Stem Cell Reports
Repor t
Xeno-Free and Defined Human Embryonic Stem Cell-Derived
Retinal Pigment Epithelial Cells Functionally
Integrate in a Large-Eyed Preclinical Model
Alvaro Plaza Reyes,1,5 Sandra Petrus-Reurer,1,2,5 Liselotte Antonsson,1 Sonya Stenfelt,1 Hammurabi Bartuma,2
Sarita Panula,1 Theresa Mader,1 Iyadh Douagi,3 Helder André,2 Outi Hovatta,1,4,6 Fredrik Lanner,1,6,*
and Anders Kvanta2,6
1Department
of Clinical Sciences, Intervention and Technology, Karolinska Institutet, 14186 Stockholm, Sweden
of Clinical Neuroscience, Section for Ophthalmology and Vision, St. Erik Eye Hospital, Karolinska Institutet, 11282 Stockholm, Sweden
3Department of Medicine, Center for Hematology and Regenerative Medicine, Karolinska Institutet, 14157 Stockholm, Sweden
4Cell Therapy Department, Nova Southeastern University, Fort Lauderdale, FL 33314, USA
5Co-first author
6Co-senior author
*Correspondence: fredrik.lanner@ki.se
http://dx.doi.org/10.1016/j.stemcr.2015.11.008
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
2Department
SUMMARY
Human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells could replace lost tissue in geographic atrophy (GA) but
efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and
defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation
methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of
native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic
activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted
in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity.
We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically
compliant hESC-RPE in a large-eyed disease model.
INTRODUCTION
Age-related macular degeneration (AMD), the most common cause of severe vision loss in the Western world, occurs in wet (neovascular) and dry (degenerative) forms.
In early dry AMD, the retinal pigment epithelium (RPE)
becomes dysfunctional, whereas end-stage disease,
geographic atrophy (GA), is characterized by degeneration
of RPE and photoreceptors (Bhutto and Lutty, 2012). The
RPE is a monolayer of polarized cells that constitutes the
outer blood-retina barrier and performs central tasks in
the eye, e.g., light adsorption, secretion of growth factors,
and phagocytosis of photoreceptor outer segments (POS)
(Sparrow et al., 2010). The apical surface harbors microvilli
that interact with the light-sensitive POS, whereas the basolateral surface adheres to Bruch’s membrane (BM), which
in turn separates the RPE from the underlying choroid.
Subretinal transplantation of RPE cells derived from human embryonic stem cells (hESC) could potentially be
used as replacement therapy in GA (Schwartz et al., 2012,
2015). However, a critical question is whether donor cells
integrate into the host RPE and support the overlying photoreceptors. Experimental transplantations of hESC-RPE
have only been conducted in small-eyed rodent models
(Carido et al., 2014; Idelson et al., 2009; Lund et al.,
2006; Vugler et al., 2008). Functional effects in these
models are however non-specific and surgical techniques,
instrumentation, and imaging methods differ from those
applied in humans, limiting their use as preclinical models
(e.g., Pinilla et al., 2009). We have recently described a damage model in the large-eyed rabbit that exhibits typical GA
changes including photoreceptor loss and RPE alterations
(Bartuma et al., 2015).
Several hESC-RPE derivation protocols have been
described with the common limitation of relying on culture steps that involve xeno- or human-feeder cells or use
medium components that are either undefined or not
xeno-free (Klimanskaya et al., 2004; Lane et al., 2014; Osakada et al., 2009; Pennington et al., 2015; Vaajasaari et al.,
2011). Recently, we described a defined and xeno-free
clonal culture of hESC using recombinant human laminin
(rhLN) and E-cadherin (Rodin et al., 2014a, 2014b).
Encouraged by this work, we set out to evaluate whether
rhLN-matrix could support efficient hESC-RPE differentiation. BM, the RPE basement membrane, contains four
LNs, LN-111, LN-332, LN-511, and LN-521, that adhere
to the RPE via specific integrins (Aisenbrey et al., 2006).
In the present study, we show that rhLNs are more effective in supporting xeno-free and defined differentiation
compared with exogenous matrix such as gelatin, used in
Stem Cell Reports j Vol. 6 j 9–17 j January 12, 2016 j ª2016 The Authors 9
(legend on next page)
10 Stem Cell Reports j Vol. 6 j 9–17 j January 12, 2016 j ª2016 The Authors
ongoing clinical studies (Schwartz et al., 2012). Moreover,
we demonstrate that suspension transplantations of
rhLN-521-hESC-RPE integrate as polarized subretinal
monolayers that rescue overlying photoreceptors from
induced damage. We conclude that rhLN-521 effectively
supports differentiation of clinically compliant hESC-RPE,
and presents evidence of efficient long-term functional
integration of hESC-RPE in a large-eyed disease model.
RESULTS
Robust Induction of Primary Pigmented Cells Using
Suspension Differentiation in Defined Medium
We have recently developed a xeno-free hESC derivation
and culture methodology, using rhLN-521-based matrix
and a xeno-free and defined NutriStem hESC XF medium
containing basic fibroblast growth factor (bFGF) (Rodin
et al., 2014a, 2014b). As FGF removal is critical for RPE differentiation (Pittack et al., 1997), we decided to evaluate
whether our hESC culture methodology could efficiently
support hESC-RPE differentiation following bFGF removal.
hESCs were cultured on rhLN-521 and manually scraped
to produce a suspension culture of embryoid bodies (EBs)
in NutriStem hESC XF without bFGF (Figures 1A and 1B).
This culture robustly supported formation of pigmented
structures resembling optical vesicles (OVs) at as early as
3 weeks of differentiation (Figures 1A and 1B). At this stage,
we observed an average of 0.3 OVs per EB, which in the
subsequent weeks increased to 0.8 per EB (Figure 1C).
