal epithelial-specific cytokeratins are K12 and K3, which are firstly detected around E15.5
and E17.5, respectively [2].
Pax6 deficiency results in no eye development [3], while
Pax6 haploinsufficiency and ectopic expression of Pax6 result
in severe eye and corneal defects [4, 5], reminiscent of the
phenotype of aniridia patients that suffer from a mutation in
the PAX6 allele [6, 7]. Interestingly, rabbit corneal epithelium
transdifferentiated into a hair-forming epidermis when transplanted on mouse dermis, and this sequential process was
accompanied by loss of Pax6 expression [8, 9]. Similarly, a
Author contributions: R.S.-F.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript
writing, and final approval of manuscript; L.S.: collection and assembly of data and data analysis and interpretation; S.D.L.F.D., I.P.,
E.A., L.C., and C.G.: collection and assembly of data; O.D., C.V., and A.S.: provision of materials; J.I.-E.: financial support and
administrative support; S.A.: provision of materials and final approval of manuscript; D.A.: conception and design, data analysis and
interpretation, manuscript writing, and final approval of manuscript.
Correspondence: Ruby Shalom-Feuerstein, Ph.D., INSERM U898, Faculty of Medicine, 28, Avenue Valombrose, Nice 06107, France.
Telephone: (33) 1 60 87 89 23/81; Fax: (33) 1 60 87 89 23/81; e-mail: [email protected] Received January 1, 2012; Revised FebruC AlphaMed
ary 2, 2012; accepted for publication February 9, 2012; first published online in STEM CELLS EXPRESS February 24, 2012. V
Press 1066-5099/2012/$30.00/0 doi: 10.1002/stem.1068
STEM CELLS 2012;30:898–909 www.StemCells.com
Shalom-Feuerstein, Serror, De La Forest Divonne et al.
reduction in Pax6 expression was observed in corneal diseases
in which the cornea gains epidermal characteristics and
becomes opaque [10]. Altogether, these accumulating data
supported the hypothesis that Pax6 is directing the choice
between epidermal and corneal fate; however, the mechanism
of Pax6 deregulation remains elusive.
Eye development and homeostasis are regulated by microRNAs (miRNAs) [11–15], a group of small noncoding RNA
molecules. However, no information is available regarding the
expression or function of miRNAs during human corneal
embryogenesis. Recently, a point mutation at the seed
sequence of miR-184 was linked with eye syndromes affecting the cornea and the lens [16, 17]. However, the exact stage
and mechanism by which miR-184 acts remain unknown.
Corneal diseases affect more than 10 million people worldwide [18]. The molecular basis for the initiation and progression
of those diseases is largely unknown. New models are required
for developing diagnostic tools and specific drugs for a variety
of inherited and acquired corneal diseases. Corneal transplantation is common and on the whole successful. However, a shortage in corneal grafts limits treatment of corneal diseases, and as
the graft is allogeneic, immune rejection always remains a risk
[19]. Despite topical immunosuppression, corneal allograft
rejection is common in the long-term [19]. Therefore, alternative cell sources for autologous therapy are being widely investigated [20–23], with only partial success to date.
Human pluripotent stem cells, either embryonic stem cells
(hESCs) or reprogrammed induced pluripotent stem cells
(hiPSCs), are powerful cellular models to recapitulate in vitro
embryonic events and could provide cellular sources for therapy,
with hiPSCs derived from patients as autologous alternatives.
Previous studies have shown that hESCs can partially differentiate into corneal epithelial-like cells [24, 25]. Here, we designed
a robust protocol that recapitulates the canonical steps of corneal
epithelial embryogenesis from hair follicle (HF)-derived and
skin fibroblasts-derived hiPSC lines. It gave rise to a relatively
pure population of differentiated corneal epithelial-like cells.
This cellular model was further used to identify miRNAs profiling during corneal epithelial differentiation and revealed new
roles for two miRNAs in corneal embryonic lineage.
MATERIALS
AND
METHODS
Tissue Culture
Unless indicated otherwise, reagents were from Invitrogen. All cultures were in 37 C, 5% CO2, and humidified incubator. hiPSCs
and hESCs (H9) were grown on mitomycin-treated (Sigma, 8 lg/
ml, 3 hours) mouse embryonic fibroblasts (MEFs) (40,000 cells per
square centimeter on gelatin [0.1%]) in hiPSC medium (85% Dulbecco’s modified Eagle’s medium [DMEM]/F12 [1:1], 15% serum
replacement,1% nonessential amino acids, 0.1 mM b-mercaptoethanol, and 8 ng/ml basic fibroblast growth factor). Medium of human
corneal epithelial (HCE) cell line contained DMEM/F12 (1:1), 5%
fetal calf serum (FCS), 5 lg/ml insulin, 0.5% dimethyl sulfoxide,
and 10 ng/ml epidermal growth factor (EGF). Epithelial media
contained 60% DMEMþGlutMax, 30% F12, 10% fetal clone II,
5 lg/ml insulin, 0.5 lg/ml hydrocortisone, 10 ng/ml EGF, 0.2 mM
adenine, and 10 mM choleratoxin. Other cells were grown in
DMEM supplemented with 10% FCS. Keratinocytes were isolated
by trypsin treatment of HFs and grown in coculture on mitomycintreated NIH3T3 cells in epithelial medium. Fibroblasts were isolated by as previously described [26, 27].
