Vol.2, No.4, 176-186 (2012) Stem Cell Discovery
http://dx.doi.org/10.4236/scd.2012.24022
Low level of activin A secreted by fibroblast feeder
cells accelerates early stage differentiation of retinal
pigment epithelial cells from human pluripotent stem
cells
Heidi Hongisto1,2*, Alexandra Mikhailova1,2*, Hanna Hiidenmaa1,2, Tanja Ilmarinen1,2,
Heli Skottman1,2#
1Institute of Biomedical Technology, University of Tampere, Tampere, Finland;
#Corresponding Author:
2Institute of Biosciences and Medical Technology, Tampere, Finland
Received 6 July 2012; revised 3 August 2012; accepted 3 September 2012
ABSTRACT
Human pluripotent stem cells (hPSC) differenti-
ated to retinal pigment epithelial cells (RPE)
provide a promising tool for cell replacement
therapies of retinal degenerative diseases. The
in vitro differentiation of hPSC-RPE is still poor-
ly understood and current differentiation pro-
tocols rely on spontaneous differentiation on
fibroblast feeder cells or as floating cell aggre-
gates in suspension. The fibroblast feeder cells
may have an inductive effect on the hPSC-RPE
differentiation, providing variable signals mim-
icking the extraocular mesenchyme that directs
the differentiation in vivo. The effect of the com-
monly used fibroblast feeder cells on the hPSC-
RPE differentiation was studied by comparing
suspension differentiation in standard RPEbasic
(no bFGF) medium to RPEbasic medium condi-
tioned with mouse embryonic (mEF-CM) and
human foreskin (hFF-CM) fibroblast feeder cells.
The fibroblast secreted factors were found to
enhance early hPSC-RPE differ entiation. The on -
set of pigmentation was faster in the condition-
ed media (CM) compared to RPEbasic for both
human embryonic (hESC) and induced pluripo-
tent (iPSC) stem cells, with the first pigments
appearing around two weeks of differentiation.
After four weeks of differentiation, CM condi-
tions consistently contained higher number of
pigmented cell aggregates. The ratio of PAX6
and MITF positive cells was quantified to be
clearly higher in the CM conditions, with mEF-
CM containing most positive cells. The mEF cells
were found to secrete low levels of activin A
growth factor that is known to regulate eye field
differentiation. As RPEbasic was supplemented
with corresponding, low level (10 ng/ml) of re-
combinant human activin A, a clear increase in
the hPSC-RPE differentiation was achieved.
Thus, inductive effect provided by feeder cells
was at least partially driven by activin A and
could be substituted with a low level of recom-
binant growth factor in contrasts to previously
reported much higher concentrations.
Keywords: Retinal Pigment Epithelial Cell; Human
Pluripotent Stem Cell; Conditioned Medium; Human
Foreskin Fibroblast; Mouse Embryonic Fibroblast;
Activin A; Cell Differentiation
1. INTRODUCTION
Retinal pigment epithelium (RPE) is a highly pola-
rized and specialized monolayer of cells located between
the neural retina and choroid at the back of the eye. RPE
has several vitally important functions as a part of the
blood-retina-barrier and in supporting photoreceptor
function and survival [1,2]. RPE degeneration has a ma-
jor role in pathogenesis of retinal diseases including age-
related macular degeneration (AMD) and retinitis pig-
mentosa. The degeneration of RPE cells leads to the
degradation of photoreceptors and as a consequence to
either partial or total loss of vision. Currently, functiona-
lity of destroyed RPE cells can be restored only with cell
transplantation, setting high demands to develop novel
cell sources for replacement therapy. Transplantation of
RPE cells has been studied extensively in animal models
and also in humans [2-5]. Several cell sources have been
*Equal contribution.

H. Hongisto et al. / Stem Cell Discovery 2 (2012) 176-186 177
studied for cell therapy but currently human pluripotent
stem cells (hPSCs) are considered to be the most pro-
mising cell source of differentiating cells for tissue engi-
neering applications due to their differentiation potential
and high replicative capacity. Several research groups
have reported successful differentiation of RPE cells
from hPSCs [6-10] and first clinical studies using human
embryonic stem cell (hESC) derived RPE cells are on-
going [11].
During mammalian development, RPE and neural re-
tina are both derived from optic neuroepithelium and
share the same progenitor [12]. The neuroepithelium near
the anterior part of the neural tube evaginates laterally to
form the optic vesicles. Invagination of the distal part of
the optic vesicle leads to the formation of the optic cup in
a complex environment affected by many external sig-
nals [13]. By the sixth or seventh week of development,
the optic cup has differentiated into two epithelial sheets.
Of these, the distal layer differentiates into the neural
retina and the proximal layer develops into the RPE in
interactions with the surrounding extraocular tissue, in-
cluding the extraocular mesenchyme [12,14,15].
