EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS
Derivation of Functional Retinal Pigmented Epithelium from Induced
Pluripotent Stem Cells
DAVID E. BUCHHOLZ,a,c,d SHERRY T. HIKITA,a,c,d TEISHA J. ROWLAND,a,c,d AMY M. FRIEDRICH,a,c,d
CASSIDY R. HINMAN,a,c,d LINCOLN V. JOHNSON,a,b,c,d DENNIS O. CLEGGa,b,c,d
a
Center for Stem Cell Biology and Engineering, bCenter for the Study of Macular Degeneration, cNeuroscience
Research Institute, dDepartment of Molecular, Cellular, and Developmental Biology, University of California,
Santa Barbara, California, USA
Key Words. retinal pigmented epithelium • induced pluripotent stem cells • age-related macular degeneration • retinitis pigmentosa
ABSTRACT
Human induced pluripotent stem cells (iPSCs) have great
promise for cellular therapy, but it is unclear if they have
the same potential as human embryonic stem cells
(hESCs) to differentiate into specialized cell types. Ocular
cells such as the retinal pigmented epithelium (RPE) are
of particular interest because they could be used to treat
degenerative eye diseases, including age-related macular
degeneration and retinitis pigmentosa. We show here that
iPSCs generated using Oct4, Sox2, Nanog, and Lin28 can
spontaneously differentiate into RPE cells, which can then
be isolated and cultured to form highly differentiated RPE
monolayers. RPE derived from iPSCs (iPS-RPE) were analyzed with respect to gene expression, protein expression,
and rod outer segment phagocytosis, and compared with
cultured fetal human RPE (fRPE) and RPE derived from
hESCs (hESC-RPE). iPS-RPE expression of marker
mRNAs was quantitatively similar to that of fRPE and
hESC-RPE, and marker proteins were appropriately
expressed and localized in polarized monolayers. Levels of
rod outer segment phagocytosis by iPS-RPE, fRPE, and
hESC-RPE were likewise similar and dependent on integrin avb5. This work shows that iPSCs can differentiate
into functional RPE that are quantitatively similar to
fRPE and hESC-RPE and further supports the finding
that iPSCs are similar to hESCs in their differentiation
potential. STEM CELLS 2009;27:2427–2434
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION
The derivation of human induced pluripotent stem cells
(iPSCs) has created the potential for patient-specific, immunematched cells for regenerative medicine [1, 2]. Human iPSCs
have been derived via expression of Oct4, Sox2, Nanog, and
Lin28 by Yu et al. [1] and others and via expression of Oct4,
Sox2, cMyc, and Klf4 by Takahashi et al. [2] as well as other
investigators. Although iPSCs generated by both methods
have been differentiated into derivatives of the three embryonic germ layers, and iPSC-derived cells have shown efficacy
in animal models of disease [3–7], their differentiation abilities are relatively unexplored [8]. Functional retinal pigmented epithelial (RPE) cells have been derived from human
embryonic stem cells (hESC-RPE) and have been shown to
rescue visual function in the dystrophic rat [9–11]. More
recently, iPSCs reprogrammed using the Yamanaka factors
have been shown to give rise to ocular cells, including RPE
cells [59]. These studies support the idea that stem cell–
derived RPE are good candidates for the treatment of agerelated macular degeneration (AMD) and other degenerative
eye diseases [9, 10, 12–15].
AMD is the leading cause of blindness among people
over 60 years of age [16]. Presenting first with the formation
of drusen (deposits forming between the RPE and Bruch’s
membrane), AMD progresses from the dysfunction and death
of RPE cells to photoreceptor loss and deficits in high-acuity
vision. The RPE plays many roles in visual function: absorption of stray light with pigment granules, formation of the
blood–retina barrier with tight junctions, transport of nutrients
and ions, secretion of growth factors and transport molecules,
isomerization of retinol in the visual cycle, and phagocytosis
of rod outer segments (ROS) [17]. Antiangiogenic pharmaceuticals can slow the wet or exudative form of the disease,
but this applies to only 5% of patients diagnosed with AMD.
For the more common dry or nonexudative form of AMD,
there is no effective treatment option to date [16]. Autologous
transplantation of RPE/choroid from the periphery of the eye
to the macula has demonstrated the potential for RPE cell
Author contributions: D.E.B.: Conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript
writing, final approval of manuscript; S.T.H.: Conception and design, collection and/or assembly of data, data analysis and
interpretation, final approval of manuscript; T.J.R.: Conception and design, collection and/or assembly of data, data analysis and
interpretation, final approval of manuscript; A.M.F.: Collection and/or assembly of data, final approval of manuscript; C.R.H.: Collection
and/or assembly of data, final approval of manuscript; L.V.J.: Conception and design, financial support, final approval of manuscript;
D.O.C.: Conception and design, financial support, final approval of manuscript.
Correspondence: Dennis O. Clegg, Ph.D., Neuroscience Research Institute, University of California, Santa Barbara, California 93106,
USA. Telephone: 805-893-8490; Fax: 805-893-2005; e-mail: clegg@lifesci.ucsb.edu Received May 5, 2009; accepted for publication
C AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1002/
July 16, 2009; first published online in STEM CELLS EXPRESS August 5, 2009. V
stem.189
STEM CELLS 2009;27:2427–2434 www.StemCells.com
Derivation of Functional Retinal Pigmented Epithelium
2428
replacement as a therapy for AMD [18–24]. Use of iPSCderived RPE (iPS-RPE) as a therapy would reduce surgical
complexity and maintain the benefit of immune-matched autologous cells.
