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Efficient Stage-Specific Differentiation of Human
Pluripotent Stem Cells Toward Retinal Photoreceptor
Cells
Carla B. Mellough, Evelyne Sernagor, Inmaculada Moreno-Gimeno, David H.W.
Steel, Majlinda Lako
EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS
Efficient Stage-Specific Differentiation of Human Pluripotent
Stem Cells Toward Retinal Photoreceptor Cells
CARLA B. MELLOUGH,a EVELYNE SERNAGOR,b INMACULADA MORENO-GIMENO,c
DAVID H.W. STEEL,a,d MAJLINDA LAKOa
a
Institute of Genetic Medicine; bInstitute of Neuroscience, Newcastle University, Newcastle, United Kingdom;
c
Centro de Investigacion Principe Felipe, Valencia, Spain; dSunderland Eye Infirmary, Sunderland,

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United Kingdom

Key Words. Embryonic stem cell • Photoreceptor • Retinal pigmented epithelium • Outer retinal degeneration

ABSTRACT
Recent successes in the stem cell field have identified some
of the key chemical and biological cues which drive photo-
receptor derivation from human embryonic stem cells
(hESC) and human induced pluripotent stem cells (hiPSC);
however, the efficiency of this process is variable. We have
designed a three-step photoreceptor differentiation proto-
col combining previously published methods that direct
the differentiation of hESC and hiPSC toward a retinal lin-
eage, which we further modified with additional supple-
ments selected on the basis of reports from the eye field
and retinal development. We report that hESC and hiPSC
differentiating under our regimen over a 60 day period
sequentially acquire markers associated with neural, reti-
nal field, retinal pigmented epithelium and photoreceptor
cells, including mature photoreceptor markers OPN1SW
and RHODOPSIN with a higher efficiency than previously
reported. In addition, we report the ability of hESC and
hiPSC cultures to generate neural and retinal phenotypes
under minimal culture conditions, which may be linked to
Disclosure of potential conflicts of interest is found at the end of this article.
their ability to endogenously upregulate the expression of a
range of factors important for retinal cell type specifica-
tion. However, cultures that were differentiated with full
supplementation under our photoreceptor-induction regi-
men achieve this within a significantly shorter time frame
and show a substantial increase in the expression of photo-
receptor-specific markers in comparison to cultures differ-
entiated under minimal conditions. Interestingly, cultures
supplemented only with B27 and/or N2 displayed compara-
ble differentiation efficiency to those under full supplemen-
tation, indicating a key role for B27 and N2 during the
differentiation process. Furthermore, our data highlight an
important role for Dkk1 and Noggin in enhancing the
differentiation of hESC and hiPSC toward retinal progeni-
tor cells and photoreceptor precursors during the early
stages of differentiation, while suggesting that further
maturation of these cells into photoreceptors may not
require additional factors and can ensue under minimal
culture conditions. STEM CELLS 2012;30:673–686

complex underlying genetic etiology. In such cases, cell

INTRODUCTION
Outer retinal degeneration (ORD) is the leading cause of
blindness in the developed world. There is still no treatment
for many forms of ORD where retinal pigmented epithelium
(RPE) dysfunction and photoreceptor demise predominate.
Clinical trials indicate some protective effects of dietary sup-
plements or intraocularly injected trophic factors in slowing
disease progression, but these are not a definitive cure. Gene
therapy strategies for specific forms of hereditary retinal dys-
trophy (HRD) are under development, and the viability of this
approach for partial restoration of visual function has been
demonstrated [1–5]. Yet this approach remains ineffective in
cases where a substantial loss of photoreceptors has already
occurred and is difficult to achieve in HRD resulting from
replacement remains an important option.
A seminal paper by MacLaren et al. demonstrated that
the only cells capable of successful integration and differen-
tiation into functional rod photoreceptors in the mouse retina
were postnatal postmitotic cells that were already committed
to a photoreceptor fate [6]. By contrast, only embryonic
stage Crx-positive cells were able to differentiate into cone
photoreceptors following transplantation [7]. For photorecep-
tor replacement in humans, the equivalent donor cell type
would have to be obtained from the second trimester
embryo, which raises clear ethical concerns. A more viable
option would be to try and differentiate human embryonic
stem cells (hESC) and/or human induced pluripotent stem
cells (hiPSC) into photoreceptors in vitro prior to
transplantation.

Author contributions: C.B.M.: designed and performed research, analyzed data, wrote the paper, and approved final version of
manuscript; E.S. and D.H.W.S.: performed research and contributed to paper writing and final approval of manuscript; I.M.G.:
performed research, data writing, and final approval of manuscript; M.L.: designed and performed research and contributed to paper
writing, final approval of manuscript, and fund raising.
Correspondence: Majlinda Lako, M.Sc., Ph.D., International Centre for Life, Institute of Genetic Medicine, Newcastle University,
Newcastle NE1 3BZ, U.K. Telephone: 44-1912418688; Fax: 44-1912418666; e-mail: majlinda.lako@ncl.ac.uk Received July 7, 2011;
accepted for publication December 20, 2011; first published online in STEM CELLS EXPRESS January 20, 2012. V C AlphaMed Press