Generation of hiPSCs
Lentiviral infections for hiPSC generation were performed as previously described [27]. Emerging colonies with pluripotent stem
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cell morphology were manually isolated and cocultured on mitomycin-treated MEF in 24-well plates. Some three to four colonies
were amplified and characterized for the expression of pluripotent
markers by real-time polymerase chain reaction (PCR) analysis
and immunofluorescent staining, and their differentiation potential
was tested by the formation of embryoid bodies followed by realtime PCR analysis of various markers of embryonic lineages
[27].
Corneal Differentiation and Transfection
During Differentiation
Conditioned medium (CM) was prepared by treating confluent
corneal fibroblasts (CFs) or limbal fibroblasts (LFs) cultures with
mitomycin (8 lg/ml, 3 hours), the next day washed with phosphate buffered saline (PBS), and epithelial media (15 ml) was
added. CM (15 ml) was collected every day for 10 days and was
kept in 20 C if necessary for up to 1–2 months or at 4 C up to
7 days before filtering and using for corneal differentiation. Corneal differentiation was induced by seeding hiPSCs/hESCs (1:2)
on collagen IV (Sigma)-coated dishes [24], in CF-CM or LF-CM
that was replaced every second day. Cells were trypsinized if
necessary.
For transfections during differentiation, trypsinized cells were
resuspended in CFs-CM and reseeded (1:2) on collagen IV-coated
plates. Transfections were performed the next day or following
cell adhesion (4–6 hours) by Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer recommendations using 2 lg
K12-GFP plasmid or 50 lM pre-miR, or control oligos, or antimiR (Ambion, Saint Aubin, France).
Western Blots
Cell were lysed in RIPA buffer (TrisHCl 10 mM, 10 mg/ml deoxycholate, 1% NP40, 1% SDS, 150 mM NaCl, and protease
inhibitors cocktail [Roche, Paris, France]) and subjected to SDSpolyacrylamide gel electrophoresis, followed by immunoblotting.
Antibodies used were mouse anti-K3 (1:300) (Millipore, Molsheim, France), mouse anti-K12 (1:20) (a kind gift from Danielle
Dhouailly), rabbit anti-Pax6 (1:300) (Chemicon, Paris, France),
rabbit anti-Connexin43 (1:500) (Thermo Scientific, Auburn, AL),
rabbit anti-Oct4 (Santa Cruz), rabbit anti-Nanog (1:500) (R&D,
Lille, France), and mouse anti-b-tubulin (1:1,000) (Santa Cruz,
Heidelberg, Germany). Proteins were visualized by enhanced
chemiluminescence (Amersham Pharmacia Biotech, Piscataway,
NJ) and quantified by densitometry with Image Master VDS-CL
using TINA 2.0 software (Ray Tests).
Quantitative Real-Time PCR and TaqMan
Assay for miRNAs
cDNA was prepared from 1 lg RNA (Trizol extraction) using
iScript Reverse Transcriptase kit (BioRad, Marnes-la-Coquette,
France). Each reaction of real-time PCR contained 12.5 ll
SYBR-Green PCR Master Mix (Applied Biosystems, Carlsbad,
CA), 0.125 ll MultiScribe Reverse Transcriptase (Applied Biosystems, Carlsbad, CA), 5 ll cDNA, and 5 ll primer mix (0.5
lM, sequences available in Supporting Information Table S3),
adjusted to 25 ll reaction volume. For TaqMan assays of miRNAs, 5 ll RNA 5 ng/ll was subjected to PCR using the reverse
transcription kit and miRNA-specific primers (Applied Biosystems, Carlsbad, CA). Reaction products were further subjected to
real-time PCR using TaqMan universal master mix and TaqMan
miRNA-probes or U54 as control (Applied Biosystems, Carlsbad,
CA). The relative amounts of each mRNA or miRNA was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
or U54 reaction, respectively, and the relative expression of each
reaction was calculated as a fold change relative to control sample. Wide miRNA profiling was performed by TaqMan low density miRNA arrays (Applied Biosystems, Carlsbad, CA), according to the manufacturer’s instructions. The results were
normalized using expression of small nucleolar RNA, according
to the manufacturer’s protocol.
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Immunofluorescent Staining
Immunofluorescent staining were performed as previously
described [28, 29]. The following primary antibodies were used:
rabbit anti-K14 (1:200) (Covance, Paris, France), rabbit anti-K5
(1:200) (Covance, Paris, France), mouse anti-K18 (1:200) (Chemicon, Paris, France), rabbit anti-Pax6 (1:100) (Chemicon, Paris,
France), mouse anti-K3 (1:100) (Millipore, Molsheim, France),
mouse anti-P63 (1:100) (Santa Cruz, Heidelberg, Germany), and
mouse anti-E-Cadherin (1:100) (R&D, Lille, France).