In this report, we show that iPSCs reprogrammed using
the Thomson factors [1] spontaneously differentiate into RPE,
and we compare these iPSC-RPE to hESC-RPE and fetal RPE
(fRPE); we find a high degree of similarity in gene expression
patterns and in protein distribution. Furthermore, we show
that iPS-RPE are functionally similar to fRPE and hESC-RPE
in the phagocytosis of ROS, a process critical for proper visual function. Our results demonstrate that iPSCs generated
using the Thomson factors are similar to hESCs in their ability to differentiate into functional RPE, suggesting that iPSRPE may also be beneficial in cellular therapy. This work
was presented in abstract form at the 2008 annual Optical
Society of America Vision Meeting [25].
MATERIALS
AND
METHODS
Pluripotent Stem Cell Culture
Induced pluripotent stem cell lines iPS(IMR90)-3, iPS(IMR90)-4,
iPS(foreskin)-1, and iPS(foreskin)-2 (gift of J. Thomson, University of Wisconsin and UC Santa Barbara) were maintained in
Dulbecco’s modified Eagles medium (DMEM)/F12 containing
2 mM GlutaMAX-I, 10% knockout serum replacement, 0.1 mM
MEM NEAA, 0.1 mM b-mercaptoethanol (Invitrogen, Carlsbad,
CA, http://www.invitrogen.com), and 100 ng/ml recombinant
zebrafish basic fibroblast growth factor (bFGF; gift of J. Thomson) on a mouse embryonic fibroblast feeder layer treated with
mitomycin C (Sigma-Genosys, Cambridge, U.K., http://www.
sigmaaldrich.com/Brands/Sigma_Genosys.html) or a matrigelcoated tissue culture plate (BD Biosciences, San Diego, http://
www.bdbiosciences.com). The hESC line H9 (WiCell Research
Institute, Madison, WI, http://www.wicell.org) was maintained in
DMEM/F12 containing 2 mM GlutaMAX-I, 10% knockout serum
replacement, 0.1 mM MEM NEAA, 0.1 mM b-mercaptoethanol
(Invitrogen), and 4 ng/ml bFGF (Peprotech, Rocky Hill, NJ,
http://www.peprotech.com) on a human foreskin fibroblast (Hs27,
ATCC) feeder layer treated with mitomycin C (Sigma-Genosys).
Differentiation, Enrichment, and Culture of
Pigmented Cells
Spontaneous differentiation of pluripotent stem cells was induced
by removal of bFGF from the medium. The first signs of pigment
were seen 20--35 days after removal of bFGF. These small spots
of pigment grew into larger pigmented foci. After sufficiently
large foci of pigmented cells had developed, they were mechanically dissected and dissociated into single cells using 0.05%
Trypsin/EDTA (Invitrogen) (foci were large enough for dissection
60--90 days after removal of bFGF; see supplementary online
information for exact lengths of time before enrichment). Cell
suspensions were strained through a sterile 30-lm strainer cap
(BD Falcon, Becton, Dickinson and Company, Franklin Lakes,
NJ, http://www.bd.com) to remove clumps and seeded at a minimum of 6.3 104 cell/cm2 onto tissue culture plates coated with
0.1% gelatin for 1 hour at room temperature. Cells were maintained in DMEM (high glucose) containing 7% knockout serum
replacement, 0.1 mM MEM NEAA, 2 mM GlutaMAX I, 0.1 mM
b-mercaptoethanol (Invitrogen), and 5% standard fetal bovine serum (HyClone, Logan, UT, http://www.hyclone.com). bFGF (10
ng/ml; Peprotech, Rocky Hill, NJ, http://www.peprotech.com)
was included in the medium until cells reached confluence [9].
Cells were subcultured monthly by dissociation with 0.05% trypsin/EDTA (Invitrogen) and replated at a minimum of 6.3 104
cells/cm2; 10 ng/ml bFGF (Peprotech) was included in the
medium until the cultures reached confluence. Studies were
performed on cells at passage 0 (enrichment), passage 1, or passage 2. Three separate enrichments of iPS-RPE were examined,
one derived from iPS(IMR90)-3 and two from iPS(IMR90)-4.
Seven separate enrichments of hESC-RPE were examined, all
from H9. One additional enrichment of iPS-RPE from
iPS(IMR90)-4 was performed after 8 months in bFGF-free culture, specifically to examine the protein expression of RPE65 after this length of time.
Fetal Human RPE Cell Culture
Fetal human RPE cells from a 21-week-old donor were a kind
gift of D. Bok (University of California Los Angeles). Cells were
cultured in fetal human RPE media [26] on 0.1% gelatin-coated
tissue culture plates for 30 days prior to use; all fRPE analyses
used fRPE at passage 0.
Quantitative Real-Time Polymerase Chain Reaction
Total RNA was isolated from iPS-RPE cell cultures derived from
iPS(IMR90-3) (one replicate) and iPS(IMR90-4) (two replicates)
at passage 0 (30 days after enrichment), three replicates of hESCRPE from H9’s at passage 1, and three replicates of fRPE using
the Qiagen RNeasy Plus Mini Kit (Qiagen, Hilden, Germany,
http://www1.qiagen.com). cDNA was synthesized from 1 lg of
RNA using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules,
CA, http://www.bio-rad.com). Primer pairs were designed to create a 75–200 base pair product (Beacon Design 4.0; Premier Biosoft International, Palo Alto, CA, http://www.premierbiosoft.
com). Quantitative real-time polymerase chain reaction (PCR)
was carried out on a Bio-Rad MyIQ Single Color Real-Time
PCR Detection System using the SYBR Green method [27]. Triplicate 20-ll reactions were run in a 96-well plate with half of the
cDNA synthesis reaction used per plate. Primer specificity was
confirmed with melting temperature analysis, gel electrophoresis,
and direct sequencing (Iowa State DNA Facility, Ames, IA). Data
was normalized to the geometric mean of the ‘‘housekeeping’’
genes: glyceraldehyde phosphate dehydrogenase (GAPDH), peptidylprolyl isomerase A (PPIA), hydroxymethylbilane synthase
(HMBS) and glucose phosphate isomerase (GPI) [28]. Primer
sequences are listed in supplemental online information.