This efficiency of OV induction is well in line with previous
reports (Idelson et al., 2009), confirming that our xeno-free
culture medium efficiently supports the first stage of hESCRPE differentiation.
rhLN-521 Efficiently Supports Homogeneous
Expansion of Pigmented and Functional hESC-RPE
Endogenous BM contains four LNs: LN-111, LN-332, LN511, and LN-521. Consequently, we decided to compare subsequent expansion and maturation of primary pigmented
cells on gelatin or rhLNs found in the endogenous BM.
The pigmented OVs were mechanically cut out using a
scalpel and dissociated into single cells. Cells were seeded
through a cell strainer onto gelatin or LN-coated dishes.
Three days following plating, it was clearly observable
that LN-521 had the best performance, with 69% plating
efficiency compared with 8% in gelatin-coated cultures
(Table S1). Pigmentation was initially lost in all cultures,
but was progressively reestablished from day 21 (Figure 1D),
as previously described. Interestingly, time-lapse microscopy showed that cells on rhLN-511 and rhLN-521 were
highly migratory forming uniform monolayers throughout
the wells (Figures 1D–1F and Movie S1), while progressively
maturing into pigmented hexagonal cells. This correlates
well with a previous study showing that the same subtype
of integrin receptors recognizes LN-511 and LN-521 (Aisenbrey et al., 2006). Cells on gelatin were migratory, but
tended to stay in tight colonies and failed to fully cover
the plate even after 77 days (Figures 1D–1F and S1A).
Transcriptional analysis showed similar profiles in hESCRPE differentiated on each of the five substrates with
Figure 1. Xeno-Free and Defined RPE Differentiation of hESC to hESC-RPE
(A) Differentiation protocol scheme. Confluent hESC cultures were scraped and cultured in low-attachment plates with NutriStem /
(bFGF removed) to form EBs with Rock inhibitor during the first 24 hr. First pigmentation (OV-like structures) appears following week 3. At
week 5, pigmented structures are cut out with a scalpel and dissociated into single cells. Cells are plated on different substrates and
cultured until homogeneous pigmentation is reached (week 9). RPE cells are dissociated to a single-cell suspension for subretinal injection
in the rabbit eye.
(B) hESC-EB with pigmented OV-like structures.
(C) Quantification of pigmented OV formation per starting EB during differentiation. Bars represent means ± SD from three independent
experiments.
(D) Differentiation on gelatin and LN-111, LN-332, LN-511, and LN-521 following 21 and 32 days. See also Movie S1.
(E) Images depicting the migratory tracks of ten random cells during the first 7 days of culture on the different conditions.
(F) Bar graph showing the average length migrated by cells growing on the different substrates. Three individual wells with ten cells
analyzed in each were quantified over a period of 7 days using time-lapse imaging. Bars represent means ± SD.
(G) Transcriptional analysis of hESC-RPE differentiated on the different substrates. Values are normalized to RPLPO and GAPDH and
displayed as relative to undifferentiated hESCs. Bars represent means ± SEM from three independent experiments.
(H) Flow cytometry analysis of MITF expression on hESC-RPE cells grown on the different substrates for 29 days.
(I and J) Polarized secretion of VEGF and PEDF in hESC-RPE. Bars represent means ± SEM from three independent experiments.
(K) Phagocytosis of fluorescein isothiocyanates (FITC)-labeled POS by hESC-RPE on the different substrates. hESC-RPE cells incubated with
FITC-labeled POS at 4 C were used as negative controls. Bars represent means ± SD from three independent experiments.
(L) TER measurements of hESC-RPE cells grown on the different substrates. The TER value for undifferentiated hESCs (fully confluent plate)
is shown for comparison (dashed line). Bars represent means ± SEM from three independent experiments.
Scale bars: B, D, E, 500 mm.
See also Figure S1.
Stem Cell Reports j Vol. 6 j 9–17 j January 12, 2016 j ª2016 The Authors 11
Figure 2. Morphology and Marker Expression of rhLN-521-hESC-RPE
(A) Bright-field image of hESC-RPE cells
grown on rhLN-521 acquiring hexagonal
shape before pigmentation.
(B) Mature and highly pigmented hESC-RPE
cells displaying hexagonal morphology.
(C–F) Expression of key RPE markers: cellular
retinaldehyde-binding protein (CRALBP),
zonula occludens protein-1 (ZO-1), Na/
K-ATPase, and Bestrophin1 (Best1) by
immunostaining.
(G and H) Z-stack confocal projections
showing polarized expression of ZO-1 and
Na/K-ATPase markers on the apical side of
the hESC-RPE.
Scale bars: A, B, 100 mm; C–H, 20 mm.
reduction of pluripotency-associated transcripts OCT3/4
and NANOG, together with robust expression of neuroectoderm transcripts sex-determining region Y-box 9 protein
(SOX9) and paired box 6 (PAX6). Low expression levels of
paired box 3 (PAX3) and endothelin receptor B (EDNRB)
transcripts eliminated the possibility of contaminating
melanocytes in any of the substrates (Figure S1B). RPE differentiation was evident with expression of bestrophin 1
(BEST1), RPE-specific protein 65 kDa (RPE65), and premelanosome protein (PMEL) (Figure 1G). However, more sensitive single-cell analysis of mature RPE purity through flow
cytometry for microphthalmia-associated transcription
factor (MITF) and BEST1 showed more homogeneous
expression on all LNs compared with gelatin (Figures 1H
and S1C).