In Situ Hybridization
In situ was performed as detailed previously [30] with minor
modifications. Frozen sections were fixed (15 minutes, 4% PFA
[Electron Microscopy Sciences]), followed by PBS wash (all
washes described below were 3 5 minutes at room temperature), acetylation (15 minutes), PBS wash, proteinase K treatment
(5 minutes [Roche, Paris, France]), PBS wash, prehybridization
(1 hour), and hybridization with locked nucleic acid (LNA) detection probes (50 /30 -Dig labeled (Exiqon), at 54 C and 44 C for
miR-184 and miR-450b-5p, respectively). The next day, samples
were washed (30 minutes, 54 C, 5 saline-sodium citrate (SSC)
[BIO-LAB, Jerusalem, Israel]), washed (1 hour, 60 C, 0.2
SSC), washed (10 minutes, TBS [Bio-Rad, Marnes-la-Coquette,
France]), incubated for 30 minutes (3% H2O2), washed (TBS supplemented with 0.1% Tween 20 [Sigma, Paris, France]), blocked
(10% naı̈ve goat serum [Biological Industries, Beit Ha-Emek,
Israel]), incubated with anti-Dig antibody ([1:500], 1 hour, in
blocking solution) followed by tris-buffered saline and Tween 20
(TBST) wash, tyramide signal amplification step (Perklin Elmer)
for 10 minutes, TBST wash, and mounting in Dapi mounting gel
(Sigma, Paris, France).
Cloning and Luciferase Assay
To clone K12 promoter region (1,390 bases upstream to K12 start
codon) downstream to green fluorescent protein (GFP)-encoding
sequence, mouse DNA was used as template for PCR using specific primers (5-cttacggaggcctgccatg-3, 5-gctggtatgccagaagggg-3).
The amplified fragment was inserted into StuI and AgeI sites of
pEGFP-1 plasmid (Clontech, Mountain View, CA). Cells were
cotransfected with 1.5 lg K12-GFP plasmid and 50 lM pre-miR450b-5p or control oligos. Transfection efficiency was determined
separately by pEGFP transfection. Transfectants were subjected
to fluorescent microscopy and data were normalized and K12GFP expression was calculated as the ratio between the percentages of K12-GFP positive among transfectants (percentage of
pEGFP positive).
For generating LUC-Pax6 plasmid, the 30 untranslated region
(UTR) region of PAX6 gene was amplified (primers used: 50 tatactcgagaaaggaaaggaaauauuguguuaauuc-30 ,
50 -tatagcggccgcaaggttaaaaagaaaactgtataataaa-30 ) and cloned downstream to luciferase-encoding sequence at xhoI/NotI sites of psiCHECH2
plasmid. Site-directed mutagenesis allowed the generation of
LUC-DmiR-450b-5p, a mutated form of LUC-Pax6 plasmid in
which the miR-450b-5p binding site is disrupted, by PCR (pri50 mers
used:
50 -aacagatgaagtttatgtaacgaaaagggtaaga-30 ,
0
tcttacccttttcgttacataaacttcatctgtt-3 ) using LUC-Pax6 plasmid as
template. Luciferase activity was determined using luciferase kit
(Promega, Paris, France) in lysates of 293 human embryonik kidney (HEK) cells that were cotransfected with 0.3 lg LUC-Pax6
or LUC-DmiR-450b-5p and pre-miR-450b-5p or control oligos
(Ambion) by Fugene reagent (Invitrogen, Saint Aubin, France).
Organotypic Reconstitution of Corneal Equivalent
Tissue In Vitro
Primary CFs were embedded in gels that were prepared in 24well plates [31], incubated 4 hours at 37 C, followed by reseeding trypsinized hESCs that were differentiated for 7 days in sixwell plates, on these gels. The next day, air-liquid interphase was
miR-450b-5p and miR-184 Regulate Corneal Lineage
induced for 8 days [31] followed by tissue sectioning and staining
as detailed above.
RESULTS
Pluripotent Stem Cell Differentiation Recapitulated
Corneal-Epithelial Commitment
HF keratinocytes and dermal fibroblasts (DFs) were isolated
and reprogrammed by infection with a lentiviral vector containing a polycistronic cassette of OCT4, SOX2, CMYC, and
KLF4 (Fig. 1A). As demonstrated by real-time PCR analysis
(Fig. 1B) and immunofluorescent staining (Fig. 1C), we
selected individual clones of reprogrammed cells that displayed typical hiPSC morphology and expressed markers of
pluripotency. The ability of hiPSCs to differentiate into the
three embryonic germline lineages was confirmed by the formation of embryoid bodies followed by inspection of the
expression of various markers of the endoderm, ectoderm,
and mesoderm (Fig. 1D and [27]).
Previously, corneal epithelial differentiation was demonstrated by seeding hESCs on collagen IV-coated dishes in
LF-CM [24]. This method was relatively efficient, resulting
in some 60% of the cells expressing K3, a marker of terminally differentiated corneal epithelial cells. However, the
expression of limbal stem/progenitors cell markers (e.g., P63
and K14) was highly variable between the pluripotent stem
cell lines used ([27] and not shown). We challenged human
iPSCs to corneal fate by introducing two major modifications
on the protocol designed for hESCs. Instead of using epithelia medium conditioned by LFs that are isolated from the
limbus, a narrow band of tissue of limited amount, we used
CFs isolated from the entire cornea including the corneal periphery. In addition, we tested the effect of bone morphogenetic factor 4 (BMP4), a major regulator of embryonic epithelial commitment [32, 33]. Accordingly, hiPSCs that were
derived from HF cells (HF1-hiPSC) were seeded on collagen
IV-coated dishes in the presence of CF-CM and were treated
with BMP4 (0.5 nM) during restricted periods of differentiation (days 0–3 or 4–8 or 9–12 or for the whole period of 12
days of differentiation) (Fig. 2B, 2D). At day 12, cells were
harvested and subjected to real-time PCR, immunofluorescent
staining, or Western blotting. Among all conditions tested,
addition of BMP4 during the first 3 days efficiently enhanced
corneal epithelial differentiation, as illustrated by increased
expression of markers of corneal progenitors, DNP63 and
K14 (Fig. 2B, 2C), and markers of terminally differentiated
corneal epithelial cells, K12 (Fig. 2D) and K3 (Fig. 2E),
while constant BMP4 treatment seemed unnecessary. Corneal
epithelial differentiation was completely abrogated in the
presence of the BMP antagonist, LDN-193189 (LDN) (Fig.