In the absence of external signal molecules, the hPSCs
choose the neural differentiation pathway as a default
[16]. Most of the published hPSC-RPE differentiation
protocols rely on spontaneous differentiation in absence
of basic fibroblast growth factor (bFGF). The induction
of differentiation is based on confluent overgrowth on
feeder cells especially mouse embryonic fibroblasts (mEF)
or through embryoid body/neurospehere formation [7,
9,10]. Recently, RPE differentiation efficiency has been
enhanced with prolonged culture and growth factor/in-
hibitor based differentiation strategies. Factors, such as
activin A, transforming growth factor β1 (TGFβ1), and
nodal antagonist SB431542 [17] as well as Wnt signaling
inhibitor CKI-7 together with Dkk-1, Lefty-A, FGF an-
tagonist Y-27632 and SB431542 [18,19] have been used.
Regardless of these, many groups are using feeder cell
(mEF, foreskin fibroblasts, PA6 cells) containing meth-
ods with spontaneous differentiation method [20,21] and
first clinical studies are conducted with mEF supported
and spontaneously differentiated hESC-RPE cells [11]. It
is not clear why the removal of FGFs from feeder cell
based hPSC cultures [20] or the use of PA6 stromal
feeder cell to promote neural differ- entiation [22-24] is
sufficient to produce RPE cells but both of the differen-
tiation strategies suggest important function of external
signals provided by mesenchymal fibroblasts/stromal
cells.
We hypothesized that fibroblast feeder cells used for
the culture of undifferentiated hPSC may have an induc-
tive effect on RPE cell differentiation providing mesen-
chymal signals necessary for the key cellular decision
guiding optic cup differentiation and further cell com-
mitment towards RPE cell fate [25,26]. Moreover we
hypothesized that different feeder cells types (mEF and
human foreskin fibroblast, hFF) may provide variable
mesenchymal signals guiding RPE differentiation. In this
study, we studied the inductive effects of feeder cells
routinely used for hPSC differentiation towards RPE
cells. Human PSCs were differentiated using media con-
ditioned by two types of fibroblasts feeder cells (hFF-
CM and mEF-CM) and non-conditioned differentiation
medium (RPEbasic).
2. MATERIALS AND METHODS
All cells were cultured in 37˚C, 5% CO2 incubator
(Thermo Electron Corp., Waltham, MA, USA) and mo-
nitored regularly with Nikon Eclipse TE2000-S phase
contrast microscope (Nikon Instruments Europe B.V.,
Amstelveen, The Netherlands).
2.1. Fibroblast Feeder Cell Culture
Human FF (CRL-2429™, American Type Culture Col-
lection, ATCC, Manassas, VA, USA) were cultured in
Iscove’s Modified Dulbecco’s Medium (IMDM, Life
Technologies, Carlsbad, CA, USA) supplemented with
10% FBS (PAA Laboratories GmbH, Pasching, Austria)
and 0.5% Penicillin/Streptomycin (Lonza Group Ltd,
Basel, Switzerland). P-MEF (EmbryoMax®, Millipore,
Billerica, MA, USA) were cultured in Knock-Out Dul-
becco’s Modified Eagle Medium (KO-DMEM) supple-
mented with 10% FBS and 1% GlutaMax-I, sterile-fil-
tered prior use. Cell culture flasks for mEF were pre-
coated with 0.1% porcine gelatin (Sigma-Aldrich, St.
Louis, MO, USA) for 1 h at room temperature (RT).
Both fibroblast cell lines were purchased as frozen stocks
and cryopreserved at early passages with 5% - 10% di-
methyl sulfoxide (DMSO, Sigma-Aldrich) supplementa-
tion.
2.2. Human Pluripotent Stem Cell Culture
The human embryonic stem cell (hESC) line Regea
06/040 was derived at IBT—The Institute of Biomedical
Technology (former Regea—Institute for Regenerative
Medicine), University of Tampere, Finland. The hESC
line was derived on hFF feeder cells and cultured and
characterized as described previously [27]. Human in-
duced pluripotent stem cell (iPSC) line FiPS5-7 was es-
tablished by Professor Otonkoski’s research group at
University of Helsinki, Finland. It was generated from
human fibroblasts using four transcription factors—
OCT3/4 (POU5F1), SOX2, nanog, and LIN28 [28], and
transgene silencing was confirmed with qPCR [29]. Prior
to the experiments, both pluripotent cell lines were cul-
tured on hFF feeder cells in standard hPSC culture me-
dium consisting of KO-DMEM supplemented with 20%

H. Hongisto et al. / Stem Cell Discovery 2 (2012) 176-186
178
knock-out serum replacement (KO-SR), 2 mM Gluta-
Max-I, 0.1 mM 2-mercaptoethanol (all from Life Tech-
nologies), 1% Non-Essential Amino Acids (NEAA), 50
U/ml Penicillin/Streptomycin (both from Lonza Group
Ltd.) and 8 ng/ml human bFGF (R&D Systems Inc.,
Minneapolis, MN, USA). The culture medium was chan-
ged five times a week and undifferentiated colonies were
manually passaged onto new, γ-irradiated (40 Gy) feeder
cell layers once a week.