1066-5099/2012/$30.00/0 doi: 10.1002/stem.1037

STEM CELLS 2012;30:673–686 www.StemCells.com

674
Several key publications have shown that it is possible to
drive the differentiation of hESC and hiPSC toward photorecep-
tors and RPE using various approaches ([8–15] and Supporting
Information Table S1). It has been suggested that early retinal
progenitor cells (RPCs) can be obtained from ventral
neural populations derived from differentiating hESC and hiPSC
cultures in the absence of growth factors; however, this process
is heavily dependent on the cell line-specific endogenous signal-
ing, especially those related to Dkk1 and Noggin [11, 16].
The RPE, which can be generated during the spontaneous
differentiation of hESC and hiPSC upon removal of basic fibro-
blast growth factor (bFGF; Supporting Information Table S1),
is critical to the normal function and survival of the light-
sensitive photoreceptors; indeed the two cell types are interde-
pendent on each other. It is likely that in many forms of ORD,
the replacement of both cell types will be required, highlighting
the need to identify new renewable cell sources that can be used
for restorative purposes. The restorative potential of hESC- and
more recently hiPSC-derived RPE has already been demon-
strated in the dystrophic Royal College of Surgeons (RCS) rat
[17–21]. RPE transplantation alone, however, although protec-
tive for remaining photoreceptors, does not act to replace photo-
receptors that have already been lost. Additionally, the potential
of hESC-derived photoreceptors for cell replacement has been
demonstrated [22, 23], yet photoreceptor differentiation proto-
cols remain variable and in some cases require extensive culture
periods (Supporting Information Table S1). Two studies [22,
23] have shown that hESC- and hiPSC-derived retinal cell
grafts can express photoreceptor markers and integrate within
the outer nuclear layer in intact newborn and adult retina. How-
ever, following subretinal transplantation into Crx
/
mice (an
animal model of Leber’s Congenital Amaurosis), these cells
failed to develop photoreceptor outer segments, indicating that
their final maturation and functional integration within the com-
promised retina remains incompletely understood. Thus, devel-
opment of protocols aimed at efficiently generating enriched
population of photoreceptor precursors at the correct ontoge-
netic stage is key prior to any translation of cell-based restora-
tive therapies for the treatment of photoreceptor loss.
With this aim, following on from previous reports
which demonstrate the differentiation of retinal cell types from
pluripotent cells, and by mimicking the chemical and cytokine
microenvironmental signals known to guide retinal histogenesis
during normal development, we have developed an adapted
differentiation protocol to promote photoreceptor differentiation
from hESC and hiPSC cultures. This method combines some
aspects of previously published protocols on hESC and
hiPSC differentiation toward the retinal lineage [8, 12], which
we have then further modified with additional supplements that
were added on the basis of reports from retinal and eye field
developmental papers. We show that our serum and feeder-free
differentiation method results in the generation of photoreceptor
progenitors and RPE from hESC and hiPSC with higher effi-
ciency than what has previously been achieved. Our data also
suggest that at least some hESC and hiPSC lines are endowed
with an endogenous potential to generate neural and retinal cells
under minimal culture conditions that can be enhanced and/or
accelerated by the addition of specific mitogens and metabolites
(Dkk1, B27, and Noggin) during the initial phase of differentiation.
MATERIALS AND METHODS
hESC and hiPSC Culture and Differentiation
hESC (line H9, WiCell, passage 34-45) and hiPSC generated
from human neonatal dermal skin fibroblasts (hiPSC-NHDF,
Efficient Photoreceptor Generation from Human Stem Cells
[24]) were grown under atmospheric oxygen conditions on
mitotically inactivated mouse embryonic fibroblasts (MEFs) in
hESC medium containing knockout-Dulbecco’s modified
Eagle’s medium (DMEM), 1 mM L-glutamine, 100 mM nones-
sential amino acids (NEAA), 20% knockout serum replacement
(KOSR, Gibco), 1% penicillin-streptomycin and 8 ng/ml bFGF
(Invitrogen). hESC/hiPSC medium was changed daily and
hESC/hiPSC colonies were passaged every 4-5 days at a ratio of
1:3-1:4 by incubation in 1 mg/ml collagenase IV. Prior to hESC/
hiPSC differentiation, MEFs were removed by culture of hESCs
and hiPSCs on hESC-qualified mouse matrigel (BD Biosciences,
New Jersey) for two passages. Differentiation experiments were
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initiated using the protocol outlined in Figure 1A. hESC/hiPSC
clumps were harvested following collagenase treatment and cul-
tured in low adherence bacteriological plates (Fisher Scientific,
Loughborough, UK) to form embryoid body (EB) cultures. Con-
trol cultures were differentiated in a basal ventral neural induc-
tion media (VNIM) (Fig. 1A, blue dashed line) consisting of
DMEM/Ham’s F-12 (DMEM/F-12, PAA, Linz, Austria) contain-
ing decreasing concentrations of KOSR (20% for the first 5 days,
15% until day 9 then 10% until day 37) supplemented with L-glu-
tamine, NEAA, and B27 (1:50, PAA). On day 30 of differentia-
tion, EB suspension cultures were transferred to attachment cul-
ture conditions on tissue culture were coated with poly-L-
ornithine (dissolved at 10 lg/ml in sterile dH2O, Sigma, Dorset,
UK) and laminin (5 lg/ml in phosphate buffered saline [PBS],
Sigma) for the remaining 30 days. On day 37, culture medium
was changed to a basal KOSR-free formula (Fig. 1A, blue
dashed line) consisting of DMEM/F12/L-glutamine/NEAA/B27
and N2 (1:100, Gibco, Paisley, UK).
To drive photoreceptor induction from hESC/hiPSC (which
we have termed PR-induced cultures, shown by the red dashed
line in Fig. 1A), hESC/hiPSC were differentiated as for control
cultures but with VNIM that was supplemented with recombi-
nant mouse (rm) Noggin (1 ng/ml, R&D Systems, Abingdon,
UK), recombinant human (rh) Dickkopf-1 (Dkk1, 1 ng/ml, R&D
Systems), Insulin-like growth factor 1 (rhIGF-1, 5ng/ml, Sigma)
rhLefty A (500 ng/ml, R&D Systems), Human Sonic Hedgehog
(Shh, 30 nM, Peprotech, London, UK) and 3,30 ,5-triiodo-L-thyro-
nine (T3, 40 ng/ml, Sigma) until day 37. KOSR-free media was
then supplemented with rmNoggin (10 ng/ml), rhDkk1 (10 ng/
ml), rhIGF-1 (10 ng/ml), rhbFGF (bFGF, 5 ng/ml), retinoic acid
(500 nM, Sigma), T3 (40 ng/ml), taurine (2 lm, Sigma), and Shh
(12 nM) until day 60. During days 37-41 (Fig. 1A, black dashed
line), PR-induction medium was supplemented with Human
Activin-A (100 ng/ml, Peprotech), to encourage the exit of pho-
toreceptor progenitor cells from the cell cycle and their further
maturation [25]. This induction method combines growth factors
at defined concentrations shown to induce photoreceptor differ-
entiation from two previous studies [8, 12] plus our own addition
of Activin A, Shh (known to cause an increase in the number of
retinal progenitors and photoreceptor cells) and T3 (known to
promote cone photoreceptor differentiation in human fetal retinal
cultures; [26]). Two different concentrations were used for Shh;
30 nM during the initial stages of differentiation (previously
defined as optimal for inducing ventral forebrain specification;
[27, 28]) and 12 nM in the last stage of differentiation (shown to
promote differentiation of embryonic rat retinal cultures toward
a photoreceptor phenotype; [29]). Minimal control cultures
[con(
)] consisted of hESC/hiPSC population that were differen-
tiated in basal media alone (Fig. 1A, green dashed line), in the
absence of B27/N2/additional factors. For more information,
refer to Supporting Information.
Analyses
Quantitative Reverse Transcription-Polymerase Chain
Reactions. Total RNA was extracted using TRIzol reagent
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Figure 1. Three-step induction protocol and examples of the resulting culture morphology. (A): Schematic showing the different stages of the
60-day differentiation protocol. Colored broken lines represent the different stages of media supplementation (Materials and Methods). (B–J):
The morphology of human embryonic stem cells differentiating under (B–D, I, J) control and (E–G, H) photoreceptor-induction conditions at (B,
E) 35 days, (C, F) 45 days, and (D, G, H) 60 days of differentiation. (H): Cells differentiating under conditions designed to induce photoreceptor
production generated neural-like populations with a proportion of cells exhibiting long neurites. (I, J): The typical appearance of retinal pig-
mented epithelial-like cells in control cultures on day 35 of differentiation under (I) phase microscopy and (J) following confocal microscopy
counterstained with Hoechst 33342. KOSR, knockout serum replacement. Scale bars: B, C, E, F ¼ 200 lm, D, G, H ¼ 100 lm, and I, J ¼ 50
lm. Abbreviation: PR, photoreceptor.