Functionally, all cultures showed polarized secretion of
vascular endothelial growth factor (VEGF) and pigment
epithelium-derived factor (PEDF), as well as active phagocytosis of POS (Figures 1I–K and S1D–S1G). hESC-RPE
only secreted PEDF from week 5 and not earlier (data not
shown). We found that hESC-RPE growing on LN-332
and gelatin displayed lower levels of PEDF secretion
compared with those growing in all the other tested conditions. Also, interestingly, transepithelial electrical resistance (TER) measurements proved the functional tight
12 Stem Cell Reports j Vol. 6 j 9–17 j January 12, 2016 j ª2016 The Authors
junction integrity of our hESC-RPE monolayer on LN111, LN-511, and LN-521 in a time-dependent manner,
but not on LN-332 and gelatin (Figure 1L). This observation
is in line with the fact that RPE cells did not manage to form
a continuous monolayer when growing on these two substrates (Figures 1D and S1A). hESC-RPE seeded on LN-521
reached values of 180 U cm2 after 31 days, indicative
of a functionally mature monolayer. Extended analysis
confirmed that rhLN-521-hESC-RPE cultures acquired a
pigmented and hexagonal morphology (Figures 2A and
2B), and they were also shown to be uniformly positive
for cellular retinaldehyde-binding protein (CRALBP) and
BEST1 with clear apical polarization of zonula occludens
protein 1 (ZO-1) and Na/K-ATPase (Figures 2C–2H).
hESC-RPE Transplantation into Albino Rabbits
For transplantation, we chose the albino rabbit with an eye
size approximately 70% of that the human eye. All major
retinal and subretinal layers were clearly detectable using
cross-sectional spectral domain optical coherence tomography (SD-OCT) (Figures S2A and S2B).
We next transplanted suspensions of rhLN-521-hESCRPE into the subretinal space. Pigmentation was not
evident by ophthalmoscopy but a thickened and irregular
RPE/BM layer was detected by SD-OCT 1 week after
Figure 3. In Vivo Integration of rhLN521-hESC-RPE
(A) H&E-stained section 1 week after
transplantation demonstrates the presence
of lightly pigmented cells as a continuous
monolayer (arrows) between the POS and
the underlying choriocapillaris.
(B) Subretinal human (NuMA-positive) cells
detected in the injected region. Panels
show NuMA (B1), DAPI (B2) and NuMA with
DAPI (B3).
(C) A heavily pigmented subretinal monolayer of transplanted cells is detected
in the injected region 8 weeks after transplantation.
(D and E) 8 weeks after transplantation, the
integrated NuMA-positive rhLN-521-hESCRPE cells are polarized with basolateral BEST1
expression. BF, bright field. Panels show
BEST1 with BF (D1) and BEST1 only (D2).
(F–H) 34 weeks after transplantation, a
heavily pigmented monolayer is present in
the injected region displaying RPE65-positive cells and engulfment of rhodopsinpositive particles. Dashed lines represent
individual nuclei.
(I) The non-pigmented native RPE layer
(arrows) of an untreated area is shown for
comparison.
Scale bars: A–C, 10 mm; D, 100 mm; E–I,
10 mm.
transplantation (Figure S2C). Histologic analysis demonstrated a monolayer of lightly pigmented cells that integrated into the host RPE overlaid by well-preserved photoreceptors (Figure 3A). Positive immunostaining for human
nuclear mitotic apparatus protein (NuMA) confirmed the
human origin of the cells (Figure 3B). Eight weeks after
transplantation, monolayers of cells had become heavily
pigmented and acquired a polarized phenotype as demonstrated by basolateral expression of BEST1 in the injected
area (Figures 3C–3E and S2E). Importantly, all NuMA-positive cells were also pigmented and BEST1-positive. Pigmented rhLN-521-hESC-RPE monolayers with preservation of the neurosensory retina were further observed for
up to 34 weeks (Figures 3F, S2C, and S2D). Donor cells
were positive for the specific RPE marker RPE65 and cyto-
plasmic rhodopsin suggestive of maintained phagocytic activity (Figures 3G and 3H).
In Vivo Photoreceptor Rescue by hESC-RPE
In the albino rabbit, subretinal injection of PBS alone creates a GA-like phenotype including photoreceptor loss
(Bartuma et al., 2015). We therefore tested whether transplanted rhLN-521-hESC-RPE possessed photoreceptor
rescue capacity. The outer nuclear layer (ONL), normally
consisting of five to six layers, was reduced to a single layer
in PBS-injected eyes (Figure 4A), whereas eyes transplanted
with rhLN-521-hESC-RPE had preserved ONL and POS (Figure 4B). Integration of transplanted cells was further necessary for ONL rescue as outer retinal thickness (ORT) measurements in eyes with minimal or no integration were
Stem Cell Reports j Vol. 6 j 9–17 j January 12, 2016 j ª2016 The Authors 13
Figure 4. Photoreceptor Rescue by Transplanted rhLN-521-hESC-RPE
(A) PBS-induced photoreceptor degeneration is shown as a hyperreflective circle and
outer retinal thinning by infrared-confocal
scanning laser ophthalmoscopy (A1) and
SD-OCT (A2) respectively. Green arrow, SDOCT scan plane. A H&E stained section of the
bleb transition zone (arrowhead, A3) is
shown with the normal (A4) and degenerated (arrowheads, A5) photoreceptor
layer magnified. ORT is indicated (arrow).
(B) Two months after transplantation,
large pigmented areas are shown (B1) with
integrated rhLN-521-hESC-RPE shown as a
hyperreflective band (between arrowheads,
B2). The overlying neurosensory retina is
well preserved as demonstrated by SD-OCT
(B2) and histology (B3, B4). ORT is indicated (arrow).
(C–E) ORT is significantly greater in eyes
with integrated (defined as a pigmented
area >2.5 mm2) than with non-integrated
(defined as a pigmented area <2.5 mm2)
rhLN-521-hESC-RPE. The ORT for PBS injection alone is adapted from Bartuma et al.
(2015) and shown for comparison (dashed
line, E). Bars represent means ± SD from
14 injected eyes. See also Supplemental
Experimental Procedures.