2.3. Collection of Conditioned Media
Both hFF (passage 6 - 11) and mEF (passage 4 - 5) were
harvested at confluency with TrypLE™ Select (Life Te-
chnologies) at 37˚C, 15 min, and mitotically inactivated
with γ-radiation (40 Gy). Irradiated fibroblasts were seed-
ed onto 0.1% gelatin-coated culture dishes (cell density
3.6 × 104/cm2) and left to adhere overnight. The cells
were adapted to serum-free culture conditions by se-
quential addition of RPE differentiation medium (RPE-
basic) the day after irradiation. RPEbasic included the
same reagents as described above for hPSC culture me-
dium, but supplemented with 15% KO-SR and lacking
bFGF. For a period of 10 days, 2 ml/cm2 of RPEbasic
was collected daily from the culture dishes and replaced
with equal amount of fresh medium. Collected media
were centrifuged at 1000 rpm, 4 min, transferred to new
tubes and stored at −70˚C. After collection, CM for each
fibroblast type was thawed, pooled, and stored at −70˚C
in aliquots until used for differentiation experiments. Four
different batches of CM were similarly prepared for both
fibroblast types.
2.4. Differentiation Culture
Undifferentiated hPSC colonies (Regea06/040 and
FiPS5-7) were manually dissected, and the pieces trans-
ferred to low cell-bind cell culture plates (Corning Inc.,
Corning, NY, USA) in RPEbasic, mEF-CM or hFF-CM.
The media were changed five times a week. Human ESC
line (Regea06/040) was used for differentiation experi-
ments at passage levels 31 - 91 and hiPSC line (FiPS5-7)
at passage levels 48 - 117. The differentiation experi-
ments were repeated six times in total. Influence of ac-
tivin A on RPE differentiation was tested with hESC line
(Regea06/040) (passages 37 - 42) using RPEbasic sup-
plemented with 10 ng/ml activin A (Peprotech, London,
England). The activin A supplementation test was re-
peated three times. The workflow of the study and ana-
lyses performed are summarized in Figure 1.
After six to seven weeks in suspension culture, pig-
mented areas of cell aggregates were selectively replated
to adherent cultures, in order to create purified popula-
tions of hPSC-RPE. Pigmented areas were selected,
washed with Dulbecco’s Phosphate Buffered Saline
(DPBS, Lonza Group Ltd.) and dissociated with 1× Tryp-
Figure 1. Workflow of the study. RPE differentiation was per-
formed using four test media: RPEbasic, hFF and mEF condi-
tioned media and RPEbasic supplemented with activin A. Ana-
lyses performed in different time points are presented.
sin-EDTA (Lonza Group Ltd.) for 20 - 35 min at 37˚C
with repeated trituration. Trypsin was inactivated with
10% human serum (PAA Laboratories), and cells col-
lected to appropriate culture medium through 40 µm cell
strainers. Dissociated cells were plated either on 24-well
plate wells (Corning Cellbind, Corning Inc.) coated with
5 µg/cm2 human placental collagen IV (Sigma-Aldrich)
for 3 h at 37˚C, or to permeable 0.3 cm2 BD BioCoat™
mouse collagen IV cell culture inserts (Becton, Dickin-
son and Company, Franklin Lakes, NJ, USA). Adherent
cultures were maintained using appropriate media that
were changed three times a week.
2.5. Analysis of Pigmentation
The onset of pigmentation was followed daily and the
day of appearance of the first pigmented cells in each
medium was recorded. The appearance of first pigmenta-
tion was recorded from five individual differentiation
experiments for hESCs, four experiments for hiPSCs,
and three activin A supplementation experiments. To
assess the amount of pigmentation after four weeks of
differentiation, the ratio of cell aggregates containing
pigment in relation to total number of aggregates was
counted after 28 days of differentiation. This was done
in three individual differentiation experiments for both
studied cell lines and for all three activin A supplementa-
tion experiments. Results were plotted using Microsoft
Excel 2003 and figures edited with Adobe PhotoShop
CS4.
2.6. Quantitative Real-Time Polymerase
Chain Reaction
Differences in expression levels of genes related to
RPE differentiation: retina and anterior neural fold ho-
meobox (RAX), paired box gene 6 (PA X 6) and mi-
crophthal-mia-associated transcription factor (MITF),
were studied with qPCR. Gene expression was evaluated
for hPSCs differentiated in the three test media: RPE-
basic, mEF-CM and hFF-CM, after 7, 14, and 28 days in

H. Hongisto et al. / Stem Cell Discovery 2 (2012) 176-186 179
differentiation culture. Additionally the expression of
neural retina markers ceh-10 homeodomain containing
homolog (CHX10) and cone-rod homeobox protein (CRX)
was studied after 28 days of differentiation.