(Invitrogen, Paisley, UK) according to manufacturer’s instructions.
Following DNase treatment using RQ1 DNaseI (Promega, South-
ampton, UK), cDNA was synthesized using SuperScript III
Reverse Transcriptase (Invitrogen). Quantitative reverse transcrip-
tion-polymerase chain reactions (qRT-PCR) analysis was carried
out using SYBR GreenER PCR master mix (Invitrogen). All reac-
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tions were carried out and samples analyzed using an AB7900HT
real-time analyzer. All primers used in this study are listed in the
Supporting Information Table S2. Data were analyzed using qBase
v1.3.5 and the comparative threshold cycle (Ct) method. In all
samples, the results were normalized to the geometric mean of
three housekeeping genes GAPDH, SDHA, and RPL13A, and
676
referenced to hESC, 7-day differentiated hESC, human retina or
human RPE in accordance with the gene of interest.
Array-Comparative Genomic Hybridization. To ensure that
the hESC line (H9) used in this study had not acquired any
chromosomal abnormalities during ex vivo expansion, we per-
formed array-comparative genomic hybridization (aCGH) as
outlined in Supporting Information. The analysis did not show
significant abnormalities (Supporting Information Fig. S1A–
S1C), with the exception of two close amplifications of 186
and 458 kb at 14q23.2 and 14q23.3, which correspond to
known copy number variations (Database of Genomic Var-
iants at http://projects.tcag.ca/variation/). We also noticed a
deletion of 2MB at 6p22.2-p22.1; however, in-depth analysis
suggested that the deletion observed at chromosome 6 and
amplifications observed at chromosome 14 were not signifi-
cant as they did not meet the criteria of significant genomic
aberrations, which is the presence of three consecutive probe
alterations (at chromosome 6p22.2-p22.1, three nonconsecu-
tive deleted probes were found in a region comprising 77
probes [Supporting Information Fig. S1B], while at 14q23.2
and 14q23.3, only two consecutive probes were amplified,
respectively [Supporting Information Fig. S1C]). We also
investigated the copy numbers of 61 reported stem cell-associ-
ated genes [30] and this analysis showed no significant differ-
ences in DNA copy number over the entire chromosomal
regions. In summary, our aCGH analysis indicated that H9
hESC used in this study did not show genomic instabilities
that could impact upon their pluripotency.
Fluorescence Immunocytochemistry and Microscopy. Cells
were fixed in 4% (w/v) paraformaldehyde for 15 minutes.
Permeabilization (0.2% (v/v) Triton X-100 in PBS) was
performed prior to staining with antibodies for internal cell
markers. A blocking step was performed by incubation in
antibody diluent (1% (w/v) bovine serum albumin, Sigma,
with 5% (v/v) normal goat serum, Invitrogen) prior to staining
for 30 minutes. Cells were incubated with anti-rhodopsin
(Sigma), anti-opsin blue (OPN1LW, Millipore), anti-opsin
red/green (OPN1SW, Millipore, Watford, UK), anti-RPE65
(AbCam, Cambridge, UK) antiprimary antibodies for 1 hour
and secondary antibodies for 30-60 minutes. Nuclei were
counterstained with 10 lg/ml Hoechst 33342 then cultures
coverslipped using Vectashield (Vector Laboratories, Peter-
borough, UK). Bright-field and fluorescent images were
obtained using a Zeiss inverted microscope and AxioVision
software. Immunopositive cells were quantified manually by
randomly sampling and photographing 10 fields of view at
20 magnification. All Hoechst-labeled nuclei and immuno-
positive cells within these confines were counted.
Flow Cytometric Analysis. Differentiating cells were disso-
ciated by treatment with Accutase (Gibco) and then processed
in 35 lm Medicons using a Medimachine (Becton Dickinson,
New Jersey). Cells were collected by centrifugation at 250g
for 5 minutes and fixed first in 0.5% paraformaldehyde at
37 C for 10 minutes, then by treatment with 90% methanol
for 30 minutes on ice. Cells were washed in PBS then trans-
ferred to incubation buffer (0.2% BSA and 5% goat serum in
PBS) for 10 minutes at room temperature followed with un-
conjugated anti-Chx10, anti-CRX, anti-rhodopsin, anti-
OPN1LW, anti-OPN1SW, or anti-RPE65 primary antibody
for 1 hour before incubation with fluorescein (FITC) second-
ary antibody (1:800, Sigma) for 30 minutes at room tempera-
ture. Samples were washed in PBS and protected from light
prior to analysis. Analysis was performed using the FACSCa-
Efficient Photoreceptor Generation from Human Stem Cells
libur system (BD Biosciences) with CellQuest Pro software
(BD Biosciences). At least 10,000 events were analyzed in
each experiment.
Statistical Analysis
Two-tailed paired Student’s t test was used to analyze results
between groups at various time points during the differentia-
tion process and between control and PR-induced groups at
the same stage of differentiation. Results were considered
significant if p < 0.05.
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RESULTS
To design a more efficient photoreceptor differentiation proto-
col for pluripotent cell types, we have combined some aspects
of previously published protocols on hESC and hiPSC differ-
entiation toward a retinal lineage [8, 12] and then made addi-
tional modifications on the basis of reports from retinal and
eye field developmental papers. Akin to previously published
methods [8, 12], our differentiation protocol (Fig. 1A) was
designed to guide the transition of cells from a pluripotent
state to that of the retinal lineage, more specifically, a photo-
receptor fate by sequentially exposing cells to antagonists and
cytokines known to direct: (a) a ventral neural fate; (b) eye
field/retinal determination; and (c) photoreceptor specification
and maturation. hESC/hiPSC were differentiated under this
defined culture protocol (named PR-induction, red line in
Fig. 1A) for a period of 60 days and analyzed by qRT-PCR,
flow cytometry, and immunocytochemistry. Control cultures
were set up in parallel in the absence of growth factors and/or
culture supplements and named appropriately in each figure
(blue and green lines in Fig. 1A).
The Loss of Pluripotency and Acquisition of
a Neural Lineage
Differentiation of hESC under control and PR-induction condi-
tions yielded no discernable differences in culture morphology
at the EB stage. qRT-PCR analysis of gene expression demon-
strated the rapid downregulation of pluripotency markers
NANOG and OCT4 over the first 30 days, indicating the loss
of pluripotency (Fig. 2). Following transfer to adherent culture
conditions, EBs attached to generate proliferative cultures (Fig.
1B–1H) with many cells displaying neural-like morphology.
Neural cells exhibit a variety of morphology depending on
their phenotype and stage of maturation. Neural progenitor
cells are often bipolar in shape with a small soma and shorter,
less developed neurites than their mature neuronal and glial
counterparts (e.g., Fig. 1D). Late stage neurons can differ
vastly in their soma size as well as their polarity (ranging from
unipolar to multipolar), dendritic arborization and axon length,
some cells exhibiting axons which traverse great distances
(e.g., as in Fig. 1D–1H). Pigmented foci, indicating the emer-
gence of RPE cells, appeared within floating EB cultures over
days 20-30 and following transfer to adherent culture condi-
tions generated sheets of pigmented epithelial cells resembling
native RPE (Fig. 1I and 1J). This occurred with higher fre-
quency in control cultures, possibly due to the addition of
bFGF in PR cultures, which has been shown to suppress RPE
induction [10, 13].
qRT-PCR analysis of PR-induced cultures revealed low
levels of trophoctoderm (CDX2), endoderm (GATA4, AFP),
and mesoderm (BRACHYURY) markers after day 30 of differ-
entiation and an upregulation of the primitive ectodermal-
associated gene FGF5 (Fig. 2, red bars). This occurred
alongside an increase in SOX2 and NCAM1 gene expression
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Figure 2. Pluripotency and germline gene expression in differentiating human embryonic stem cells (hESC) over 60 days. Assessment of
pluripotency markers NANOG and OCT4 and lineage markers from all three germ layers in differentiating control (blue bars) and PR-induced
population (red bars) over 60 days by quantitative reverse transcription-polymerase chain reactions. All data (Y-axis) are presented as the normal-
ized ratio of gene expression to housekeeping genes GAPDH, SDHA, and RPL13A. NANOG and OCT4 are calibrated to hESC (hESC ¼ 1), all
other genes to hESC that had been differentiated for 7 days (H9d7 ¼ 1). Data ¼ mean 6 SEM, n ¼ 3. Abbreviations: FGF, fibroblast growth
factor; PR, photoreceptor.