2B, 2D), indicating that endogenous BMP signaling is essential for corneal epithelial commitment of hiPSCs. As shown
in Supporting Information Figure S1, epithelial media that
was conditioned by either LFs or CFs induced corneal epithelial lineage commitment of hiPSC to a similar manner.
To follow corneal epithelial commitment, corneal epithelial lineage gene expression was recorded at different time
points during HF1-hiPSCs differentiation by real-time PCR
analysis (Fig. 3A). Elevation in ectodermal marker K18 (and
K8, not shown) appeared already within 2–4 days, along with
the expression of Pax6 (Fig. 2A), reminiscent of their coexpression in vivo in the embryonic lens placode cells. Markers
of corneal epithelial progenitors emerged at day 8 by the
expression of DNP63 and K14, while markers of terminally
Shalom-Feuerstein, Serror, De La Forest Divonne et al.
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Figure 1. Generation of hiPSCs from HF keratinocytes and DFs. (A): Schematic representation of hiPSCs generation from HF keratinocytes
and DFs. An example of real-time polymerase chain reaction (PCR) analysis (B) or immunofluorescent staining and AP assay (C) that were performed for screening the expression of the indicated markers of individual clones that were identified as hiPSCs. (D): To assess pluripotency of
colonies, hiPSCs were subjected to differentiation through formation of embryoid bodies as described in Materials and Methods, followed by
RNA extractions and real-time PCR analysis of endodermal (AFP), ectodermal (K14), and mesodermal (CD31) markers. Data were normalized to
GAPDH and represents the relative fold change in gene expression as compared to undifferentiated hiPSCs. Abbreviations: AFP, alpha-fetoprotein (AFP); AP, alkaline phosphatase; DF, dermal fibroblast; hESCs, human embryonic stem cells; HF, hair follicle; hiPSCs, human-induced pluripotent stem cells.
differentiated corneal epithelium (K3, K12) peaked at day 14
(Fig. 3A). This method was efficient for hESCs and for
hiPSCs derived from HFs and DFs with relatively minor variability (Supporting Information Fig. S2A). At day 8, the vast
majority of cells expressed K18, while few cells were already
positive for K5, an epithelial progenitor cell marker (Fig. 3B).
In addition, first sign of corneal epithelial differentiation was
already evident at day 8 by the coexpression of K3 and K14.
Increasing expression of K5, K14, and K3 was observed by
day 14 (Fig. 3B). Typical corneal epithelial characteristic was
also evident by the expression of E-Cadherin, K3, and Pax6
(Fig. 3C). This data were confirmed by Western blot analysis,
showing that K3, K12, and Connexin 43 increased during differentiation, while Oct4 and Nanog declined and disappeared
within 6–8 days of differentiation (Supporting Information
Fig. S2B). The robustness of corneal epithelial-like cell production was quantified at day 14 by fluorescence-activated
cell sorting (FACS) analysis. As shown in Figure 3D, some
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20%–25% of the cell population was K14-positive progenitor
cells, while most cells expressed K3 (>90%). We further generated a GFP-K12 construct carrying the corneal epithelialspecific promoter of K12 upstream to the green fluorescent
protein (GFP) encoding sequence. Specificity of the construct
was evaluated by transfecting different cell types with the
K12-GFP construct followed by FACS analysis. As demonstrated in Figure 3E, the K12 promoter was silent in noncorneal cells (HeLa, HaCaT, and undifferentiated hESCs) but
active in corneal epithelial cell line (HCE), and significantly
expressed by hESCs, which were differentiated as described
for 12 days.
To test the ability of these cells to stratify, pluripotent
cells were differentiated into corneal epithelial fate for a
week and then trypsinized and seeded on the top of a stroma
equivalent made of CF embedded into collagen gel for a
three-dimensional organotypic reconstitution assay. As illustrated in Figure 3F, differentiated cells were able to stratify
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miR-450b-5p and miR-184 Regulate Corneal Lineage
Figure 2. Differentiation of hiPSCs into corneal epithelial cells. (A): Schematic representation of culture method. hiPSCs were seeded on collagen
IV-coated dishes in the presence of corneal fibroblast-conditioned media (CF-CM) and supplemented with BMP4 at days 0–3, as described in Materials and Methods. (B, D): Corneal differentiation was performed on collagen IV and CF-CM. BMP4 or LDN (a BMP inhibitor) (þ) or vehicle ()
were supplemented during the indicated periods. Real-time polymerase chain reaction analysis of the indicated genes (B, D) was performed at day
12. Immunofluorescent staining (C) and Western blotting (E) of the indicated proteins were performed at day 12 of differentiation in the presence of
BMP4 (supplemented at days 0–3) or vehicle (Ctl). Scale bar in (C) ¼ 20 lm. Abbreviations: BMP, bone morphogenetic factor; LDN, LDN193189; hiPS, human-induced pluripotent stem.