Scale bars: A1, A2, B1, B2, 200 mm; A3, A4,
A5, B3, B4, 50 mm. **p < 0.01 (2-sided
Student’s t test).
See also Figures S2 and S3.
similar to those with PBS treatment alone (Figures 4C–4E).
Importantly, undifferentiated hESCs and fibroblasts that
formed transient cell aggregates were not protective of
photoreceptor loss (Figures S3A and S3B). Thus, the photoreceptor rescue model showed evidence of both specificity
(i.e., non-RPE cells were ineffective) and sensitivity (i.e.,
only integrated RPE cells were effective) for functional RPE.
DISCUSSION
Our differentiation method based on rhLN-521 completely
eliminates undefined and animal-based products
throughout the process from derivation of hESCs to differentiated functional hESC-RPE. Using our approach, we can
avoid microbial contamination, including new agents that
have not yet been identified. In addition, we can prevent
the identified risk of rejection potentially brought by
14 Stem Cell Reports j Vol. 6 j 9–17 j January 12, 2016 j ª2016 The Authors
non-human proteins into the cells during culture and/or
differentiation (Aisenbrey et al., 2006). Key to our methodology is the combined use of xeno-free and defined culture
medium with a relevant extracellular matrix. BM supporting the RPE contains collagen types I, II, and IV, fibronectin, heparin sulfate proteoglycans, and LNs: LN-111,
LN-332, LN-511, and LN-521 (Campochiaro et al., 1986;
Martin et al., 2005). As recombinant mouse LN-111
has been shown to support RPE differentiation (Rowland
et al., 2013) and we have shown that LN-521 supports derivation and culture of hESCs (Rodin et al., 2014b), we set out
to evaluate LNs in a xeno-free and defined differentiation
to functional RPE. In accordance with previous observations, we found that rhLN-511 and rhLN-521 supported
the highest degree of migration (Aisenbrey et al., 2006),
which allowed the cells to spread out evenly in the culture
plates. Interestingly, TER assessment from human adult
retinal explants and immortalized RPE cell lines has been
reported to reach 148 and 100 U cm2, respectively (Hornof
et al., 2005). In our culture, cells seeded on LN111, LN511,
and especially on LN521 appear to be as good as human eye
explants and superior to immortalized RPE cell lines in
creating highly polarized monolayers. Gelatin-coated cultures instead generated tight and irregularly shaped colonies, which failed to fully cover the plate and displayed
a poor monolayer functional integrity. All substrates generated cells with similar transcriptional in vitro profiles, but
single-cell analysis of MITF and BEST1 revealed significant
heterogeneity in gelatin cultures compared with all LN cultures. Expanding cells in suboptimal culture conditions
have the inherent risk of selecting for growth-promoting
genetic abnormalities. It was therefore encouraging that
seeding efficiency on rhLN-521 was 69%, compared with
8% on gelatin. From these in vitro observations, we
conclude that rhLN-521 not only eliminates the need for
undefined or xeno-derived matrix components but is in
fact a more supportive and suitable culture matrix for
hESC-RPE.
Upon transplantation, integrated cells initially displayed
reduced pigmentation, which was progressively reestablished, similar to what was observed during in vitro
hESC-RPE derivation. Concomitantly, integration of donor
cells varied both between animals and between eyes of the
same animal. A putative explanation is xeno-graft rejection, as failure of integration correlated with signs of immunoreaction including subretinal cell infiltration, retinal
atrophy, and donor cell loss. Optimization of our immunosuppressive protocol may thus overcome this variation. In
addition, integration may depend on the state of the native
RPE as transplanted cells could adhere better to a denuded
BM as shown in mice with sodium iodate-induced RPE
atrophy (Carido et al., 2014). Accordingly, we have shown
that subretinal injection alone causes disturbed RPE
morphology and partial RPE loss (Bartuma et al., 2015).
In this study, we transplant hESC-RPE to a large-eyed animal. Human and rabbit eyes are of similar size, the main
advantage compared with previously studied mouse and
rat models (Carido et al., 2014; Lund et al., 2006). Transplantation in rodents requires high cell concentrations
(i.e., typically 50,000 cells/ml or more) and a transscleral
approach through the choroid (Vugler et al., 2008). The
outer blood-retinal barrier is thus compromised, potentially triggering an inflammatory response. Moreover,
high cell concentrations and limited surgical control may
cause misdirection, multilayering, and clumping of transplanted cells. Indeed, several publications have shown
that photoreceptor rescue is neither RPE specific nor correlated with an intact donor cell layer (eg. Pinilla et al., 2009).
The large-eyed rabbit allowed us to perform a surgical technique with instrumentation identical to a clinical setting
(el Dirini et al., 1992). The model also permitted high-reso-
lution in vivo tracking of transplanted cells and monitoring of the overlying neurosensory retina through time.
Using this methodology, we demonstrated subretinal
monolayers of rhLN-521-hESC-RPE that remained for up
to 8 months. Furthermore, integrated cells possessed
in vivo functionality including phagocytic activity and
rescue of photoreceptors from induced degeneration.
Importantly, this effect showed both specificity and sensitivity as non-RPE and non-integrated RPE cells were
ineffective.
Suspension transplants have been frequently used in rodent models (Carido et al., 2014; Idelson et al., 2009; Lund
et al., 2006; Vugler et al., 2008) and ongoing clinical studies
(Schwartz et al., 2012, 2015). Concerns about this delivery
method were raised with the main criticism being possible
multilayering of donor cells leading to inefficient integration (Schwartz et al., 2012). As an alternative, transplantation of cells as prepolarized sheets with or without a supporting biomatrix has been suggested (Diniz et al., 2013;
Stanzel et al., 2014). However, these large transplants are
surgically demanding and may lead to retinal scarring
and outer retinal degeneration (Kamao et al., 2014; Stanzel
et al., 2014). In the present study, we noted minimal retinal
scarring and a well-preserved neurosensory retina overlying the transplanted monolayer. Moreover, by using
suspension transplants, we obtained monolayers up to
ten times the size of RPE sheets with a typical size of
2–2.5 mm2 (Kamao et al., 2014; Stanzel et al., 2014). We
demonstrate that a minimally invasive surgical procedure
in a large-eyed disease model can achieve high-yield functional long-term hESC-RPE integration with photoreceptor
preservation. These findings have important implications
for ongoing and future clinical studies for the development
of a safe and efficient cell replacement therapy for GA.