over the differentiation period (Fig. 4A), indicating the emer-
gence of neuroectodermal derivatives. In control cultures
however, some expression of AFP, CDX2, and GATA4 was
maintained over the 60 days of differentiation (Fig. 2, blue
bars), with observed levels of FGF5 expression lower than in
mitogen-treated population, indicating spontaneous differentia-
tion toward other germ layers.
The Emergence of a Retinal Lineage
We went on to assess the emergence of eye field and retinal
progenitor markers in cultures differentiating under our
PR-induction and control regimens (Figs. 3, 4). NESTIN
expression remained constant throughout the differentiation
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period while the early retinal marker PAX6 increased in PR-
induced cultures over time (p ¼ .048) from day 45 to reach a
prominent peak at day 60 (Fig. 4A). Flow cytometric analysis
detected the greatest number of cells expressing the retinal
progenitor marker CHX10 on day 15 of differentiation in the
PR-induced group (Fig. 4B, red line) showing an almost two-
fold increase over control population at this time (expressed
in 25% of control population and 43% of PR-induced). After
day 30, both population follow a similar expression course
and decline from 13% of cells at day 45 (Fig. 3A) to only a
fragment of the population expressing this antigen by day 60
(<1%, Fig. 4B). No significant changes were, however,
detected in CHX10 transcript levels during the differentiation
678 Efficient Photoreceptor Generation from Human Stem Cells

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Figure 3. The expression of retinal and photoreceptor-specific markers in differentiating human embryonic stem cells by immunofluorescence
histochemistry and flow cytometry. (A): Flow cytometric analysis of CHX10, CRX, Opsin blue (OPN1SW), Opsin red/green (OPN1LW), and
RHODOPSIN under control (con) and photoreceptor-induction conditions (PR) at 45 and 60 days. (B–D): Immunofluorescent staining of (B, C)
RHODOPSIN (green) and ZO-1 (red) counterstained with Hoechst (blue) and (D) cone-specific OPN1SW (red) near cells immunopositive for the
RPE marker RPE65 (green) at 45 days. Scale bars ¼ 50 lm. (E): CRX, OPN1SW, and RHODOPSIN expression as detected by flow cytometry
in H9 cells differentiated under control (con) and PR-induction conditions at 45 days. Values equal mean 6 SEM (n ¼ 3). Both cultures demon-
strated expression of photoreceptor-specific markers at this time point. Abbreviation: RPE, retinal pigmented epithelium.