and form two to three layers of cells coexpressing K3 and
Pax-6.
miRNA Profiling of hESCs During Corneal
Epithelial Differentiation
Although miRNAs are essential for eye development and corneal integrity [3, 14, 17], the distribution and function of
human miRNAs during embryonic commitment into the corneal epithelial lineage are unknown. We thus performed a
wide miRNA profiling during corneal epithelial differentiation
of pluripotent stem cells at days 2, 6, and 10 (Fig. 4A). The
array data were confirmed by TaqMan assays for four miR-
NAs using the same RNA samples that were used for the
array (Fig. 4B) and also by examining RNA preparations
from two additional independent experiments (not shown). In
agreement with the array data, the levels of miR-24, miR27b, and miR-184 gradually increased during corneal epithelial differentiation, while the levels of miR-450a decreased at
days 2 and 6 and then increased at day 10 (Fig. 4B), thus
confirming the reliability of the array data. Comparable
expression profiles were shown for these four miRNAs in corneal epithelial differentiation of two clones of hiPSCs derived
from HFs (Supporting Information Fig. S3 and not shown).
Next, we compared our data with a list of 21 miRNAs that
were previously identified as miRNAs with the highest
Shalom-Feuerstein, Serror, De La Forest Divonne et al.
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Figure 3. Human-induced pluripotent stem cell (hiPSC) differentiation recapitulated major steps of corneal epithelial embryogenesis. (A--D):
Corneal epithelial differentiation of hair follicle-derived hiPSCs was performed as in Figure 1A, cells were harvested at the indicated days and
subjected to real-time polymerase chain reaction analysis (A), immunofluorescent staining (B, C), or flow cytometer analysis (D) of the indicated
markers. (E): The indicated cell lines or hiPSCs that were differentiated for 12 days (Day 12) were transfected with K12-GFP plasmid followed
by flow cytometry, as described in Materials and Methods. Data represent the percentage of K12-GFP-positive cells among transfectants (calculated as described in Materials and Methods). (F): Organotypic reconstitution of pluripotent stem cell-derived cells followed by immunofluorescent staining of Pax6 and K3 (F). Inset shows enlargement of the annotated region. Scale bar in B, C, F ¼ 20 lm. Abbreviations: GFP, green
fluorescent protein; HCE, human corneal epithelial.
expression levels in adult mouse corneal epithelium [34].
Notably, 86% of these miRNAs was significantly induced
(>2) at day 10 of corneal epithelial differentiation (Fig. 4A),
suggesting that miRNA profile of pluripotent stem cellderived corneal epithelial-like cells shares a high degree of
similarity with that of corneal epithelial cells in vivo.
miR-450b-5p Represses Pax6 Expression by
Binding to Its 30 -Untranslated Region
Pax6 is a major corneal transcription factor and mutations in
the PAX6 gene are associated with aniridia, a rare eye dystrowww.StemCells.com
phy affecting the iris and cornea, leading to blindness [35].
Interestingly, inductions of Pax6 protein (Fig. 5A) and mRNA
(Fig. 5B) levels were shown at day 2 of differentiation,
peaked around day 6, and then moderately dropped down at
day 14. Surprisingly, miRNAs related to Pax6 regulation have
not been described yet. We thus used the cellular model
described here to identify miRNAs that may potentially inhibit Pax6 expression. In silico analysis of the 30 -untranslated
region (30 UTR) of Pax6 messenger RNA by ‘‘TargetScan’’
software (http://www.targetscan.org/vert_50/) allowed the prediction of 51 potential binding sites for different miRNAs.
We next screened the expression pattern of the 51 candidates
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Figure 4. MicroRNA (miRNA) profiling during human embryonic
stem cell (hESC) differentiation into corneal epithelial lineage. (A):
miRNA array was performed using RNA samples obtained from
hESC differentiation for the indicated days. Heatmap presentation
shows the fold change in the expression of 21 miRNAs that were previously found to have the highest levels in the adult mouse corneal
epithelium [34], listed in a descending order according to their
expression. (B): TaqMan assay using specific probes for the indicated
miRNAs. Data represent the relative fold change of each miRNA as
compared to the levels in undifferentiated hESCs (A, B).
in our array data to identify miRNAs that display reciprocal
expression profile relative to Pax6. miR-450b-5p was clearly
positive for this criterion (Fig. 5B). The match between the
seed sequence of miR-450b-5p and positions 570-576 of Pax6
30 UTR and to upstream sequences is illustrated in Figure 5C.
The sequence of the 30 UTR of Pax6 gene is highly evolutionarily conserved among vertebrates, and the seed sequence of
miR-450b-5p is conserved between mouse and human but not
all mammals (Supporting Information Table S1).
At E14.5, Pax6 was expectedly detected in the ocular surface epithelium and absent in the adjacent epidermis of the
miR-450b-5p and miR-184 Regulate Corneal Lineage
eye lids (Fig. 5D, left). Remarkably, in situ hybridization
revealed that miR-450b-5p was distributed throughout the presumptive epidermis, at the surface ectoderm that is negative
for Pax6, while it was absent in the ocular regions that are
Pax6 positive (Fig. 5D). It was difficult to specifically detect
miR-450b-5p at earlier embryonic stages. This reciprocal
expression could suggest that Pax6 repression by miR-450b5p in the presumptive epidermis prevents ocular epithelial
commitment of the ectoderm.