Ten to fifteen differentiated cell aggregates were col-
lected from each test medium. In addition, pieces of un-
differentiated colonies of both hPSC lines were collec-
ted for control material prior to the beginning of the ex-
periment. Total RNA was extracted using the NucleoSpin
RNA XS kit (Macherey-Nagel, GmbH & Co., Düren,
Germany), according to the manufacturer’s protocol. The
RNA quality and concentration were determined using
NanoDrop-1000 spectrophotometer (NanoDrop Tech-
nologies, Wilmington, DE, USA). Complementary DNA
(cDNA) was synthesized from 200 ng of each RNA sam-
ple, using MultiScribe Reverse Transcriptase in the pre-
sence of RNase inhibitor (High-capacity cDNA RT kit,
Applied Biosystems Inc., Foster City, CA, USA), ac-
cording to the manufacturer’s instructions. The synthesis
of cDNA was carried out in PCR MasterCycler (Eppen-
dorf AG, Hamburg, Germany): 10 min at 25˚C, 120 min
at 37˚C, 5 min at 85˚C, and finally cooled down to 4˚C.
FAM-labeled TaqMan® Gene Expression Assays (Ap-
plied Biosystems Inc.) were used for qPCR reactions:
RAX (Hs00429459_m1), PA X 6 (Hs00240871_m1), MITF
(Hs01115553_m1), CRX (Hs01549131_m1) and CHX10
(Hs01584048_m1). Glyceraldehyde 3-phosphate dehy-
drogenase, GAPDH (Hs99999905_m1) was used as en-
dogenous control. Each reaction mixture consisted of 7.5
µl TaqMan® Universal PCR Master Mix (2×), 0.75 µl
Gene Expression Assay (20×), 3 µl of cDNA (diluted 1:5
with sterile water) and sterile water to the total volume of
15 µl. All samples and controls were run as triplicate
reactions using the 7300 Real-time PCR system (Applied
Biosystems Inc.) as follows: 2 min at 50˚C, 10 min at
95˚C, and 40 cycles of 15 s at 95˚C, and 1 min at 60˚C.
Results were analyzed using 7300 System SDS Software
(Applied Biosystems Inc.). Based on the CT-values given
by the software, the relative quantification of each gene
was calculated using the 2–ΔΔCt method [30] and Micro-
soft Excel 2003.
The values for each sample were normalized to ex-
pression levels of GAPDH. The expression level of un-
differentiated hPSC sample was set as the calibrator (fold
change equals 1). Results were plotted using Microsoft
Excel 2003 and figures edited with Adobe PhotoShop
CS4. For visualization of down-regulation, the fold
change values <1 are presented as the negative inverse of
the value, calculated as –1/(fold change). Standard devia-
tions were calculated for each set of technical replicates,
and presented as error bars.
2.7. Immunofluorescence
Differences in protein expression of PA X 6 and MITF
after 28 days of differentiation were studied using im-
munofluorescence. The cell aggregates were dissociated
to single cells as described above. Single-cell suspen-
sions containing 1.6 – 3.5 × 105 cells/ml were prepared in
DPBS, and 150 µl samples were centrifuged onto 15-mm
glass cover slips at 600 rpm, 5 min, using Shandon Cy-
tospin 2 cytocentrifuge (Thermo Fisher Scientific, Wal-
tham, MA, USA). Cells were fixed immediately with 4%
paraformaldehyde (PFA, Sigma-Aldrich) for 15 min at
RT. Cells were permeabilized with 0.1% Triton X-100
(Sigma-Aldrich) in DPBS at RT for 10 min, and unspe-
cific binding blocked with 3% bovine serum albumin
(BSA, Sigma-Aldrich) at RT for 1 h. Incubation with
primary antibodies was carried out either overnight at
4˚C or for 1 h at RT with the appropriate antibody: 1:200
dilution of mouse anti-PA X 6 (Developmental Studies Hy-
bridoma Bank, University of IOWA, Department of Bio-
logy, Iowa City, IA, USA) or 1:350 dilution of rabbit
anti-MITF (Abcam, Cambridge, UK). Secondary anti-
bodies were diluted 1:1500 in 0.5% BSA-DPBS and cells
incubated 1 h at RT in either Alexa Fluor 568-conjugated
goat anti-mouse IgG or goat anti-rabbit IgG (both from
Molecular probes, Life Technologies). Cell nuclei were
stained with VectaShield mounting medium (Vector La-
boratories Inc., Burlingame, CA, USA) containing 4’,
6’-diamidino-2-phenylidole (DAPI). Cells were imaged
with Olympus BX60 microscope (Olympus, Tokyo, Ja-
pan) using a 40× objective. The images were captured
using same exposure time within each experiment and
imaged areas were selected randomly. Minimum 700
cells were counted from each condition. PA X 6 and MITF
expression was quantified using Image J Image Process-
ing and Analysis Software [31]. For each experiment, the
threshold for positive expression was set by analysing
several randomly selected images. Intensity threshold
was adjusted for each image within an experiment and
label to normalize the levels of background intensity.