Immunocytochemistry
iPS-RPE at passage 1 or passage 2 were grown on gelatin-coated
chambered slides for 1 month. For fixation, slides were washed
with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 15
minutes at 4 C, and stored in PBS at 4 C until labeling. Slides
were washed with PBS, blocked with PBS containing 1% BSA,
0.1% NP40, and 1% normal goat or donkey serum in PBS for 1
hour at 4 C, and probed with anti--ZO-1 (Zymed Labs, Invitrogen), anti-Otx2 (10 lg/ml; R&D Systems Inc., Minneapolis, MN,
http://www.rndsystems.com), anti-pigment epithelium-derived factor (PEDF) (10 lg/ml; Lifespan Biosciences, Inc., Seattle, WA,
http://www.lsbio.com), anti-EMMPRIN (1:50; Zymed Labs, Invitrogen), or anti-av integrin (1:100; Santa Cruz Biotechnology
Inc., Santa Cruz, CA, http://www.scbt.com) for 1 hour (ZO-1) or
overnight (all others) at 4 C. Slides were incubated with an
appropriate Alexa Fluor (Invitrogen)–conjugated secondary antibody (1:200) for 30 minutes at 4 C, stained with Hoechst (2 lg/
ml) (Invitrogen) for 5 minutes at room temperature, washed with
PBS, and then imaged at room temperature using an
Olympus BX51 fluorescence microscope or an Olympus Fluoview
500 confocal microscope (Olympus, Tokyo, http://www.
olympus-global.com).
Immunoblot Analysis
iPS-RPE protein cell lysates derived from iPS(IMR90)-4 at passage 0 (30 days in culture) (lysis buffer: 50 mM Tris-Cl (pH 7.6),
150 mM NaCl, 5 mM EDTA, 1% IGEPAL, 0.2% SDS, and
EDTA-free protease inhibitor cocktail set V (Calbiochem, San
Diego, http://www.emdbiosciences.com)) were quantified using a
Buchholz, Hikita, Rowland et al.
2429
Micro BCA Protein Assay Kit (Pierce, Rockford, IL, http://
www.piercenet.com). Total protein was separated via 8% SDSPAGE, transferred to nitrocellulose membranes (Bio-Rad),
blocked for 1 hour at room temperature in tris-buffered saline
tween-20 containing 5% milk, then probed with anti-Mitf (1:100;
Lab Vision, Fremont, CA, http://www.labvision.com), anti-bestrophin (1:1,000; Novus Biologicals, LLC, Littleton, CO, http://
www.novusbio.com), anti-tyrosinase (1:200; Santa Cruz Biotechnologies), or anti-RPE65 (1:5,000; Chemicon, Temecula, CA,
http://www.chemicon.com) for 1 hour at room temperature in
block solution. To visualize labeling, blots were incubated with
secondary anti-mouse Ig conjugated to HRP (1:10,000; GE
Healthcare, Piscataway, NJ, http://www.gehealthcare.com), followed by antigen detection via chemiluminescence (Pierce) and
exposure of the membranes to radiographic film. Lysates for
RPE65 labeling were obtained from iPSCs allowed to differentiate for 8 months in iPSC media in the absence of bFGF. Pigmented and nonpigmented cells were present in these cultures,
and pigmented cells were enriched just prior to protein extraction.
Melanocyte protein lysate was collected from MeWo cells (HTB65; American Type Culture Collection, Manassas, VA, http://
www.atcc.org). Adult human RPE/choroid protein lysate was a
kind gift from the Center for the Study of Macular Degeneration
(University of California Santa Barbara).
ROS Phagocytosis
ROS phagocytosis assays were performed as previously described
[29]. Bovine eyes were obtained fresh from a local slaughterhouse; ROS were purified from retinal extracts and fluorescently
labeled using the FluoReporter FITC Protein Labeling Kit (Invitrogen). Cells were seeded in quadruplicate on gelatin-coated
wells in a 96-well plate at a concentration of 25–50,000 cells per
well and allowed to grow to confluence for 3–4 weeks. Cells
were then challenged with 1 106 FITC-labeled ROS per well
with or without 50 lg/ml anti-avb5 (ab24694; Abcam, Cambridge, U.K., http://www.abcam.com) or 50 lg/ml IgG1 control
(ab9404; Abcam) for 5 hours at 37 C in 5% CO2. Wells were
then vigorously washed five times with warm PBS to remove
unbound ROS. Photomicrographs of total ROS uptake (ROS
bound and internalized) were obtained using an Olympus IX70
inverted microscope equipped with a FITC excitation filter. To
determine the level of ROS internalization, an equal volume of
0.4% trypan blue was added to the PBS for 10 minutes to quench
extracellular fluorescence, followed by four gentle washes with
PBS. The internalized ROS was then documented in fluorescence
photomicrographs. Fluorescence intensity was quantified with
pixel densitometry using ImageJ software (NIH, Bethesda, MD)
for photomicrograph analysis. Photomicrographs from three wells
for each condition were averaged within each assay. Assays were
performed in triplicate on iPS-RPE, hESC-RPE (four replicates),
fRPE, and human foreskin fibroblast (Hs27) cells. Separate
experiments were normalized to the positive control ARPE-19
cell line, which was assayed in each experiment.