To further test this hypothesis, we examined whether
miR-450b-5p can bind to and repress Pax6. We cloned the
30 UTR region of Pax6 (1,023 bases) downstream to a luciferase gene as illustrated in Figure 6A. Additionally, by sitedirected mutagenesis, we generated a mutated construct
(DmiR-450b-5p) in which the binding site of miR-450b-5p
was disturbed (illustrated in Fig. 6A and underlined mutated
sequence in Fig. 5C). 293HEK cells were cotransfected with
the wild-type construct (LUC-Pax6) or the mutated construct
(LUC-DmiR-450b-5p) along with increasing concentrations of
pre-miR-450b-5p or with scrambled oligonucleotide as control. As shown in Figure 6B, a significant and dose-dependent
decrease in the luciferase activity was observed in the presence of pre-miR-450b-5p. This effect was specific to premiR-450b-5p as cotransfection with pre-miR-450a, which is
clustered with miR-450b-5p and shares high sequence similarities with miR-450b-5p but yet contains altered seed
sequence, had no such effect (Fig. 6B). Moreover, this inhibitory effect of miR-450b-5p on luciferase activity was dependent on the integrity of the predicted binding site, as miR450b-5p had almost no effect on the mutated luciferase construct (Fig. 6B). This data indicated that miR-450b-5p can
specifically bind to the 30 UTR of Pax6 and may inhibit Pax6
expression.
To further assay the effect of miR-450b-5p on PAX6 gene
expression, we transfected HCE cells by 50 nM anti-miR450b-5p (AM), pre-miR-450b-5p (PM), or control oligos (Ctl)
followed by Western blotting (Fig. 6C). Transfection was confirmed by TaqMan assay coupled with real-time PCR analysis
(Supporting Information Fig. S3A). As shown in Figure 6C,
Pax6 expression significantly decreased in the presence of
pre-miR-450b-5p, and an inverse effect was shown in the
presence of specific antago-miR.
We next addressed the question of whether the decrease
in miR-450b5p during early corneal epithelial differentiation
(Fig. 5B) was directly linked to an increase in Pax6 protein
(Fig. 5A, 5B). To prevent the decline of miR-450b-5p during
commitment, hESCs were transfected by pre-miR-450b-5p at
day 1 of corneal epithelial commitment (transfection validation is shown in Supporting Information Fig. S3B), and Pax6
expression was determined by Western blot analysis. As
expected, Pax6 expression was attenuated by the presence of
pre-miR-450b-5p (Fig. 6D), confirming that Pax6 is a target
of miR-450b-5p, and indicating that the decrease in the levels
of miR-450b-5p is contributing to the induction in Pax6 protein expression during early neuroectodermal commitment of
hESCs [36].
The in situ hybridization analysis shown in Figure 5D
could suggest that by repressing Pax6 at the presumptive epidermis, miR-450b-5p was preventing the ocular epithelial
fate. To further test this hypothesis, cotransfection of K12GFP and pre-miR-450b-5p or control oligos was performed at
day 7 of corneal epithelial differentiation, and cells were analyzed at day 10 of differentiation. As shown in Figure 6E,
transfection of pre-miR-450b-5p significantly reduced K12
promoter activity. Altogether, these data indicate that miR450b-5p inhibits Pax6 expression and regulates corneal epithelial fate.
Shalom-Feuerstein, Serror, De La Forest Divonne et al.
905
Figure 5. Inverse correlation in the expression of miR-450b-5p and Pax6. hESCs were differentiated into corneal epithelial fate for the indicated periods followed by Western blotting of Pax6 and b-tubulin (b-tub) (A), real-time PCR analysis of Pax6 (B), or TaqMan assay of miR450b-5p (B). (C) In silico analysis (by TargetScan (http://www.targetscan.org/)) showing the predicted binding of miR-450b-5p (seed sequence in
red) and Pax6-30 UTR. Below is the mutated form of Pax6-30 UTR that is described in Figure 6A, 6B (mutated sequence is underlined). (D): In
situ hybridization of miR-450b-5p using cryosections of mouse embryos at E14.5. Basement membrane is annotated by a dashed line. Right inset
is an enlargement of the dashed region, showing a staining of miR-450b-5p in the corneal stroma but not epithelium. Below (left) is immunofluorescent staining of Pax6 in an adjacent section showing that Pax6 expression is restricted to the presumptive conjunctiva and cornea. White
arrows indicate positive staining of miR-450b-5p and no staining of Pax6 in the epidermis. Red arrows demonstrate positive staining of Pax6 and
negative staining of miR-450b-5p at the conjunctiva and ocular surface. Scale bar ¼ 50 lm. Abbreviations: el, eye lid; ep, corneal epithelium; st,
stroma.
miR-184 Is Essential for Corneal-Epithelial
Lineage Commitment
miR-184 showed the highest hybridization signal among all
adult mouse corneal epithelial miRNAs listed in vivo [34]. In
silico analysis revealed that the mature sequence of miR-184
is extremely conserved in vertebrate’s evolution (Supporting
Information Table S2). miR-184 expression, which gradually
elevated concomitantly with markers of epithelial progenitors
at days 6–8, markedly increased (>100-fold increase) during
terminal differentiation stages (Fig. 4A, 4B). Previously, miR184 was detected in the corneal epithelium at postnatal stages
[13]. In situ hybridization performed at E11.5 revealed that
miR-184 is detectable at low levels in the head surface ectoderm and at higher levels in the newly formed lens vesicle.