EXPERIMENTAL PROCEDURES
Cell Culture and Differentiation
hES line HS980 was established and cultured under xeno-free
and defined conditions on rhLN-521, and passaged as previously
described (Diniz et al., 2013; Rodin et al., 2014b). For differentiation, cells were scraped and cultured in low-attachment plates,
5–7 3 104 cells/cm2 in custom-made NutriStem hESC XF medium
without bFGF and transforming growth factor b. Rho-kinase inhibitor was included during the first 24 hr. Following differentiation,
pigmented areas were cut out using a scalpel, dissociated, passed
through a 20G needle, and plated at a density of 0.6–1.2 3 104
cells/cm2.
Subretinal Transplantation and In Vivo Imaging
Dissociated hESC-RPE cells were injected (50 ml, 50,000 cells) subretinally using a transvitreal pars plana technique. SD-OCT and
confocal scanning laser ophthalmoscopy was performed to obtain
Stem Cell Reports j Vol. 6 j 9–17 j January 12, 2016 j ª2016 The Authors 15
horizontal cross-sectional b scans and en face fundus in vivo
images, respectively.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental
Procedures, three figures, one table, and one movie and can be
found with this article online at http://dx.doi.org/10.1016/j.
stemcr.2015.11.008.
AUTHOR CONTRIBUTIONS
A.P.R., S.P.R., H.B. I.D., and A.K. performed the experiments. L.A.,
S.S., S.P., and T.M. contributed to the in vitro differentiation methodology. H.B. and H.A. contributed to the animal work. A.P.R.,
S.P.R., O.H., F.L., and A.K. planned the experiments, analyzed the
data, and wrote the manuscript.
ACKNOWLEDGMENTS
This work was supported by grants from the Swedish Research
Council (VR), Ragnar Söderberg Foundation, Swedish Foundation
for Strategic Research, Stockholm County Council (ALF project),
Ögonfonden and Cronqvist Foundation. This study was performed at the WIRM flow cytometry facility, supported by Knut
och Alice Wallenbergs Stiftelse (KAW) and the Live Cell Imaging
Unit/Nikon Center of Excellence, supported by KAW, VR, Centre
for Innovative Medicine, and the Jonasson donation.
Diniz, B., Thomas, P., Thomas, B., Ribeiro, R., Hu, Y., Brant, R.,
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Received: February 21, 2015
Revised: November 16, 2015
Accepted: November 18, 2015
Published: December 24, 2015
Lane, A., Philip, L.R., Ruban, L., Fynes, K., Smart, M., Carr, A., Mason, C., and Coffey, P. (2014). Engineering efficient retinal pigment
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Stem Cell Reports j Vol. 6 j 9–17 j January 12, 2016 j ª2016 The Authors 17
Stem Cell Reports, Volume 6
Supplemental Information
Xeno-Free and Defined Human Embryonic Stem Cell-Derived
Retinal Pigment Epithelial Cells Functionally
Integrate in a Large-Eyed Preclinical Model
Alvaro Plaza Reyes, Sandra Petrus-Reurer, Liselotte Antonsson, Sonya
Stenfelt, Hammurabi Bartuma, Sarita Panula, Theresa Mader, Iyadh Douagi, Helder
André, Outi Hovatta, Fredrik Lanner, and Anders Kvanta
SUPPLEMENTAL FIGURES AND LEGENDS
Figure S1. Morphology of hESC-RPE on gelatin and quantification of phagocytic
activity, related to Figure 1.
(A) hESC-RPE growing on gelatin fail to cover the whole surface even after long-term
cultures. hESC-RPE on gelatin after 7 days (A1), 35 days (A2) and 77 days (A3) in culture.
(B) Transcriptional analysis for melanocytic markers PAX3 (C1) and EDNRB (C2) of hESCRPE differentiated on the different substrates. Values are normalized to RPLPO and GAPDH
and displayed as relative to undifferentiated hESC. Normal human epidermal melanocytes
(NHEM) were used as a positive control. Bars represent mean±SEM, from three independent
experiments.
(C) Flow cytometry analysis of BEST1 expression on hESC-RPE cells grown on the different
substrates for 47 days.
(D-E) Phagocytosis of FITC-labeled POS by hESC-RPE growing on rhLN-521, after
overnight incubation at 4°C (D) and 37°C (E). The former used as negative control, since
active-phagocytosis is temperature-dependent. (D1 and E1) Maximum intensity projection
image depicting internalized FITC-labeled POS in green and phalloidin staining of hESCRPE in red. (D2 and E2) Output image of phagocyted FITC-labeled POS generated by the
CellProfiler software. (D3 and E3) Z-stack confocal projections of the area delimited by
arrows in D1 and E1.
(F-G) Phagocytosis of FITC-labeled POS by hESC growing on rhLN-521, after overnight
incubation at 4°C and 37°C. The former used as negative control, since active-phagocytosis
is temperature-dependent. The images correspond to maximum intensity projections
depicting internalized FITC-labeled POS in green (not present) and hESC bounderies shown
in red by wheat germ agglutinin staining.
Scale bars: (A1) = 200μm (A2, A3) = 500μm; (D1, D2, E1, E2, F, G) = 100μm; (D3, E3) =
20μm.