period and also between control and PR-induced groups. This
might be explained by differences between quantification
methods, the sensitivity of each method and the measurable
outcome (protein vs. transcript) with flow cytometric analysis
assessing the percentage of CHX10 immunopositive cells
(and not total level of CHX10 protein expression) and qRT-
PCR measuring the total level of the CHX10 transcript.
RPE65 is a cytoplasmic protein involved in retinoid me-
tabolism [31, 32]. Flow cytometric and qRT-PCR analysis
revealed an early peak in RPE65 expression at day 30 in the
PR-induced group (Fig. 4A, 4B). In control cultures, we
observed the emergence of RPE cells over the first 30 days
which, unlike PR-induced cultures, continued to rise until day
60 (Fig. 4A, RPE65, SILV) consistent with published litera-
ture, which indicates spontaneous emergence of these cells in
the absence of exogenous growth factors [10, 13, 15]. The
expression of eye field and retinal progenitor markers PAX6,
CHX10, and RPE-specific RPE65 alongside the appearance of
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Figure 4. The expression of differentiation-associated genes and antigens. (A): Quantitative reverse transcription-polymerase chain reaction analy-
sis of neuroectodermal (SOX2, NCAM1, NESTIN, and NEUROD), retinal progenitor (PAX6 and CHX10), photoreceptor progenitor (CRX), phototrans-
duction pathway (RECOVERIN, PDE6b, OPSIN, and ARR3), and retinal pigmented epithelial (RPE65 and SILV) gene expression in control (blue
bars) and mitogen-treated population (PR, red bars) over the 60 days of differentiation. Data (Y-axis) is normalized to housekeeping genes and
referenced to human embryonic stem cells (hESC; SOX2, NCAM1, NEUROD, NESTIN, and PAX6, hESC ¼ 1), human retina (CHX10, CRX, RECOV-
ERIN, PDE6b, OPSIN, and ARR3, human retina ¼ 100), or human RPE (RPE65 and SILV, RPE ¼ 1). Values equal mean 6 SEM (n ¼ 3). (B): Flow
cytometric analysis of PR-induced (red line) and control cultures differentiated with B27/N2 (con, blue line) against control(
) cultures differentiated
in the absence of B27/N2 (con(
), green line) revealed reduced immunopositivity for CHX10, RPE65, and OPN1SW in con(
) cultures and an
important role for B27/N2 in the levels of retinal-specific gene expression previously observed in control population (A, blue bars) in the absence of
any other mitogens or antagonists. Values equal mean 6 SEM (n ¼ 3). Abbreviations: PR, photoreceptor; RPE, retinal pigmented epithelium.

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680
pigmented foci over the first 30 days of differentiation indi-
cates the emergence of early retinal-specific phenotypes from
differentiating hESC.
The Emergence of a Photoreceptor Phenotype
Following the transfer of differentiating hESC to adherent cul-
ture conditions, we assessed the expression of photoreceptor
markers by qRT-PCR, flow cytometry and immunofluorescent
histochemistry (Figs. 3, 4). Flow cytometric analysis on day
45 of differentiation confirmed the persistence of some
CHX10þ cells (up to 13%) and a similar number of cells
were immunopositive for the early postmitotic photoreceptor
precursor marker CRX (up to 16%, Fig. 3A). Over days 45-
60, a marked increase was observed in CRX gene expression
under control induction conditions (Fig. 4A). This led us to
test whether any differentiating cells within these populations
might be expressing other photoreceptor markers. This
included the neuronal calcium-binding protein RECOVERIN,
ARRESTIN 3 which is a marker of outer segments of red,
green, and blue cone photoreceptors, and cone-specific light-
sensitive G-protein-coupled receptors, or opsins. Indeed,
from days 45 to 60 of differentiation, an increase in the
expression of ARRESTIN 3 was detected under control condi-
tions (Fig. 4A).
Flow cytometric analysis revealed the highest expression
of photoreceptor markers RHODOPSIN, cone-specific opsin
blue (OPN1SW) and opsin red/green (OPN1LW) at day 45 of
differentiation (found in 18%, 52%, and 60% of cells cultured
under PR-inducing conditions, respectively, Fig. 3A, 3E). By
day 60, this proportion had declined, resulting in almost com-
plete loss of RHODOPSIN (1.1%) and only a small number
of OPN1SW ( 4%) immunopositive cells remaining, while
13% of the population remained OPN1LWþ at this time
(Fig. 3A). The expression of photoreceptor-specific markers
was confirmed following immunocytochemical analyses and
manual cell counts (Fig. 3B–3D).
Detection of the postmitotic photoreceptor marker CRX
alongside late-stage photoreceptor-specific markers indicates
the emergence of hESC-derived photoreceptors within cul-
tures differentiated using our protocol. Comparison of control
and PR-induced cultures by qRT-PCR, however showed only
one statistically significant increase in the expression
of OPSIN in PR-induced population observed at day 60 (p ¼
.035, Fig. 4A). In addition, flow cytometric analysis from day
30 onward showed no significant changes in the expression of
photoreceptor-specific markers (Figs. 3E, 4B) and, in most
cases, control and PR-induced populations followed a similar
time course with regard to the onset of neuroectodermal, reti-
nal progenitor and photoreceptor-specific markers and levels
of gene expression, suggesting that perhaps an endogenous
and compensatory growth factor signaling pathway is active
in control cultures, corroborating findings reported by Meyer
et al. [11].
Further Investigation into the Conditions Enabling
Retinal Specification from Differentiating hESC
Reveals an Important Role for B27 and N2
As shown above, we observed that control populations differ-
entiated under basal media conditions were capable of exhib-
iting neural, eye field, retinal and photoreceptor gene expres-
sion over time. This was an unexpected result and we set out
to address the role of specific media components during
hESC differentiation. Human ESCs express the receptors for
fibroblast growth factor (FGF), activin A, epidermal growth
factor (EGF), nerve growth factor (NGF) and retinoic acid;
however, the ligands for these are all absent in commercially
Efficient Photoreceptor Generation from Human Stem Cells
available KOSR [33]; therefore, it is not likely to be the pres-
ence of KOSR during the first 37 days of differentiation
which enabled these effects. Some constituents of the neural
supplement B27 are demonstrated to promote neuralization,
enhancing neural progenitor proliferation and differentiation
as well as the development of the machinery necessary for
phototransduction [34–36]. This suggests that B27, a compo-
nent of our basal ventral neural induction medium and there-
fore present in control cultures during differentiation, may
play a role in the specific differentiation of hESC towards ret-
inal cells. We therefore questioned whether the removal of
this supplement over the first 37 days of differentiation fol-
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lowed by the combined removal of B27 and N2 in the final
window of differentiation (days 37-60) would affect differen-
tiation potential.
To investigate this, we introduced an additional control
into our experiments, by differentiating hESC cultures in the
absence of B27 or N2 in a minimal media [named control(
)
cultures, refer to Figure 1A green line]. hESC differentiating
under these conditions lost their pluripotency marker expres-
sion in a similar way to control and PR-induced groups (Sup-
porting Information Fig. S2). The removal of B27, followed
by combined B27 and N2 removal from the differentiation
media resulted in a reduction in early expression levels of the
retinal progenitor marker CHX10 when compared with sup-
plemented cultures; but this was, however, followed with by a
steady increase in CHX10 over days 15-60 (Fig. 4B), reach-
ing by day 60 the same expression level as observed in B27/
N2 supplemented cultures at day 15 of differentiation. Similar
results were also observed for the RPE marker RPE65, where
the same expression levels were observed for PR-induced,
control and control(
) groups on day 60 of differentiation
(Fig. 4B). Despite this, hESC differentiating under control(
)
conditions failed to reach the peak OPN1SW expression
observed at day 45 in PR-induced and control cultures. Both
sets of results shown in Figures 3E and 4B suggest no signifi-
cant differences in early and mature photoreceptor marker
expression between control and PR-induced populations.
Given that B27 (present throughout differentiation) and N2
(added in the final window of differentiation after day 37,
refer to Fig. 1A) are the only common culture components
between PR-induced and control groups and are both missing
from the control(
) group, this suggests a role for these two
components in enhancing and accelerating the endogenous
capacity of hESC cultured under minimal culture conditions
to undergo neural and retinal differentiation.
Similar results were obtained during the differentiation of
hiPSC-NHDF cells (Supporting Information Fig. S3). Expres-
sion of both cone-specific opsin blue (OPN1SW) and opsin
red/green (OPN1LW) at day 45 of differentiation was similar
across control and PR-induced cultures, but lower in con-
trol(
) cultures, while the opposite was observed for early
retinal marker, PAX6 (Supporting Information Fig. S3).
Together, these results indicate an endogenous, although
somewhat delayed, capability of differentiating hESC cultures
to produce certain neural and retinal phenotypes during
prolonged culture in the absence of specific inductive cues.
The Endogenous Expression of Key Signaling
Factors in hESC
The endogenous expression of the Wnt antagonist DKK1 and
FGF signaling has previously been reported in hESC differen-
tiating toward the eye field [11]. Endogenous SHH signaling
in mouse ESC (mESC) can promote a ventral neural fate [37]
and, although endogenous SHH is very low in hESCs, levels
of SHH become enriched upon EB differentiation [38]. We
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Figure 5. Assessment of endogenous growth factor and antagonist gene expression. (A): Quantitative reverse transcription-polymerase chain
reactions (qRT-PCR) analysis of EGF, NGF, SHH, NODAL, and DKK1 expression was performed at 15-day intervals over the differentiation
period in control (with B27/N2 only, blue line), minimal control (no B27/N2/mitogen supplementation, green line) and in B27/N2/mitogen-
supplemented PR-induced population (PR, red line). (B): qRT-PCR analysis showing delayed onset of NEUROD expression in control(
)
cultures compared with control and PR-induced population. (C): qRT-PCR analysis revealed that, of all conditions tested, the highest expression
of CRX and RECOVERIN on day 60 was seen in control and PR-induced cultures. (A–C): Data (Y-axis) are normalized to housekeeping genes
and calibrated to human embryonic stem cells ¼ 1 for panel A and human retina for panels B and C (human retina ¼ 1). Values equal mean 6
SEM (n ¼ 3). Abbreviations: EGF, epidermal growth factor; NGF, nerve growth factor; PR, photoreceptor; and SHH, human sonic hedgehog.