Cells below the set threshold level were considered
negative. The total number of cells in each image was
determined by counting nuclei counterstained with DAPI.
The numbers of cells expressing PA X 6 or MITF in rela-
tion to the total amount of cells were counted for hESC-
RPE from two individual experiments and two activin A
supplementation experiments. Results were plotted as bar
charts using Microsoft Excel 2003 and figures edited
with Adobe PhotoShop CS4.
Monolayers of hESC-RPE maturated on mouse colla-
gen IV cell culture inserts were analyzed with immu-
nofluorescence for the expression and correct localiza-
tion of RPE-related proteins: MITF, cellular retinal-de-
hyde-binding protein (CRALBP), Bestrophin, and tight
junction protein zona occludens-1 (ZO-1). Detailed pro-
tocol has been published previously [21]. Images were
taken either with Olympus BX60 microscope or LSM
700 confocal microscope (Carl Zeiss, Jena, Germany)

H. Hongisto et al. / Stem Cell Discovery 2 (2012) 176-186
180
using a 63× oil immersion objective. All images were
edited using ZEN 2009 Light Edition (Zeiss) and Adobe
Photoshop CS4.
2.8. Reverse Transcriptase PCR
Monolayers of hPSC-RPE maturated on human colla-
gen IV were analyzed for expression of RPE specific
genes by reverse transcription polymerase chain reaction
(RT-PCR). Expression of the following genes was as-
sessed: RPE precursor markers MITF and orthodenticle
homeobox 2 (OTX2), and mature RPE-specific markers
retinal pigment epihelium-specific protein 65 kDA (RPE65),
bestrophin (BEST1), pre-melanosomal protein 17 (PMEL
17), pigment epithelium-derived factor (PED F ) and ty-
rosinase (TYR). GAPDH was used as endogenous control.
Total RNA was extracted and 40 ng was reverse- tran-
scribed to cDNA as described above. Genomic control
reactions excluding the reverse transcriptase enzyme
(-RT) for each RNA sample were performed. RT-PCR
was carried out using 1 µl of cDNA as template. Detailed
protocol and primer sequences used have been previ-
ously published [21].
2.9. Growth Factor Secretion Analysis
The three differentiation media: RPEbasic, mEF-CM
and hFF-CM were analyzed for concentrations of TGF-β1,
activin A and bFGF growth factors with enzyme-linked
immunosorbent assay (ELISA). The following comer-
cial ELISA kits were used: Human TGF-β1 Immunoas-
say, Human/Mouse/Rat Activin A Immunoassay, human
FGF basic Immunoassay (all from Quantikine®, R&D
Systems, Minneapolis, MN, USA). The Human TGF-β1
Immunoassay and human FGF basic Immunoassays have
been previously shown to detect the growth factor con-
centrations also from mEF-CM [32]. All assays were
performed according to manufacturer’s instructions. All
standards and samples were tested in duplicates. For the
activin A immunoassay, each sample was diluted 1:5 and
1:25, with the diluent supplied in the kit. Optical densi-
ties were measured using Wallac Victor2TM
1420 Multi-
label counter (Perkin Elmer-Wallace, Norton, OH, USA).
Using optical densities of the standard series, standard
curves were created using Microsoft Excel 2003 and
concentrations of the samples calculated accordingly.
Standard deviations were calculated from the concentra-
tions of duplicates of each tested sample, and were pre-
sented as error bars. The measurements were repeated
twice from two different batches of CM.
2.10. Ethical Considerations
The study of human embryos at University of Tampere
has been approved by National Authority for Medicole-
gal Affairs Finland (TEO) (Dnro 1426/32/300/05). We
have a supportive statement of Ethical Committee of
Pirkanmaa Hospital District to derive, culture, and dif-
ferentiate hESC lines from surplus human embryos
(Skottman/R05116). No new cell lines were derived for
this study.
3. RESULTS
3.1. Appearance of Pigmentation Was
Accelerated in the Conditioned Media
Human ESCs and iPSCs were differentiated in suspen-
sion as floating cell aggregates in three different media:
standard RPEbasic, hFF-CM and mEF-CM. Differentia-
tion rate of hPSC-RPE was monitored by recording the
appearance of first pigmented cells in each medium. The
appearance of pigmented cells was faster in the CM
compared to the RPEbasic for both hESCs and iPSCs.
On average, the hESCs pigmented fastest in mEF-CM
(day 13), next in hFF-CM (day 15) and slowest in RPE-
basic (day 16) (Figure 2(A)). Human iPSCs generated
pigmented cells on average at day 16 in both CM and at
day 18 in RPEbasic (data not shown).