Figure S2. In vivo imaging of the normal albino rabbit posterior segment and longterm integration of hESC-RPE, related to Figure 3.
(A) En-face IR-cSLO reflectance shows the choroidal vasculature (A1). Green arrow = SDOCT scan plane. The corresponding cross-sectional SD-OCT b-scan of the neurosensory
retina and choroid is shown (A2).
(B) On higher magnification the individual layers of the neurosensory retina, RPE/BM, and
underlying choroid are distinguished (B1). The corresponding histologic structures are shown
(HE staining) (B2). GCL (ganglion cell layer), IPL (inner plexiform layer), INL (inner nuclear
layer), OPL (outer plexiform layer), ONL (outer nuclear layer), OLM (outer limiting
membrane), EZ (ellipsoid zone), OS (outer segments), RPE (retinal pigment epithelium), BM
(Bruch’s membrane), CC (choriocapillaris).
(C) Pigmentation is absent in the subretinal bleb area (dashed circle) one week after
transplantation (C1). Green arrow = SD-OCT scan plane. Simultaneous SD-OCT scan
shows a thickened hyperreflective RPE/BM (between closed arrowheads). Magnification of
the boxed area shows an irregular layer representing putative rhLN-521-hESC-RPE (open
arrows) beneath the hyporeflective outer segments. Twelve weeks (C2), and 34 weeks (C3)
after
transplantation
of
rhLN-521-hESC-RPE multicolor-cSLO
and SD-OCT
scans
demonstrate stable pigmented donor cell integration (between arrowheads) and a wellpreserved overlying photoreceptor layer. Green arrow = SD-OCT scan plane. *Indicates the
corresponding region of the multicolor-cSLO images in C2 and C3.
(D) HE staining of the corresponding histologic section confirms presence of a preserved
neurosensory retina (D1) and pigmented hESC-RPE monolayer (D2).
(E) 8 weeks after transplantation, uninjected areas do not present pigmented cells (E1) and
show negative staining for both NuMA and BEST1 (E2). (BF = bright field).
Scale bars: Scale bars: (A1, A2, C) = 200µm; (B1) = 100µm; (B2, D) = 50µm; (E1, E2) =
10µm.
Figure S3. Undifferentiated hESC or fibroblasts do not rescue photoreceptors, related
to Figure 4.
(A-B) Transplanted undifferentiated hESC (A) or human fibroblasts (B) form transient
hyperreflective subretinal aggregates as demonstrated by SD-OCT. ORT (arrows) overlying
the transplanted bleb area is reduced as depicted by comparing the respective magnified
(boxed) areas 1 week and 4 weeks after transplantation. Note that cell aggregates have
disappeared after 4 weeks. The bleb margin is indicated (arrowheads).
Scale bars = 200µm.
SUPPLEMENTAL MOVIES
Movie S1, related to Figure 1.
Time-lapse phase contrast imaging of the hESC-RPE growing on gelatin, rhLN-111, -332, 511 or -521. Videos are the result of time-lapse stack images taken every hour during the
first 21 days after cell seeding.
SUPPLEMENTAL TABLES
Supplemental Table S1, related to Figure 1.
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Cell Culture
Human embryonic stem cell lines HS980, HS975 and HS983a were derived and cultured
under xeno-free and defined conditions according to the previously described method. [Rodin
2014b]. The cells were maintained by clonal propagation on LN-521 (Biolamina) in NutriStem
hESC XF medium (Biological Industries), in a 5% CO2/5% O2 incubator and passaged
enzymatically at 1:10 ratio every 5-6 days. For passaging, confluent cultures were washed
twice with PBS without Ca2+ and Mg2+ and incubated for 5 min at 37°C, 5% CO2/5% O2 with
TrypLE Select (GIBCO, Invitrogen). The enzyme was then carefully removed and the cells
were collected in fresh pre-warmed NutriStem hESC XF medium by gentle pipetting to obtain
a single cell suspension. The cells were centrifuged at 1100 rpm for 4 min, the pellet
resuspended in fresh prewarmed NutriStem hESC XF medium and cells plated on a freshly
rhLN-521 coated dish. Two days after passage the medium was replaced with fresh
prewarmed NutriStem hESC XF medium and changed daily subsequently.
GFP-labeled H9 human embryonic stem cell (hESC) line cells were maintained in mTESR
media (StemCell Technologies) on Matrigel (BD Biosciences) coated plates (1:60) until
confluence.
Normal human dermal fibroblasts (NHDF) from adult origin (Lonza) were maintained in
DMEM media (LifeTechnologies) with 10% FBS (LifeTechnologies) until confluence.
In vitro differentiation
Pluripotent stem cells were cultured to confluence on rhLN-521 and manually scraped to
produce embryoid bodies (EBs) using a 1000 µl pipette tip. The EBs were then cultured in
suspension in low attachment plates (Corning) at a density of 5-7x104 cells/cm2.
Differentiation was performed in custom-made NutriStem hESC XF medium in which bFGF
and TGFβ have been eliminated with media change twice a week. 10 μM Rho-kinase
inhibitor (Y-27632, Millipore) was added to the suspension cultures only during the first 24h.
Following five weeks differentiation, pigmented areas were mechanically cut out of the EBs
using a scalpel. Cells were then dissociated using TrypLE Select, followed by flushing
through a 20G needle and syringe. Cells were seeded through a cell strainer (ø 40 μm, BD
Bioscience) on LN-coated dishes at a cell density of 0.6-1.2x104 cells/cm2 and fed twice a
week with the same differentiation medium referred above.
Time-lapse microscopy
The behavior of hESC-RPE on the different substrates was monitored using the Cell-IQ live
imaging system (Chip-Man Technologies Ltd.) equipped with a 10x phase contrast objective,
an automated stage and an integrated incubator (37°C, 5% CO2).