therefore investigated the expression of various factors in
hESCs differentiating under our protocol. qRT-PCR results
indicated that endogenous levels of EGF, NGF, and SHH rose
in hESC cultures over the differentiation period, NODAL was
strongly expressed on day 15 but then rapidly declined, while
expression of DKK1 remained relatively constant after this
time point. We observed no significant difference in the en-
dogenous expression of EGF, NGF, SHH, NODAL, or DKK1
between control and PR-induced cultures (Fig. 5A, blue and
red lines).
When these results were compared with control(
) popu-
lation, some differences emerged (Fig. 5A, green lines). Akin
to our observations of the delayed upregulation of early reti-
nal markers in control(
) cultures, increased endogenous
www.StemCells.com
expression of EGF, NGF, NODAL, and SHH was observed
from day 45 to day 60 in these cultures, reaching levels that
were greatly elevated in comparison with control and PR-
induced population (Fig. 5A). DKK1 levels were also higher
in control(
) cultures suggesting elevated endogenous Dkk1
signaling in hESC differentiating under minimal culture con-
ditions and corroborating previously published data [11].
We then went on to assess the effects of each of our three
culture conditions; control(
), control, and PR-induced on the
expression of neuroectodermal, retinal, and photoreceptor
genes. Our analysis demonstrated that hESC differentiating
for 60 days without B27 and N2 resulted in a delay in the
onset of neuroectodermal gene expression when compared
with normal controls and PR-induced population (Fig. 5B),
682 Efficient Photoreceptor Generation from Human Stem Cells

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Figure 6. Further analysis of the individual contribution of B27/N2 supplements and added factors on endogenous gene expression and embry-
oid body morphology. (A): Morphological differences were observed between EBs from (a) control(
) and (b) control cultures as well as during
supplementation with individual factors. T3 treatment promoted the formation of small, highly pigmented EBs (c, d), LeftyA induced the forma-
tion of polarized EBs with a prominent axis (e), while in IGF-1-treated population disc-shaped EBs emerged which contained numerous embed-
ded optic cup-like structures (f, g) only rarely seen to contain pigmented cells (g). (B): PAX6 and CHX10 expression at days 15 and 30 was
enhanced in cultures treated with Dkk1, noggin, or T3 during the first 30 days of differentiation. Data (Y-axis) are normalized to housekeeping
genes. Results for PAX6 and CHX10 are calibrated to human retina (human retina ¼ 1). Values equal mean 6 SEM (n ¼ 3). Abbreviations: IGF,
insulin-like growth factor; shh, human sonic hedgehog; and T3, 3,30 ,5-triiodo-L-thyronine.