3.2. hPSCs Expressed Marker Genes for Eye
Field and RPE Precursor Cells during
Differentiation
Gene expression of early eye field markers PAX 6 and
RAX, and RPE precursor marker MITF was analyzed
with relative qPCR after 7, 14 and 28 days of differentia-
tion. The gene expression levels were compared to un-
differentiated hPSCs. For hESCs, expression of PA X6
increased substantially during differentiation, suggesting
that differentiation progressed to eye field direction in all
studied media (Figure 2(B)). Expression levels of RAX
increased during the first two weeks of differentiation,
and decreased by day 28 in cells differentiated in both
CM (Figure 2(C)). This decrease in RAX expression was
accompanied by a 10-fold increase in the expression of
RPE-specific MITF (Figure 2(D)) which indicates pro-
gress toward RPE fate. In RPEbasic, expression of RAX
further increased by day 28, but the pattern of MITF ex-
pression was similar to that of CM. In addition, the ex-
pression of neural retina markers CHX10 and CRX were
analyzed at day 28 and found to be substantially de-
creased for both CM conditions compared to RPEbasic.
Especially the early neural retina marker CHX10 expres-
sion was 15 times higher in the RPEbasic condition
compared to mEF-CM and 9 times higher compared to
hFF-CM (data not shown). This expression pattern indi-
cated increased differentiation toward neural retina di-
rection in RPEbasic and toward RPE fate in CM. The
studied genes showed a similar expression pattern also in

H. Hongisto et al. / Stem Cell Discovery 2 (2012) 176-186 181
(A)
(B)
(C)
(D)
Figure 2. Analysis of early-stage hESC-RPE differ-
entiation. The day of first pigmentation observed in
replicate experiments, as well as the average, shown
for each medium (A). Gene expression of the early
eye-field markers PA X 6 (B) and RAX (C) as well as
early RPE marker MITF (D), relative to undifferen-
tiated stem cells (d0) was analysed with qPCR.
hiPSC differentiation but with lower relative expression
levels (data not shown).
3.3. Conditioned Media Contained More RPE
Cells after Four Weeks of
Differentiation
After four weeks of differentiation, the number of
pigmented cell aggregates to total number of aggregates
was calculated for each medium. CM consistently conta-
ined higher percentage of pigmented cell aggregates
compared to RPEbasic for both hESCs (Figure 3(A))
and hiPSCs (data not shown) in each of the three re-
plicate experiments. Typically, the pigmented areas were
also larger in CM compared to RPEbasic (Figure 3(B)),
indicating higher number of pigmented cells within the
areas. In addition, the number of PA X 6 and MITF ex-
pressing cells in the differentiated cell aggregates in each
medium were quantified at 28 day time point. After dis-
sociation to single cells and immunostaining, the number
of positive cells was calculated. In two replicate ex-
periments, the ratios of PA X 6 and MITF positive cells
were clearly higher in CM compared to RPEbasic
(Figure 3(C)) with mEF-CM containing highest per-
centage of positive cells. On average, over 90% of cells
expressed PA X 6 and MITF in both CM, whereas in
RPEbasic only 61% (±8%) of cells were positive to
PA X6 and 74% (±8%) to MITF . Representative images of
cells immunolabeled for PA X 6 and the same cells coun-
terstained with DAPI are shown in Figure 3(D).
3.4. Mature hPSC-RPE Cells Possessed RPE
Morphology and Expressed
RPE-Specific Genes and Proteins
After selective plating of pigmented areas to adherent
cultures on collagen IV, pigmentation and RPE-like cell
morphology were initially lost. Cells acquired fibro-
blast-like morphology and proliferated to confluence,
after which cobblestone morphology and pigmentation
began to reappear within two weeks of culture. Mature
cells were pigmented and possessed regular hexagonal
arrangement typical to RPE (Figure 4(A)).
Expression of RPE-specific markers was studied at the
protein level with immunofluorescence. Cells co-expre-
ssed MITF in the nuclei and CRALBP in the cytoplasm
and cell membranes (Figure 4(B)). Moreover, expression
of Bestrophin (Figure 4(C)) and tight junction protein
ZO-1 (Figure 4(D)) confirm the maturity of hESC-de-
rived RPE cells. The maturated hPSC-RPE cells were
analysed for RPE-specific gene expression with RT-PCR.
Cells in all three test media were shown to express RPE
precursor genes MITF and OTX2, as well as genes spe-
cific to mature RPE, namely RPE65, BEST1, PMEL17,
PEDF and TYR, confirming differentiation to RPE fate.
The maturated hESC-RPE (Figure 4(E)) and hiPSC-
RPE (data not shown) cells showed identical gene ex-
pression profile.