The expression of miR-184 in advanced embryonic stages
was punctuated or variable (Fig. 7A). At day 8 after birth
(P8), prior to eye lid opening and first corneal stratification,
www.StemCells.com
miR-184 was clearly detected in the corneal epithelium,
which comprises one to two layers, and became restricted to
the corneal epithelial basal layer following stratification (at
P14, not shown). Intense miR-184-signal was observed in the
lens, while unclear signals were occasionally found in the corneal stroma or endothelium at P8 (Fig. 7A). Remarkably,
miR-184 was also expressed in the epidermis and in late anagen-staged HFs (Fig. 7A). No significant staining was shown
with scrambled probes (not shown). Altogether, this expression profile suggested that miR-184 plays a role in early eye
development in the lens and the cornea, in line with the early
onset of severe corneal and lens abnormalities in patients with
miR-184 mutation [16, 17]. In addition, the specific expression of miR-184 in the skin and HF suggests that it plays a
general role in ectodermal derivatives.
We next addressed the question of whether the increase in
the level of miR-184 contributes to embryonic commitment
into the corneal epithelial lineage. hESCs were transfected at
906
day 7 of corneal epithelial differentiation (as described in
Materials and Methods) with a synthetic antagonizing antimiR-184 oligonucleotides or scrambled sequences as control
and analysis was performed at day 14. The marked elevation of
miR-184 expression during differentiation was significantly
attenuated in the presence of antago-miR-184 (AM) (Fig. 7B).
Importantly, the knockdown of miR-184 resulted in reduced
levels of K3 and Pax6, as demonstrated by immunofluorescent
staining (Fig. 7C). In agreement, FACS analysis confirmed that
inhibition of miR-184 expression prevented the corneal epithelial fate of committed hESCs (Fig. 7C). This data suggest that
miR-184 is essential for corneal epithelial embryogenesis. The
temporal concomitant appearance of miR-184 during early epi-
miR-450b-5p and miR-184 Regulate Corneal Lineage
thelial commitment in vitro and in vivo implies that it may first
act at this particular developmental stage.
DISCUSSION
We demonstrated here that pluripotent stem cells are useful
for modeling corneal epithelial embryogenesis. By seeding
hiPSCs on collagen IV-coated dishes in the presence of CFCM allowed corneal differentiation that was augmented by
the presence of BMP4. Indeed, the major steps of corneal development were recapitulated in vitro and miRNA profiling
during embryonic commitment into the corneal epithelial fate
was obtained, and allowed the identification of miR-450b-5p
and miR-184 as essential for this lineage.
Pax6 must be tightly regulated for appropriate eye development [4–6], while abnormal expression of Pax6 is associated with eye diseases [7, 10]. Therefore, Pax6 regulators
have significant importance. In this study, we revealed the
first described role for miR-450b-5p, as a Pax6-repressing
miRNA. The sharp reciprocal correlation between Pax6 and
miR-450b-5p at E14.5 suggested that miR-450b-5p acts as a
molecular switch that directs cell fate decision of the ectoderm. Accordingly, miR-450b-5p is repressing Pax6 in the
developing epidermis and thereby allowing initiation of epidermal morphogenesis, while the absence of miR-450b-5p is
required for Pax6 expression in the developing ocular surface.
When so, the knockout of miR-450b-5p at the surface ectoderm could possibly result in ectopic eye formation, while the
uncontrolled expression of miR-450b-5p in the ectoderm is
expected to interfere with lens placode formation and/or eye
development.
Given that Pax6 also regulates early neuroectodermal
commitment [36], embryonic and adult neurogenesis [37], and
pancreatic b-cell function [38], this regulatory mechanism
may have a broader relevance in various tissues. Future studies using genetically modified mice should allow tracing the
expression and assessing the roles of miR-450b-5p in different
tissues in which it may modulate Pax6. Strikingly, mutations
in two rare genetic syndromes [39, 40] were linked with a
defined region at chromosome X, which encompasses the
locus of MIR450B (Xq26.3). Cerebello-trigeminal-dermal
dysplasia is a rare genetic disorder that is accompanied by
several defects including mental retardation, corneal opacity,
Figure 6. miR-450b-5p represses Pax6 by binding to the 30 UTR.
(A): Schematic representation of cloning into luciferase vector of the
wild-type Pax6-30 UTR (1,023 bases length) and a mutated form
(referred as DmiR-450b-5p, sequence indicated in Fig. 5C) in which
the miR-450b-5p binding site (positions 570-576) was disrupted. (B):
Luciferase assay of 293HEK cells cotransfected with a luciferase construct containing wild-type Pax6-30 UTR (LUC-Pax6) or its mutated
form (LUC-DmiR-450b-5p) and increasing concentrations (25–50
nM) of pre-miR-450b-5p or pre-miR-450a (50 nM) or scrambled oligos as control. Data represent the relative fold change in luciferase
activity as compared to control transfectant. (C): Western blot analysis of Pax6 and b-tubulin (b-tub) in HCE cells transfected with premiR-450b-5p (PM) or anti-miR-450b-5p (AM) or control sequence
(Ctl). hESCs were transfected at day 1 of corneal differentiation, harvested at day 5, and subjected to Western blot analysis of Pax6 and
b-tubulin (D). (E): hESCs were differentiated for 7 days and cotransfected with K12-GFP plasmid and pre-miR-450b-5p or control oligos
followed by flow cytometry at day 10. Data represent the percentage
of K12-GFP-positive cells among transfectants (calculated as
described in Materials and Methods). Abbreviations: HCE, human
corneal epithelial; hESC, human embryonic stem cell; UTR, 30 untranslated region (30 UTR).