consolidating the flow cytometry data analysis shown in
Figure 4B. Nonetheless, by qRT-PCR analysis, the cultures
demonstrating the best CRX and RECOVERIN expression on
day 60 across all groups were the control (with B27/N2) and
PR-induced populations (Fig. 5C) corresponding with the data
shown in Figure 4B and once more suggesting that at least
B27 and/or N2 (present in both control and PR-induced
cultures) are two important media components that enhance
the induction of photoreceptor-specific gene expression in dif-
ferentiating hESC.
This suggests that, in the absence of external cues, a pro-
portion of hESCs follow an intrinsic neural and retinal differ-
entiation pathway, which is associated with the delayed onset
of neural and retinal markers under minimal culture condi-
tions. This capability, as seen in minimal controls, may be
due to the upregulation of endogenous signaling factors start-
ing as early as the first 15 days (DKK1) but from our observa-
tions in most cases between days 30 and 60 (EGF, NGF,
NODAL, and SHH). Nonetheless, the induction of
photoreceptor marker expression remained more efficient in
control and PR-induced cultures.
Further Analysis of the Individual Contribution of
Each Factor During hESC Differentiation
To test the individual contribution of each component of the
PR-induction media, we supplemented differentiation media
with isolated factors during floating EB culture for up to 30
days. There were some apparent differences in gross EB
morphology between normal controls (with B27) and mini-
mal controls (no B27) at this stage of differentiation
(Fig. 6A; panels a and b, respectively) with more cystic EBs
appearing in the minimal control group. In addition, some
interesting morphological differences emerged following
treatment with individual factors. The addition of T3 pro-
moted the formation of small, highly pigmented EBs
(Fig. 6A, panels c and d), while LeftyA induced the forma-
tion of polarized EBs containing a prominent axis (Fig. 6A,
panel e). Interestingly, differentiation with IGF-1, shown
specifically to induce eye formation in Xenopus and an
enhancer of retinal progenitor gene expression [39], resulted
in the appearance of disc-shaped EBs containing numerous
visible internal neural rosette-like structures (Fig. 6A, panels
Mellough, Sernagor, Moreno-Gimeno et al. 683

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Figure 7. Further analysis of the directed differentiation of human embryonic stem cells (hESC) with different combinations of supplements and
mitogens revealed variance in the expression of mitogens, mature retinal genes, and culture morphology. Treatment of hESC with B27/N2 and all fac-
tors for 37 days and then with isolated factors for the remainder of the differentiation period induced variation in (A) endogenous gene expression and
(B) photoreceptor and RPE-specific genes on day 60 of differentiation. All treatments generally yielded heterogeneous neural-like cultures (C–I) with
the presence of RPE sheets displaying distinct boundaries (D, E). Cells within adherent EBs commonly generated long processes, some of which
extended hundreds of microns (C). Optic-cup-like structures that were observed following IGF-1 treatment during floating culture were maintained
following transfer to adherent conditions in PR-induction media (F). These were also observed in population differentiated with PR-induction media
over the first 37 days and then with either T3 (G) or taurine (H), and in lower frequency in cultures grown under PR-induced conditions for the full dif-
ferentiation period (I). Data are normalized to housekeeping genes and referenced to hESC (panel A, hESC ¼ 1), human retina (panel B, human retina
¼ 1), or human RPE (RPE ¼ 1, RPE65, panel B). Values equal mean 6 SEM (n ¼ 3). Scale bars: C–E, G, I ¼ 100 lm and F, H ¼ 400 lm. Abbrevia-
tions: bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; FGF, fibroblast growth factor; IGF-1, insulin-like growth factor 1; NGF,
nerve growth factor; PR, photoreceptor; RPE, retinal pigmented epithelium; SHH, human sonic hedgehog; and T3, 3,30 ,5-triiodo-L-thyronine.