H. Hongisto et al. / Stem Cell Discovery 2 (2012) 176-186
182
(A) (B)
(C) (D)
Figure 3. Degree of RPE differentiation at the 28 day
time-point. The ratio of pigmented cell aggregates to total
number of aggregates in each medium shown for three re-
plicate experiments, n = total number of counted cell aggre-
gates (A). Representative images of pigmented cell ag-
gregates in each medium, scale bars 500 µm (B). Average
percentage of cells expressing PA X6 and MITF in two repli-
cate experiments quantified by cell counting. Standard de-
viations as error bars, n = total number of counted cells (C).
Illustrative images of cells labelled with anti-PA X 6 for cell
counting before and after thresholding (D).
3.5. Feeder Cells Secreted Activin A and
TGF-β1
Concentrations of bFGF, activin A and TGF-β1 were
measured in both CM and RPEbasic with ELISA. Con-
centration of bFGF was undetected in all tested media.
Concentration of TGFβ in RPEbasic (15% KO-SR) was
67 pg/ml. Both fibroblast types secreted low levels of
TGF-β: mEF-CM contained 207 pg/ml and hFF-CM 549
pg/ml. In addition, mEFs secreted substantially more ac-
tivin A compared to hFFs—mEF-CM contained 7.1 ng/ml
of activin A, whereas hFF-CM contained 1.0 ng/ml. Ac-
tivin A was undetected in RPEbasic, meaning that practi-
cally all the activin A present in CM was secreted by the
fibroblasts.
3.6. Activin A Supplementation Accelerated
hESC-RPE Differentiation
Based on the results of the growth factor analyses, in
ductive effect of activin A was tested by supplementing
RPEbasic with 10 ng/ml of recombinant human activin
A. In all three separate repeats, addition of activin A had
Figure 4. Analysis of mature hESC-RPE cells.
Maturated cells possessed appropriate RPE mor-
phology and pigmentation (A). Protein expres-
sion of CRALBP (green) and MITF (red) (B), Be-
strophin (C), and ZO-1 (D) was confirmed with
immunofluorescence, Scale bars 10 µm. Similar
results were obtained in each test medium. Re-
presentative images in (A)-(D) are of cells cul-
tured in mEF-CM. Gene expression profile of
several RPE-related genes shown for hESC-RPE
differentiated in the three test media, -RT = ge-
nomic control (E).
a pronounced effect on the early-stage RPE differentia-
tion. Activin A accelerated the onset of pigmentation
from an average of day 16 to day 11 (Figure 5(A)), and
by day 28 of differentiation enhanced the degree of pig-
mentation from 30% to 70% of pigmented cell aggre-
gates (Figure 5(B)). Furthermore, differentiation cultures
treated with activin A showed higher expression of PA X 6
and MITF, quantified from immunofluorescence samples.
On average, 96% (±1%) of cells were positive for PA X 6
and 71% (±14%) for MITF after activin A treatment,
while corresponding values in RPEbasic were 74% (±
16%) and 57% (±13%) (Figure 5(C)). After adherent
maturation culture, the cells in RPEbasic supplemented
with 10 ng/ml activin A showed mature RPE phenotype
with corresponding pigmentation, morphology and pro-
tein expression (Figures 5 (D)-(G)).
4. DISCUSSION
During early eye development, RPE is surrounded by the

H. Hongisto et al. / Stem Cell Discovery 2 (2012) 176-186
183
(A) (B) (C)
Figure 5. Differentiation efficacy of medium supplemented with activin A. Addition of activin A to the RPEbasic
medium had a positive effect on the onset of pigmentation (A). Similarly, ratio of pigmented cell aggregates after 28
days of differentiation was enhanced, n = total number of cell aggregates counted (B). Percentage of PA X 6 and MITF
positive cells with and without activin A supplementation (C). Mature hESC-RPE cells cultured in RPEbasic sup-
plemented with activin A possessed appropriate cell morphology and pigmentation (D) and expressed CRALBP
(green) and MITF (red) (E), Bestrophin (F), and ZO-1 (G). Scale bars 10 µm.
and CH X10 demonstrating the early neural precursors’
progress towards RPE cell fate instead of neural retina.
Similar but moderated effect was seen with cells differ-
entiated in hFF-CM. Most importantly, both of the CM
conditions were verified to contain substantially more
PA X6 and MITF expressing cells compared to non-con-
ditioned RPEbasic at the protein level, using quantitative
cell counting. After selective plating of pigmented clu-
sters to adherent culture, the cells showed mature RPE
morphology and expression of RPE-specific markers,
both at gene and protein level. Taken together, the induc-
tion of RPE differentiation with feeder cell CM had a
positive effect on hPSC-RPE differentiation.
extraocular mesenchyme, while the ectoderm faces the
neural retina. RPE cell differentiation is known to be
regulated by two key regulatory transcription factors
MITF and OTX2. Expression of these transcription fac-
tors is controlled by interactions with the surrounding ex-
traocular tissue, including the extraocular mesenchyime
[12].