RESULTS
Differentiation and Enrichment of Putative RPE
from iPSCs
To test the ability of iPSCs to differentiate into RPE, we
removed bFGF from the medium and allowed the cells to
overgrow and spontaneously differentiate. Using this technique, RPE cells are reproducibly generated from hESC cultures after 6–8 weeks in bFGF-free conditions [9]. Depending
on the iPSC line used, pigment onset typically occurred 20–
35 days after removal of bFGF. The iPS(IMR90) lines,
derived from fetal lung fibroblasts, usually pigmented 1–3
weeks earlier than the iPS(foreskin) lines, which were derived
www.StemCells.com
Figure 1. iPSC differentiation to RPE. Phase contrast photomicrographs of undifferentiated iPS(IMR90-3) (A), iPS(IMR90-4) cells
after 35 days without bFGF (arrows indicate pigmented colonies) (B),
iPS-RPE (passage one) (C), and fRPE (D) are shown. Timeline of
RPE differentiation from bFGF removal (E). Bar ¼ 100 lm (A,C,D);
10 mm (B). Abbreviations: bFGF, basic fibroblast growth factor;
fRPE, fetal retinal pigmented epithelium; iPS-RPE, retinal pigmented
epithelium derived from induced pluripotent stem cells; iPSC, induced
pluripotent stem cell; RPE, retinal pigmented epithelium.
from postnatal foreskin fibroblasts. Over time, pigmented cell
foci expanded and exhibited an epithelial phenotype (Fig. 1).
Pigmented foci were large enough for mechanical dissection
after 2–3 months of bFGF-free culture (see supplemental
online information for exact timelines). Pigmented areas in
iPS(foreskin) lines rarely expanded to a size sufficient for
enrichment, so only iPS(IMR90) lines were used in these
studies.
Upon single-cell dissociation, enriched cells seeded on
gelatin-coated tissue culture plates lost their epithelial morphology and pigment until confluence was reestablished, consistent with observations of both hESC-derived RPE and primary RPE culture [9, 30]. In addition to reestablishment of
morphology and pigment, confluent cultures began to form
domes, indicative of fluid transport through an epithelial sheet
of cells with tight junctions and seen with primary RPE culture [31]. Cells could be serially passaged using trypsin for
several passages (4–5), regaining epithelial morphology and
pigment after each passage. After 4–5 passages, the ability of
iPS-RPE to regain a pigmented epithelial phenotype declined
and they became more fibroblastic in morphology. As discussed in Methods and Materials, characterization of iPS-RPE
was performed on cells derived from iPS(IMR90), no later
than passage 2. hESC-RPE for comparison were derived in
the same manner, as previously reported [9].
iPS-RPE Express RPE Genes and Proteins
and Are Polarized
Quantitative PCR was used to examine expression of genes
involved in crucial RPE functions, as well as those important
in both the parental iPS(IMR90-4) cell line and fRPE
(Fig. 2).
2430
Derivation of Functional Retinal Pigmented Epithelium
Figure 2. iPS-RPE expression of RPE gene transcripts. mRNAs were quantified from undifferentiated iPSCs (IMR90-4), iPS-RPE, hESC-RPE,
and fRPE, normalized to the geometric mean of four housekeeping genes. Bars represent standard error of the mean. Abbreviation: hESC-RPE,
RPE derived from human embryonic stem cells.
We first examined transcription factors that reflect the
states of pluripotency (Oct4, Nanog), early eye field development (Pax6, Rax, Six3), or differentiated RPE (Mitf, Otx2).
iPS-RPE did not express transcripts for the pluripotency transcription factors Oct4 or Nanog, but low levels of Rax and
Six3, transcription factors involved in early eye field development, were seen in iPS-RPE, hESC-RPE, and fRPE. Interestingly, Pax6 transcripts were seen in iPS-RPE and hESC-RPE
but not in fRPE. Pax6 is expressed during RPE development
but is turned off as the RPE matures, suggesting that iPS-RPE
and hESC-RPE may be similar to immature RPE [32]. The
transcription factors Mitf and Otx2 are necessary for RPE differentiation [32] and are expressed at similar levels in iPSRPE, hESC-RPE, and fRPE. Otx2 is also expressed in undifferentiated iPSCs, as has been found in hESCs [33].
The expression of genes involved in the many functions
of differentiated RPE was also examined. Tyrosinase, Tyrp1,
Tyrp2, and SILVER, genes involved in pigment synthesis [34],
were expressed in iPS-RPE at levels equal to or higher than
levels in hESC-RPE and fRPE. CRALBP and RPE65 are
genes involved in the visual cycle and are expressed by terminally differentiated RPE [35]. Transcripts for these genes
were present at lower levels in iPS-RPE and hESC-RPE than
fRPE, again suggesting that these cells may represent immature RPE. The genes encoding the tight junction proteins claudin and ZO-1 were expressed at similar levels in iPSCs, iPSRPE, hESC-RPE, and fRPE. Transthyretin and PEDF are both
secreted apically towards the neural retina by RPE cells, the
former for binding of retinoids, the latter as an antiangiogenic
neurotrophin [36, 37]. Transcripts for both these proteins
showed high levels in iPS-RPE, hESC-RPE, and fRPE.
mRNAs encoding bestrophin and EMMPRIN, proteins localized to the membrane of the RPE [38], were also expressed at
similar levels in iPS-RPE, hESC-RPE, and fRPE. Transcripts
between the three enriched iPS-RPE cell lines were similar
but not identical (supplemental online information).
To confirm that iPS-RPE expressed RPE proteins along
with their transcripts, immunoblotting and immunocytochemistry were used to examine expression and localization (Fig.
3). The tight junction protein ZO-1 was detected on the lateral
margins of most cells, as expected (Fig. 3A). Expression was
not uniform throughout the entire population, which was
attributed in part to blocking of fluorescence by highly pigmented cells (Fig. 3A, arrowheads). EMMPRIN was diffusely
expressed, consistent with membrane localization. PEDF
staining was both diffuse and punctuate, as has been previously reported [39]. Expected apical localization of the integrin av subunit, relative to nuclear Otx2, is shown in Figure
3B. The transcription factor Mitf was detected in immunoblots of iPS-RPE as a doublet at 65–70 kDA (Fig. 3C), consistent with isoforms found in RPE [40]. In contrast, MeWo
cells (a melanocyte cell line) express shorter isoforms, with a
doublet detected at 52–56 kDA (Fig. 3C). Bestrophin and
tyrosinase proteins were both expressed by iPS-RPE.