Shalom-Feuerstein, Serror, De La Forest Divonne et al.
907
Figure 7. Contribution of miR-184 to corneal epithelial differentiation of human embryonic stem cells (hESCs). (A): In situ hybridization of
miR-184 was performed using cryosections of mouse tissues of the indicated stages. Positive staining at the developing ocular surface is highlighted by arrows. Scale bar ¼ 50 lm. (B): hESCs were transfected at day 7 of corneal differentiation with anti-miR-184 oligonucleotides (AM)
or scrambled sequences (Ctl), and cells were harvested at day 14 of differentiation (B--D). Transfection validation by TaqMan assay is presented
in (B). Immunofluorescent staining (C) and flow cytometer analysis (D) of K3 and Pax6 is shown. Scale bar ¼ 20 lm. Abbreviations: el, eye lid;
le, lens; re, retina; se, surface ectoderm; st, stroma.
hypoplasia of the cerebellum [39]. The X-linked split-head/
split-foot malformation 2 is associated with developmental
defects in ectodermal structures and is characterized by the
absence of medial digital rays, syndactyly, and median cleft
of the hands and feet [40]. It would be therefore essential to
examine whether these phenotypes, which could hypothetically be owing to aberrant modulation of Pax6, involve mutation in miR-450b-5p.
Recently, it was shown that Pax6 is involved in early
ectodermal cell fate determination [36] in line with the
observed increase in Pax6 at early differentiation stages of
hiPSCs. Our data indicated that the decrease in miR-450b-5p
is an essential molecular switch of Pax6 at early ectodermal
commitment of pluripotent stem cells. It is possible that
Pax6-repression by miR-450b-5p contributes to the pluripotent
state of hiPSCs and its decrease allows the exit from this
state, similar to the role of miR-302 that has been shown to
be a guardian of pluripotency [41] and ameliorates the effiwww.StemCells.com
ciency of somatic cell reprogramming [42]. Accordingly, it
would be interesting to examine whether pre-miR-450b-5p
has similar roles.
Very recently, a point mutation in miR-184 was linked
with early onset of eye defects involving the corneal epithelium, corneal endothelium, and the lens [16, 17], in line with
the expression pattern of miR-184 by in situ hybridization.
Corneal diseases such as keratoconus and limbal stem cell
deficiency lack appropriate cellular models to allow the characterization of the etiology and the underlying molecular
defects. Our model suggests that mutations in miR-184 in
patients could be a primary embryonic defect, affecting normal corneal epithelial commitment. The knockdown of miR184 resulted in a decrease in Pax6 and K3, which are both
important for corneal integrity and their deregulation was
associated with corneal diseases [10, 43]. Since the 30 UTRs
of K3 and Pax6 do not contain predictable binding sites for
miR-184, this effect was most probably indirect. Recent
miR-450b-5p and miR-184 Regulate Corneal Lineage
908
studies have identified genes that are repressed by miR-184.
However, the miR-184-binding sites of these four genes identified in the fly [44] and mouse [45]) are not evolutionarily
conserved. The stringent conservation of miR-184 among vertebrates suggests that the function of miR-184 in the eye is
indispensable for the viability and/or fitness of these species.
Additionally, miR-184 regulates neural stem cells [45], germline cells [44], and epidermal cells (our unpublished data).
These functions need to be examined in mouse models or
human diseases.
CONCLUSIONS
We have demonstrated that a highly enriched population of corneal epithelial-like cells could be obtained from pluripotent
stem cells of different origins. Corneal therapy by means of
hiPSC-derived autologous transplantation may circumvent the
major problems of current allogeneic therapies namely graft
rejection and a shortage of corneal donors. This remarkable
therapeutic potential of hiPSCs must be further investigated in
vivo. Moreover, derivation of hiPSCs from somatic cells of
patients with defined genetic alterations may provide new models for identifying the abnormal molecular circuitry of a given
disease and developing appropriate treatments. Indeed, we have
recently generated hiPSCs from patients that have a mutation in
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ACKNOWLEDGMENTS
We acknowledge Thierry Virolle and Virginie Virolle for the
help in miRnome experiment. This work was partially supported
by the Agence Nationale de Recherche (ANR-08-GENOPAT024) and the Israel Ministry of Science and Technology (MOST
3-6494 for DA) to D.A., J.I.E., and A.S., a ‘‘Poste Vert’’ fellowship of INSERM, a ‘‘Chateaubriand’’ fellowship of the Embassy
of France in Israel to R.S.F., and a Ph.D. fellowship from the Israeli Ministry of Integration to L.S.
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CONFLICTS OF INTEREST
The authors declare no conflict of interest.
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