After OVs dissociation, hESC-RPE were seeded in triplicates on the different substrates:
Gelatin (0.1%, Sigma), rhLN-111, -332, -511, and -521 (all 20μg/mL, BioLamina). On day 1
after seeding, the plates were transferred to the live cell imaging equipment. Images were
acquired for both the center (2x2 image grids) and the periphery (single images) of every
well. Every region of interest was monitored every hour for 21 days.
Cell migration was assessed for every time-lapse image stacks using NIS-Elements v.4.0
(Nikon). For each stack 10 cells were randomly chosen and manually tracked during the first
7 days of imaging. The length and trajectory of the tracks followed by the different cells was
used to compare the migration capabilities of hESC-RPE among the different substrates.
Results are presented as mean ±SD (standard deviation).
Quantitative real-time PCR
Total RNA was isolated using the RNeasy Plus Mini Kit and treated with RNase-free DNase
(both from Qiagen). cDNA was synthesized using 1 µg of total RNA in 20 µl reaction mixture,
containing random hexamers and Superscript III reverse transcriptase (GIBCO Invitrogen),
according to the manufacturer’s instructions.
Taq-polymerase together with Taqman probes (Life Technologies) for RPLPO (cat. no.
4333761F),
GAPDH
(cat.
no.
4333764F),
NANOG
(cat.
no.
Hs02387400_g1),
POU5F1/OCT4 (cat. no. Hs03005111_g1), SOX9 (cat. no. Hs01001343_g1), PAX6 (cat. no.
Hs01088112_m1), BEST1 (cat. no. Hs00188249_m1), RPE65 (cat. no. Hs01071462_m1)
PMEL (cat. no. Hs00173854_m1), PAX3 (cat no. Hs00240950_m1) and EDNRB (cat no.
Hs00240747_m1) were used. Samples were subjected to real-time PCR amplification
protocol on StepOne™ real-time PCR System (Applied Biosystems). Biological triplicates
were performed for every condition and technical duplicates were carried for each reaction.
Results are presented as mean ±SEM (standard error of the mean).
Flow Cytometry
hESC-RPE growing on the tested substrates were dissociated into single cells using TrypLE
Select after 1 month in culture. Cells were stained with violet LIVE/DEAD fixable stain kit
(Invitrogen) following the manufacturer instructions. Samples were then fixed for 15 min in
4% methanol free formaldehyde (Polysciences) and permeabilized with 0.1% Triton X-100
(Sigma) for 15 min. Mouse anti-MITF (Abcam ab3201, clone [D5]) and mouse anti-BEST1
(Millipore, MAB5466) primary antibodies were used at a concentration of 10 μg/mL, diluted in
2% FBS, 0.1% Tween-20 (Sigma). Cell were incubated with the primary antibodies on ice for
30 min. Indirect immunostaining was completed using Alexa Fluor 488 Donkey anti-mouse
IgG secondary antibody at a concentration of 2 μg/mL (Life Technologies A21202) on ice for
another 30 min. Fluorescence minus one (FMO) controls were included for each condition to
identify and gate negative and positive cells. Stained cells were analyzed using a BD
LSRFortessa flow cytometer equipped with 488 nm, 561 nm, 405 nm and 640 nm lasers (BD
Biosciences). Analysis of the data was carried out using FlowJo v.10 software (Tree Star).
Enzyme-Linked Immunosorbent Assay (ELISA)
hESC-RPE were cultured on Transwell membranes (0.33 cm2, Millipore) coated with different
substrates. Supernatants from both the hESC-RPE apical and basal sides (meaning upper
and lower compartments of the Transwell, respectively) were collected 60 hours after the
medium was changed. VEGF and PEDF secretion levels were measured in triplicates for
each condition with commercially available human VEGF and PEDF ELISA Kits (VEGF:
Cat#DVE00, R&D Systems; PEDF: Cat#RD191114200R, BioVendor), in accordance with the
instructions of the manufacturers, after 4.5 and 7 weeks of culture, respectively. The optical
density
readings
were
measured
using
SpectraMax
250
Microplate
Reader
(MolecularDevices). Results are presented as mean ±SEM (standard error of the mean).
Phagocytosis assay
In order to assess the phagocytic activity of our hESC-RPE, an in vitro assay using FITClabelled photoreceptor outer segments (POS) was performed. FITC-labelled bovine POS
were isolated and kindly given by Dr. E.F. Nandrot from Institut de la Vision, Paris (Parinot et
al., 2014). For that purpose, hESC-RPE were cultured on Transwell membrane (0.33 cm2,
Corning) coated with different substrates for one month after seeding. Cells were incubated
at 37°C or 4°C for 16 hours with 2.42x106 thawed POS/Transwell diluted in DMEM or CO2
independent media (both from LifeTechnologies), respectively. After incubation, cells were
quenched with Trypan Blue Solution 0.2% (GIBCO, Invitrogen) for 10 minutes at room
temperature, fixed with 4% methanol free formaldehyde (Polysciences) at room temperature
for 10 min and permeabilized with 0.3% Triton X-100 in DPBS for 15 min. Rhodamine
phalloidin staining (1:1000, 20 min at room temperature, Life Technologies) was used to
visualize the cell boundaries. Nuclei were stained with Hoechst 33342 (1:1000, 20 min at
room temperature, Invitrogen). Undifferentiated hESC were used as a negative control.
hESC were cultured under the same conditions and incubated also at 37°C and 4°C with
2.42x106 thawed POS/Transwell diluted in DMEM or CO2 independent media. Wheat Germ
Agglutinin-Alexa Fluor® 594 Conjugate (1:200, 20 min at room temperature, Invitrogen) was
used in to visualize the cell boundaries of hESC.