Although low levels of mRNA were present for the visual
cycle gene RPE65 in 1-month-old cultures, protein expression
could not be detected at this time point. However, RPE65
protein expression was observed in iPS-RPE when cells were
cultured for 8 months in the absence of bFGF and without
enrichment (Fig. 3C).
iPS-RPE Internalize ROS
Daily phagocytosis of shed photoreceptor outer segments is
an essential function of the RPE [17]. An ROS phagocytosis
assay [29] was used to see if iPS-RPE could perform this
function (Fig. 4). Isolated bovine ROS were fluorescently labeled with FITC and incubated with iPS-RPE, hESC-RPE,
fRPE, or the human fibroblast cell line Hs27 for 5 hours. Free
Buchholz, Hikita, Rowland et al.
2431
Figure 3. Expression and localization of RPE proteins in iPS-RPE. (A) Immunofluorescence images of ZO-1, EMMPRIN, PEDF (green), and
nuclei (Hoechst, blue), are shown with corresponding bright-field images. Bar ¼ 100 lm. (B) A confocal micrograph depicts a 21-lm Z-stack
with integrin av (green) and Otx2 (red) staining. Bar ¼ 20 lm. (C) Immunoblots for Mitf, bestrophin, tyrosinase, and RPE65 are shown. Abbreviation: PEDF, pigment epithelium-derived factor.
outer segments were washed away, and external fluorescence
was quenched with trypan blue to visualize internalized outer
segments. iPS-RPE phagocytosis of ROS was as efficient as
hESC-RPE and fRPE and approximately three times more
efficient than the negative control Hs27 cells.
RPE are known to utilize integrin avb5 in the initial binding of outer segments, prior to internalization [29, 41]. To
study the role of integrin avb5 in the phagocytosis of ROS by
iPS-RPE and hESC-RPE, we coincubated the cells and ROS
with either a control IgG antibody or an inhibitory antibody
(P1F6) against integrin avb5. Addition of the P1F6 antibody
blocked ROS internalization by all cell types, while addition
of the control antibody did not affect ROS internalization.
This indicates that, like primary fRPE, the mechanism of iPSwww.StemCells.com
RPE and hESC-RPE phagocytosis is integrin avb5–dependent.
The efficiency of ROS internalization between the three
enrichments of iPS-RPE cell lines was similar but not identical (supplemental online information).
DISCUSSION
We have derived RPE from induced pluripotent stem cells
that are highly similar to primary fetal human RPE and RPE
from human embryonic stem cells. These cells spontaneously
arise from differentiating iPSCs generated with the Thomson
factors [1] after 20–35 days, a timeline slightly faster than
2432
Figure 4. ROS phagocytosis by iPS-RPE. Levels of ROS internalization as determined by pixel analysis of photomicrographs are
shown for iPS-RPE, hESC-RPE, fRPE, and negative control
fibroblast cells (Hs27). Bars represent standard error of the mean.
Abbreviations: anti-avB5, blocking anti-integrin avb5 antibody; IgG,
isotype-matched control antibody; ROS, rod outer segment.
normal human RPE development in utero (approximately 40
days to pigmentation) and similar to spontaneous differentiation from human embryonic stem cells [9, 42]. Gross morphology of iPS-RPE is highly similar to that of primary RPE
cultures as well as adult human RPE [43, 44].
iPS-RPE express RPE gene transcripts at levels similar to
those of cultured fetal human RPE and hESC-RPE. Immunocytochemistry and immunoblotting show that RPE proteins
are also expressed by iPS-RPE, in a polarized manner, as
observed in normal RPE [43, 44]. Immunocytochemistry
showed nonuniform labeling, likely due in part to fluorescence blocking by pigment granules, but also potentially due
to heterogeneity in maturity states. Like hESC-RPE [9], iPSRPE dedifferentiate and lose pigmentation upon passage and
then regain maturity upon confluence.
It is interesting to note that protein expression of RPE65
is only found after long-term culture (8 months), although
mRNA is consistently present under shorter culture conditions
(30 days). RPE65 is an essential gene in visual function,
mutations of which cause Leber’s congenital amaurosis [45,
46]. Similar reports of low/absent RPE65 protein expression,
despite mRNA expression in primary RPE cell culture, were
made upon discovery of the gene, suggesting post-transcriptional regulation for the mRNA by surrounding ocular tissues
[47, 48]. We observed long-term culture-dependent (8 month)
RPE65 protein expression when iPS-RPE cells were not isolated from other differentiated cells. It is possible that neural
retinal cells were in these cultures, regulating RPE65 protein
expression. Length of time in culture also appears to have an
important role in RPE65 expression as enriched cultures of
hESC-RPE grown for greater that 7 months without passage
had marked increases in RPE65 protein expression [Hikita
et al., manuscript in preparation]. It is unclear whether the
increase in RPE65 protein expression in our 8-month unenriched iPS-RPE culture was caused by signaling from other
cells or by length of time in culture. It is also unclear whether
Derivation of Functional Retinal Pigmented Epithelium
RPE cultured for long time periods are functionally different
from RPE from shorter cultures, aside from RPE65 protein
expression. We expect that long-term RPE cultures in the
presence of appropriate periocular tissue will be the most terminally differentiated. It will be interesting to see whether
transplantation of iPS-RPE cells into animal models increases
RPE65 protein expression. We believe this is likely the case,
as we have seen that transplantation of iPS-RPE into the
Royal College of Surgeon’s (RCS) rat, a model of retinal dystrophy [46, 47], rescues visual function [Carr et al., manuscript in preparation]. Because RPE65 is required for visual
function, it is likely that these cells produce the functional
RPE65 protein in this model, although further testing is
required to show this.
Daily phagocytosis of shed photoreceptor outer segments
by the RPE is essential for the removal of proteins damaged
by ultraviolet radiation and recycling of important nutrients
[17]. Loss of this RPE function by mutation of the gene for
the internalization receptor MerTK is the cause of blindness
in the RCS rat [49, 50]. iPS-RPE can perform this critical
function in vitro as effectively as fetal human RPE, acting
through the same integrin avb5--dependent mechanism.