In the present study we hypothesized that fibroblast
feeder cells used for the culture of undifferentiated hPSC
may provide variable mesechymal signals having an in-
ductive effect on spontaneous RPE cell differentiation in
vitro. The results of this study clearly demonstrated the
inductive effect of the two most commonly used fibro-
blast feeder cell types, mEFs and hFFs, on RPE cell dif-
ferentiation both from hESC and iPSCs. In the presence
of soluble factors secreted by feeder cells, both the onset
of pigmentation and its rate were clearly enhanced. As
expected, there was considerable biological variation in
the appearance and amount of pigmentation between the
replicate experiments typical for suspension culture
methods. However, a clear correlating trend was ob-
served. Along with the appearance of pigmented cells,
eye field transcription factor genes RAX and PAX 6 were
expressed. After four weeks of differentiation, expression
of RPE-specific transcription factor MITF was the high-
est in cells differentiated in mEF-CM, accompanied by
decreased expression of RAX and a low expression of CRX
Fibroblast feeder cells in general are known to secrete
various factors promoting or inhibiting the growth and
differentiation of hPSC cells [33-37]. To elucidate the
inductive effect of CM in RPE differentiation, we further
studied the secretion of bFGF, TGF-β1 and activin A,
known factors regulating eye field differentiation, by the
feeder cells. As a result we found that secretion of activin
A was substantially higher by mEFs compared to hFFs.
In contrast, secretion of TGF-β was higher for hFFs
compared to mEFs. This is consistent with our previous
studies showing that mEFs secrete more activin A and
hFFs secrete more TGFβ [38]. We were not able to detect
any measurable levels of bFGF from either CM thus
possible effect of difference in bFGF concentration was
excluded. Similar trend in fibroblast growth factor secre-
OPEN ACC ESS

H. Hongisto et al. / Stem Cell Discovery 2 (2012) 176-186
184
tion has been confirmed by another research group [32].
The extraocular mesenchyme secretes TGF-β1 super-
family growth factors such as activin A, activates the
expression of MITF and down-regulates CHX10 expres-
sion directing RPE cell fate differentiation in vivo. Simi-
lar effects of activin A inducing MITF expression have
been shown [26]. Activin A has also been shown to in-
duce hESC-RPE differentiation in vitro, but only after
pretreatment with nicotinamide [17,39]. The superior
secretion of activin A by mEF feeder cells could thus be
one of the key factors enhancing the early RPE differen-
tiation and reduction of the RAX, CRX, CHX10 expression.
To study the effect of activin A secretion by mEF, we
supplemented the RPEbasic medium with 10 ng/ml ac-
tivin A and concluded that addition of activin A at this
low level had a pronounced effect on the early-stage RPE
differentiation. In previously published studies, relatively
high activin A concentrations of 140 ng/ml between day
14 - 28 of differentiation [17] and 100 ng/ml between day
20 - 40 [40] have been used. On the contrary, we were able
to induce early RPE differentiation with substantially
lower activin A concentration. However, in addition to
activin A both mEF-CM and hFF-CM may contain a pool
of other possible factors inducing RPE cell differentia-
tion. Both fibroblast types secrete various ECM compo-
nents like collagens I and IV, nidogen I, and fibronectin
as well as proteins involved in TGFβ, BMP, Wnt and IGF
signaling [33]. In addition mEFs secrete the neurotrophic
pigment epithelium derived factor (PEDF) [33,34] leav-
ing the field open to identify other important players.
5. CONCLUSION
In this study, we confirmed the inductive effect of
commonly used fibroblast feeder cells on hPSC differ-
entiation towards RPE cells. Human PSCs were differen-
tiated using media conditioned by two types of fibro-
blasts originated from mouse embryos and neonatal hu-
man foreskin tissue. Both feeder cell type CM increased
RPE differentiation as compared to the non-conditioned
medium (RPEbasic). The growth factor activin A, known
inductive agent of RPE fate, was concluded to be an im-
portant factor present especially in mEF-CM. Conse-
quently, supplementation of RPEbasic medium with a
low concentration of activin A increased the differentia-
tion rate of RPE cells to comparative level achieved with
CM. Thus, inductive effect provided by feeder cells was
at least partially driven by activin A.
6. ACKNOWLEDGEMENTS
We thank Professor Timo Otonkoski’s group at University of Hel-
sinki for the kind gift of the hiPSC line FiPS5-7. We thank Outi Melin,
Hanna Koskenaho and Elina Konsén for technical assistance. The study
was financially supported by Academy of Finland (218050; 133879),
The Competitive Research Funding of the Tampere University Hospital
(9H114; 9M098), Päivikki and Sakari Sohlberg foundation, Finnish
Cultural Foundation and Tampere Graduate Program in Biomedicine
and Biotechnology. The PA X 6 antibody developed by Kawakami A was
obtained from the Developmental Studies Hybridoma Bank (DHSB)
developed under auspices of the NICHD and maintained by the Uni-
versity of IOWA, Department of Biology, Iowa City, IA, 52242.
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