www.StemCells.com
684
f and g), which have also been reported under minimal cul-
ture conditions [16].
Our quantitative RT-PCR expression analysis detected the
best CHX10 expression in cultures exposed to B27 plus Dkk1
or B27 plus noggin (Fig. 6B). In cultures supplemented with
B27 and one other factor only, PAX6 expression was also
found to be higher than PR cultures differentiated with all
factors together (Fig. 6B). Endogenous Dkk1 signaling has
been implicated in acquisition of early retinal markers (PAX6
and RAX) during differentiation of hESC [11]. To investigate
this further in our differentiation protocol, we performed flow
cytometric analysis in Dkk1-supplemented control cultures
(but devoid of all other added factors) and observed a 76.6%
increase in number of CRX expressing cells and a 7.8%
decrease in number of cone-specific opsin blue (OPN1SW)
expressing cells (data not shown), suggesting an important
role for Dkk1 in enhancing the specification of photoreceptor
precursors but not later stage photoreceptor cells.
In cultures exposed to PR-induction conditions for the first
37 days then swapped to minimal media either alone or
supplemented with isolated factors, we observed that some
factors elicited similar results (Fig. 7A). For example, hESC
differentiated under minimal conditions (no B27/N2/factors)
from days 37 to 60, or in minimal media supplemented with
Dkk1, N2, or T3 resulted in the elevated endogenous expres-
sion of FGF2, SHH, DKK1, EGF, and NGF (Fig. 7A). We
went on to assess the effect of each media component on the
expression of retinal and photoreceptor genes. Of mitogen-
treated groups, our analysis demonstrated the best expression
of OPSIN, PDE6b, and ARRESTIN3 on day 60 in cultures
that had been differentiated under PR-induction conditions for
37 days and then either with no factors or supplemented with
N2, Dkk1, or T3 (Fig. 7B). These qRT-PCR results were con-
firmed by flow cytometry data, which showed an increase in
the number of CRXþ (84%), OPN1SWþ (89.2%), and
OPN1LWþ (34.6%) cells in cultures that had been differenti-
ated under PR-induction conditions for 37 days and subse-
quently under minimal conditions compared to those
subjected to PR-inducing conditions for the entire differentia-
tion period (data not shown). Together these data suggest an
important role for B27, Dkk1, and Noggin in the first 37 days
of differentiation. In addition, they confirm that although the
addition of N2, Dkk1, or T3 during the final stage of differen-
tiation can facilitate the yield of the mature photoreceptors,
this is not a requirement for further cell maturation to occur.
DISCUSSION
The ability to generate expandable populations that can yield
a large number of photoreceptors is a prerequisite for the suc-
cessful application of cell replacement therapy for outer reti-
nal degeneration. Our strategy for photoreceptor production
from hESC and hiPSC relies upon the use of various factors
and antagonists that are known to play a role in neural induc-
tion and retinal histogenesis during development and from in
vitro studies [8, 11, 12, 21, 25, 40-42]. Our aim was to de-
velop a protocol to increase the yield of photoreceptors from
hESC and hiPSC in a shorter time frame than what has been
achieved previously.
Our results indicate that our protocol directs the stage-
specific differentiation of hESC and hiPSC toward a neural
fate, followed by retinal field and then photoreceptor lineage.
The appearance of these phenotypes occurred in a sequential
manner consistent with retinal development in vivo [43]. We
observed that CHX10þ retinal progenitors emerged during the
Efficient Photoreceptor Generation from Human Stem Cells
early phase of differentiation, while the majority of
CRXþphotoreceptor progenitors were generated over days 30-
45. The onset of mature photoreceptor markers at this time
indicated that the emergence of the two distinct photoreceptor
types, rods and cones, was also concordant with what one
might expect developmentally; cone photoreceptors are born
during the early phase of retinal histogenesis while rods
emerge later [44]. Our gene expression results support this
observation, showing elevated levels of cone-specific OPSIN
on day 30, prior to the peak of rod-specific gene expression
on day 60.
We also report an endogenous capacity of hESCs and
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hiPSC to generate neural and retinal phenotypes under
minimal culture conditions. This capability has previously
been demonstrated in hESC and hiPSC [11, 16], but is not a
widely published phenomenon and it may be due to the en-
dogenous production of various signaling molecules and
growth factors from the heterogeneous mix of cells present in
embryoid body cultures generated from pluripotent stem cells
as demonstrated by this study and others. Trophic support for
emerging retinal cell types from neighboring RPE cells is
another potential explanation for the elevation in retinal-spe-
cific markers observed in populations differentiated in the ab-
sence of mitogens or supplements [45].
A recent groundbreaking paper demonstrates that struc-
tures reminiscent of the optic vesicle can arise from mESCs
differentiated in low-concentration matrigel and minimal dif-
ferentiation medium [46], highlighting a propensity for mESC
to undergo stepwise differentiation and self-organization
under very simple culture conditions. This corroborates and
extends previously published data showing that mouse ESC
cultured under minimal conditions and without instructive sig-
naling can undergo neural differentiation [47]. We also
observed the emergence of neural rosette-like structures from
hESCs and hiPSC during the first 30 days of differentiation.
These were most commonly observed during the early stages
of differentiation in cultures treated with IGF-1, a known in-
ducer of eye formation and a potent enhancer of retinal pro-
genitor gene expression [8, 39]. During the latter stages of
differentiation, we also observed numerous rosette-like struc-
tures in populations that had been treated with all supplements
and mitogens for photoreceptor-induction over the first 37
days, followed by treatment with T3 or taurine only. Some
were also observed in cultures differentiated under our photo-
receptor-induction regime for the entire differentiation period,
albeit with less frequency. These rosette-like structures are
similar to optic vesicle-like structures reported by Meyer
et al. [16] during hESC/hiPSC differentiation, and current
work within our group is focusing on identification of the
RPC types present therein and their potential to generate func-
tionally mature photoreceptor and RPE cells.
One previous study has shown that the treatment of
hESCs with B27, Dkk1, noggin, IGF-1 and subsequently with
N2 and FGF can result in more than 80% of cells expressing
retinal progenitor markers [8]. However, although 12% of the
population expressed CRX, less than 0.01% were found to be
positive for mature photoreceptor markers. Osakada et al.
achieved CRX expression in 11% of the population following
120 days of differentiation, which increased to around 20%
by 170 days, with 5% of cells expressing Rhodopsin by day
150 [12]. Meyer et al. achieved 55.9% of CRX expressing
cells (at day 80 of differentiation), which undergo differentia-
tion to photoreceptor-like cells displaying characteristic
electrophysiological responses [16]. Each of these studies rep-
resents great achievement (Supporting Information Table S1
for more detail). Yet for this therapy to be translated to the
clinic, it will be advantageous if this process was more
Mellough, Sernagor, Moreno-Gimeno et al.
efficient. We set out to try and improve existing protocols and
from our results we report that we are able to generate 16%
of CRXþ cells and 52% of cone-like photoreceptor cells
within 45 days of differentiation. Although this differentiation
period is longer than what has been reported in one earlier
study [8], the higher efficiency of generated cells that express
mature photoreceptor markers compared to previous studies
after 45 days of differentiation (refer to Supporting Informa-
tion Table S1) strongly suggests that our three-step differen-
tiation protocol extends and improves current achievements in
the hESC/hiPSC differentiation field. Notwithstanding the
increased efficiency, our protocol has also its limitations. We
observed the rapid loss of cells committed to a photoreceptor
fate (CRXþ/OPSINþ/RHODOPSINþ) over days 45-60. This
is similar to observations in a study using cultured mouse
RPCs where the rapid loss of rhodopsin-expressing cells was
observed shortly after plating in RPC culture [48]. How to
promote the long-term survival of postmitotic photoreceptors
during prolonged culture remains an important challenge. It
seems that this will not be achieved by structural means alone
[46]; however, purification of various cell types during their
peak production period may overcome this issue somewhat.
CONCLUSION
Our three-step differentiation protocol was designed with the
aim to mimic neural and retinal development. To achieve
this, we combined serum- and feeder-free culture conditions
with known growth factors and metabolites that have been
used with some degree of success in previous hESC differen-
tiation studies [8, 12] alongside additional factors that are
implicated to play a role in retinal development from develop-
mental studies. To be able to understand the role of each of
these factors, we performed stepwise deletions of single and/
or combined factors. This detailed analysis indicated that the
most important components for achieving efficient photore-
ceptor generation within a reasonable time frame (45 days)
are B27 and/or N2. While RPC and photoreceptor precursor
generation could be enhanced by the addition of Dkk1 and
Noggin to B27-supplemented medium during the early stages
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