Importantly, phagocytosis activity in vivo has been demonstrated for hESC-RPE in the RCS rat model [10], and recently
we have shown that iPS-RPE can also rescue vision in this
animal [Carr et al., manuscript in preparation], demonstrating
that phagocytosis of photoreceptor outer segments by iPSRPE occurs in vivo.
The propensity of different hESC lines to differentiate
into specific cell types has been shown to be dependent on
the cell line [51, 52]. We have also seen a difference in the
propensity to spontaneously give rise to RPE between different iPSC lines. Specifically, the iPS(foreskin) lines had a
lower propensity to spontaneously differentiate into RPE than
the iPS(IMR90) lines. Additionally, among the different
enrichments of RPE from iPS(IMR90), different levels of
transcript expression and levels of phagocytosis were seen.
The cause for these differences between enrichments is
unclear. They may be due to the different iPS(IMR90) clones
used, the length of time prior to enrichment of RPE, or different non-RPE cell types that spontaneously arise within differentiating cultures.
For therapeutic use it will be important to establish specific protocols for derivation of RPE from stem cells that consistently give rise to cells of similar quality. In that light, it
will be necessary to have a panel of assays to determine the
quality of the cells derived. In addition to the assays used in
the current study (gene/protein expression, quantitative ROS
phagocytosis), other assays could include measurements of
transepithelial resistance [53], enzyme-linked immunosorbent
assays to detect polarized secretion of trophic factors (PEDF,
vascular endothelial growth factor) [26], and a retinoid metabolism assay [54]. Relating these assays to functional levels in
animal models will give a robust screening method for each
batch of stem cell RPE.
Multiple groups have derived RPE from hESC [9, 10, 12–
15]. Data presented here shows that iPS-RPE are similar to
previous reports of hESC-RPE in gene expression and function. We have found that hESC-RPE cell lines that we have
derived are quantitatively similar to iPS-RPE with respect to
mRNA levels of RPE markers and to phagocytosis ability.
More recently, we have used microarray analysis to compare
genome-wide transcript levels between iPS-RPE, hESC-RPE,
and fRPE, and we find that stem cell--derived RPE share a
common mRNA expression pattern (Radeke et al., manuscript
in preparation). Both iPS-RPE and hESC-RPE, similar to primary cultures of RPE [55], lose their phenotype and senesce
Buchholz, Hikita, Rowland et al.
after repeated passaging. However, we estimate that a single
six-well plate of differentiating stem cells can give rise to
approximately 107 RPE cells after two passages. Because the
macula is small, it would require RPE cells on the order of
105 to cover the entire area, so it is feasible to generate a
quantity of cells needed for therapies. We are investigating
several possible ways to increase the amount of RPE derived
from a given enrichment: improved efficiency through
directed differentiation, improved culture techniques to maintain RPE stability over multiple passages, or the use of a
larger number of undifferentiated stem cells.
Although the iPSCs used in this study are not suitable for
human trials because of transgene integration, nonintegrative
iPS cells have recently been derived [56, 57]. Although generation of patient-specific iPS lines would be a costly and challenging paradigm for future health care, iPS-RPE may be
superior to hESC-RPE because there would be less chance of
immune rejection. One challenge associated with iPSCs
derived from the patient is the possibility of reintroducing any
genetic defects that contributed to the disease. Combining iPS
technology with gene therapy is a possible solution [58]. For
AMD this could mean replacing a faulty Factor H haplotype,
which has been associated with increased propensity to
develop the disease [15]. However, any alteration of the
genome carries with it a bevy of risks that will have to be
weighed against the need. In the case of AMD and other agerelated degenerative diseases, gene therapy may not be necessary. For a disease that takes 50 years or more to progress,
reintroduction of cells harboring a slow-acting genetic defect
may be of no concern.
Degenerative diseases do carry their own challenges for
cell replacement therapy, however, the central challenge being
the window of intervention. Transplantation of cells has to be
early enough in disease progression to maintain function in a
dying tissue. Additionally, degenerative diseases may lead to
a dysfunctional microenvironment such as drusen deposits
and a dystrophic Bruch’s membrane in AMD [15]. We are
investigating synthetic substrates that could be cotransplanted
with stem cell-RPE to act as a surrogate Bruch’s membrane.
While this manuscript was in preparation, a report of RPE
derived from iPSCs was published [59]. The iPSCs used in
that report were produced using the Yamanaka transcription
REFERENCES
1
2
3
4
5
6
7
8
Yu J, Vodyanik MA, Smuga-Otto K et al. Induced pluripotent stem
cell lines derived from human somatic cells. Science 2007;318:
1917–1920.
Takahashi K, Tanabe K, Ohnuki M et al. Induction of pluripotent
stem cells from adult human fibroblasts by defined factors. Cell 2007;
131:861–872.
Xu D, Alipio Z, Fink LM et al. Phenotypic correction of murine
hemophilia A using an iPS cell-based therapy. Proc Natl Acad Sci
U S A 2009;106:808–813.
Zhang J, Wilson GF, Soerens AG et al. Functional cardiomyocytes
derived from human induced pluripotent stem cells. Circ Res 2009;
104:e30–e41.
Zhang D, Jiang W, Liu M et al. Highly efficient differentiation of
human ES cells and iPS cells into mature pancreatic insulin-producing
cells. Cell Res 2009;19:429–438.
Hanna J, Wernig M, Markoulaki S et al. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 2007;318:1920–1923.
Karumbayaram S, Novitch BG, Patterson M et al. Directed differentiation of human-induced pluripotent stem cells generates active motor
neurons. Stem Cells 2009;27:806–811.
Nishikawa S, Goldstein RA, Nierras CR. The promise of human
induced pluripotent stem cells for research and therapy. Nat Rev Mol
Cell Biol 2008;9:725–729.
www.StemCells.com
2433
factors [2], whereas our iPSCs were derived using the Thomson transcription factors [1]. Although the iPS-RPE from
Yamanaka iPSCs have not yet been fully characterized, this
indicates that iPS-RPE derivation from iPSCs is not dependent on the reprogramming mechanism.
CONCLUSION
Proper gene expression and cellular function of iPS-RPE suggests that the cells are a viable candidate for cellular therapy
to treat degenerative eye diseases such as AMD and retinitis
pigmentosa. It is still unclear which pluripotent cell type,
embryonic or induced, will prove most effective for therapies.
We show here that iPSCs generated by expression of Oct4,
Sox2, Nanog, and Lin28 are similar to hESCs in their ability
to produce functional RPE, and that iPS-RPE are remarkably
similar to primary fetal human RPE. A quantitative in vitro
phagocytosis assay shows that both iPS-RPE and hESC-RPE
are highly similar to fRPE in the levels and mechanism of
photoreceptor outer segment phagocytosis.
ACKNOWLEDGMENTS
We thank James Thomson and Jessica Antosiewicz-Bourget for
cells, reagents and advice; Dean Bok for cells; and Peter Coffey,
Don Anderson, and Monte Radeke for helpful suggestions and
advice. This work was supported by the California Institute for
Regenerative Medicine grants T300009, C4-00521-1; Army
Research Office; Millipore Corporation; Advanced Cell Technology; NIH 5R24EY014799-05; NIH NCRR Shared Instrumentation Grant 1S10RR017753-07.
DISCLOSURE
OF
OF
POTENTIAL CONFLICTS
INTEREST
The authors indicate no potential conflicts of interest.
9
10
11
12
13
14
15
16
17
Klimanskaya I, Hipp J, Rezai KA et al. Derivation and comparative
assessment of retinal pigment epithelium from human embryonic stem
cells using transcriptomics. Cloning Stem Cells 2004;6:217–245.
Lund RD, Wang S, Klimanskaya I et al. Human embryonic stem cellderived cells rescue visual function in dystrophic RCS rats. Cloning
Stem Cells 2006;8:189–199.
Lu B, Malcuit C, Wang S et al. Long-term safety and function of
RPE from human embryonic stem cells in preclinical models of macular degeneration. Stem Cells 2009 [Epub ahead of print].
Carr AJ, Vugler A, Lawrence J et al. Molecular characterization and
functional analysis of phagocytosis by human embryonic stem cellderived RPE cells using a novel human retinal assay. Mol Vis 2009;
15:283–295.
Vugler A, Carr AJ, Lawrence J et al. Elucidating the phenomenon of
HESC-derived RPE: anatomy of cell genesis, expansion and retinal
transplantation. Exp Neurol 2008;214:347–361.
Gong J, Sagiv O, Cai H et al. Effects of extracellular matrix and
neighboring cells on induction of human embryonic stem cells into
retinal or retinal pigment epithelial progenitors. Exp Eye Res 2008;86:
957–965.
Vugler A, Lawrence J, Walsh J et al. Embryonic stem cells and retinal
repair. Mech Dev 2007;124:807–829.
Gehrs KM, Anderson DH, Johnson LV et al. Age-related macular
degeneration—emerging pathogenetic and therapeutic concepts. Ann
Med 2006;38:450–471.
Strauss O. The retinal pigment epithelium in visual function. Physiol
Rev 2005;85:845–881.
Derivation of Functional Retinal Pigmented Epithelium
2434
18 Ma Z, Han L, Wang C et al. Autologous transplantation of retinal pigment epithelium-Bruch’s membrane complex for hemorrhagic agerelated macular degeneration. Invest Ophthalmol Vis Sci 2008.
19 Chen FK, Uppal GS, Rubin GS et al. Evidence of retinal function
using microperimetry following autologous retinal pigment epithelium-choroid graft in macular dystrophy. Invest Ophthalmol Vis Sci
2008;49:3143–3150.
20 Heussen FM, Fawzy NF, Joeres S et al. Autologous translocation of
the choroid and RPE in age-related macular degeneration: 1-year follow-up in 30 patients and recommendations for patient selection. Eye
2008;22:799–807.
21 MacLaren RE, Uppal GS, Balaggan KS et al. Autologous transplantation of the retinal pigment epithelium and choroid in the treatment of
neovascular age-related macular degeneration. Ophthalmology 2007;
114:561–570.
22 Treumer F, Bunse A, Klatt C et al. Autologous retinal pigment epithelium-choroid sheet transplantation in age related macular degeneration: morphological and functional results. Br J Ophthalmol 2007;91:
349–353.
23 Chen FK, Uppal GS, MacLaren RE et al. Long-term visual and microperimetry outcomes following autologous retinal pigment epithelium
choroid graft for neovascular age-related macular degeneration. Clin
Experiment Ophthalmol 2009;37:275–285.
24 Chen FK, Patel PJ, Uppal GS et al. A comparison of macular translocation with patch graft in neovascular age-related macular degeneration. Invest Ophthalmol Vis Sci 2009;50:1848–1855.
25 Buchholz DE, Hikita ST, Radeke MJ et al. Stem cell derived retinal
pigment epithelium for the treatment of age-related macular degeneration. Journal Of Vision 2008;8:47–47.
26 Maminishkis A, Chen S, Jalickee S et al. Confluent monolayers of
cultured human fetal retinal pigment epithelium exhibit morphology
and physiology of native tissue. Invest Ophthalmol Vis Sci 2006;47:
3612–3624.
27 Woo TH, Patel BK, Cinco M et al. Identification of Leptospira biflexa
by real-time homogeneous detection of rapid cycle PCR product.
J Microbiol Methods 1999;35:23–30.
28 Radeke MJ, Peterson KE, Johnson LV et al. Disease susceptibility of
the human macula: differential gene transcription in the retinal pigmented epithelium/choroid. Exp Eye Res 2007;85:366–380.
29 Lin H, Clegg DO. Integrin alphavbeta5 participates in the binding of
photoreceptor rod outer segments during phagocytosis by cultured
human retinal pigment epithelium. Invest Ophthalmol Vis Sci 1998;
39:1703–1712.
30 Zhao S, Rizzolo LJ, Barnstable CJ. Differentiation and transdifferentiation of the retinal pigment epithelium. Int Rev Cytol 1997;171:
225–266.
31 Aronson JF. Human retinal pigment cell culture. In Vitro 1983;19:
642–650.
32 Martinez-Morales JR, Rodrigo I, Bovolenta P. Eye development: a
view from the retina pigmented epithelium. Bioessays 2004;26:
766–777.
33 Sperger JM, Chen X, Draper JS et al. Gene expression patterns in
human embryonic stem cells and human pluripotent germ cell tumors.
Proc Natl Acad Sci U S A 2003;100:13350–13355.
34 Marks MS, Seabra MC. The melanosome: membrane dynamics in
black and white. Nat Rev Mol Cell Biol 2001;2:738–748.
35 Thompson DA, Gal A. Vitamin A metabolism in the retinal pigment
epithelium: genes, mutations, and diseases. Prog Retin Eye Res 2003;
22:683–703.
36 Buraczynska M, Mears AJ, Zareparsi S et al. Gene expression profile
of native human retinal pigment epithelium. Invest Ophthalmol Vis
Sci 2002;43:603–607.
37 Tombran-Tink J, Barnstable CJ. PEDF: a multifaceted neurotrophic
factor. Nat Rev Neurosci 2003;4:628–636.
38 Marmorstein AD, Marmorstein LY, Rayborn M et al. Bestrophin, the
product of the Best vitelliform macular dystrophy gene (VMD2),
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
localizes to the basolateral plasma membrane of the retinal pigment
epithelium. Proc Natl Acad Sci U S A 2000;97:12758–12763.
Tombran-Tink J, Shivaram SM, Chader GJ et al. Expression, secretion, and age-related downregulation of pigment epithelium-derived
factor, a serpin with neurotrophic activity. J Neurosci 1995;15:
4992–5003.
Bharti K, Liu W, Csermely T et al. Alternative promoter use in eye
development: the complex role and regulation of the transcription factor MITF. Development 2008;135:1169–1178.
Finnemann SC, Bonilha VL, Marmorstein AD et al. Phagocytosis of
rod outer segments by retinal pigment epithelial cells requires
alpha(v)beta5 integrin for binding but not for internalization. Proc
Natl Acad Sci U S A 1997;94:12932–12937.
O’Rahilly R. The prenatal development of the human eye. Exp Eye
Res 1975;21:93–112.
Al-Hussaini H, Schneiders M, Lundh P et al. Drusen are associated
with local and distant disruptions to human retinal pigment epithelium
cells. Exp Eye Res 2009;88:610–612.
Clegg DO, Buchholz D, Hikita S, et al. Retinal pigment epithelial
cells: development in vivo and derivation from human embryonic
stem cells in vitro for treatment of age-related macular degeneration.
In: Shi Y, Clegg DO, eds. Stem Cell Research and Therapeutics:
Springer Publishing, 2008:1–24.
Redmond TM, Poliakov E, Yu S et al. Mutation of key residues of
RPE65 abolishes its enzymatic role as isomerohydrolase in the visual
cycle. Proc Natl Acad Sci U S A 2005;102:13658–13663.
Marlhens F, Bareil C, Griffoin JM et al. Mutations in RPE65 cause
Leber’s congenital amaurosis. Nat Genet 1997;17:139–141.
Hamel CP, Tsilou E, Harris E et al. A developmentally regulated
microsomal protein specific for the pigment epithelium of the vertebrate retina. J Neurosci Res 1993;34:414–425.
Hamel CP, Tsilou E, Pfeffer BA et al. Molecular cloning and expression of RPE65, a novel retinal pigment epithelium-specific microsomal protein that is post-transcriptionally regulated in vitro. J Biol
Chem 1993;268:15751–15757.
D’Cruz PM, Yasumura D, Weir J et al. Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat. Hum Mol
Genet 2000;9:645–651.
Vollrath D, Feng W, Duncan JL et al. Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk. Proc
Natl Acad Sci U S A 2001;98:12584–12589.
Chang KH, Nelson AM, Fields PA et al. Diverse hematopoietic potentials of five human embryonic stem cell lines. Exp Cell Res 2008;314:
2930–2940.
Mikkola M, Olsson C, Palgi J et al. Distinct differentiation characteristics of individual human embryonic stem cell lines. BMC Dev Biol
2006;6:40.
Sonoda S, Spee C, Barron E et al. A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells.
Nat Protoc 2009;4:662–673.
Nikolaeva O, Takahashi Y, Moiseyev G et al. Purified RPE65 shows
isomerohydrolase activity after reassociation with a phospholipid
membrane. FEBS J 2009;276:3020–3030.
Song MK, Lui GM. Propagation of fetal human RPE cells: preservation of original culture morphology after serial passage. J Cell Physiol
1990;143:196–203.
Yu J, Hu K, Smuga-Otto K et al. Human induced pluripotent stem
cells free of vector and transgene sequences. Science 2009;324:
797–801.
Zhou H, Wu S, Joo JY et al. Generation of induced pluripotent stem
cells using recombinant proteins 2009;4:381–384.
Raya A, Rodriguez-Piza I, Guenechea G et al. Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent
stem cells. Nature 2009;460:53–59.
Hirami Y, Osakada F, Takahashi K et al. Generation of retinal cells
from mouse and human induced pluripotent stem cells. Neurosci Lett
2009;458:126–131.
See www.StemCells.com for supporting information available online.