Images were acquired with Zeiss LSM710-NLO point scanning confocal microscope. Postacquisition analysis of the pictures was performed using IMARIS (Bitplane). Total number of
engulfed FITC-POS per condition was quantified with the custom-made pipeline developed
within cell image analysis software CellProfiler (Broad Institute). Results are presented as
mean ±SD (standard deviation).
Transepithelial resistance measurements
Transepithelial electrical resistance (TER) RPE cells plated on Transwells (0.33 cm2,
Millipore) was measured using the Millicell Electrical Resistance System volt-ohm meter
(Millicell ERS-2, Millipore), according to the manufacturer’s instructions. Cultures were
equilibrated outside the incubator at room temperature for 15-20 min before the experiment.
Measurements were performed in unchanged culture media in triplicates for each condition,
at three different positions of each well. Averages were used for further analysis. The
background resistance was determined from a blank culture insert in the same media coated
with the corresponding substrate but without cells, and subtracted from the respective
experiment condition. Measurements are reported as resistance in ohms times the area in
square centimeter (Ω*cm2). Results are presented as mean ±SEM (standard error of the
mean).
Immunofluorescence
Protein
expression
of
mature
hESC-RPE
monolayers
was
assessed
with
immunofluorescence. Cells were fixed with 4% methanol free formaldehyde at room
temperature for 20 min, followed by permeabilization with 0.3% Triton X-100 in Dulbecco’s
phosphate-buffered saline (DPBS) for 10 min and blocking with 4% fetal bovine serum (FBS)
and 0.1% Tween-20 in DPBS for 1 hour. Primary antibodies were diluted to the specified
concentrations in 4% FBS, 0.1% Tween-20, DPBS solution: Bestrophin 1 (BEST1) (1:100,
Millipore MAB5466), Zonula occludens-1 (ZO-1) (1:100, Invitrogen 40-2200), cellular
retinaldehyde-binding protein (CRALBP) (1:250, Abcam ab15051, clone [B2]) and alpha 1
sodium/potassium ATPase (Na/K-ATPase) (1:200, Abcam ab7671, clone [464.6[). The
primary antibodies were incubated overnight at 4°C followed by 2 hours incubation at room
temperature with secondary antibodies: Alexa Fluor 647 donkey anti-rabbit IgG and Alexa
Fluor 488 donkey anti-mouse IgG (both from Life Technologies, A31573 and A21202,
respectively) diluted 1:1000 in 4% FBS, 0.1% Tween-20, DPBS solution. Nuclei were stained
with Hoechst 33342 (1:1000, Invitrogen). Images were acquired with Zeiss LSM710-NLO
point scanning confocal microscope. Post-acquisition analysis of the pictures was performed
using IMARIS (Bitplane).
Animals
After approval by the Northern Stockholm Animal Experimental Ethics Committee 17 New
Zealand white albino rabbits (provided by Lidköpings rabbit farm, Lidköping, Sweden) aged 5
months, weighing 3.5 to 4.0 kg, where used in this study. All experiments were conducted in
accordance with the Statement for the Use of Animals in Ophthalmic and Vision Research.
Subretinal transplantation
hESC-RPE monolayers were washed with PBS, incubated with TrypLE, and dissociated to
single cell suspension in a similar manner as here described for hESC, using a 5% CO2
incubator. Cells were then counted in a Neubauer hemocytometer chamber using 0.4%
trypan blue (GIBCO, Invitrogen). For subretinal injection, a hESC-RPE single cell suspension
was centrifuged at 2400 rpm for 4 min, the medium was carefully removed, and the cell pellet
was resuspended in freshly filter-sterilized PBS to a final concentration of 1000 cells/μL. The
cell suspension was then aseptically aliquoted into 100 μL units and kept on ice until surgery.
hESC were washed with PBS, incubated with Accutase (GIBCO, Invitrogen) for 2 min at
37°C, centrifuged at 1000 rpm for 5 min, the medium was carefully removed, and the pellet
was resuspended in freshly filter-sterilized PBS to a final concentration of 1000 cells/μL. The
cell suspension was then aseptically aliquoted into 400 μL units and kept on ice until surgery.
NHDF cells were washed with PBS, incubated with Trypsin 1x (LifeTechnologies) for 5 min at
37°C, centrifuged at 1000 rpm for 5 min, the medium was carefully removed, and the pellet
resuspended in freshly filter-sterilized PBS to a final concentration of 1000 cells/μL. The cell
suspension was then aseptically aliquoted into 400 μL units and kept on ice until surgery.
Animals were put under general anesthesia by intramuscular administration of 35 mg/kg
ketamine (Ketaminol, 100 mg/ml, Intervet) and 5 mg/kg xylazine (Rompun vet. 20 mg/ml,
Bayer Animal Health), and the pupils were dilated with a mix of 0.75% cyclopentolate / 2.5%
phenylephrine (APL). Microsurgeries were performed on both eyes using a 3-port 25G
transvitreal pars plana technique (Alcon Accurus, Alcon Nordic). 25G trocars were inserted 1
mm from the limbus and an infusion cannula was connected to the lower temporal trocar.
The cell suspension was drawn into a 1 mL syringe connected to an extension tube and a
38G polytip cannula (MedOne Surgical Inc). Without infusion or prior vitrectomy the cannula
was inserted through the upper temporal trocar. After proper tip positioning, ascertained by a
focal whitening of the retina, 50 μL of cell suspension (equivalent to 50.000 cells) was
injected slowly subretinally approximately 6 mm below the inferior margin of the optic nerve
head, forming a uniform bleb that was clearly visible under the operating microscope. Care
was taken to maintain the tip within the bleb during the injection to minimize reflux. After
instrument removal light pressure was applied to the self-sealing suture-less sclerotomies.
No post-surgical topical steroids or antibiotics were given.
Animals were treated in three separate cohorts. In the first cohort (7 animals receiving 521derived hESC-RPE) 4 animals received 2 mg (100 μL) of intravitreal triamcinolone
(Triescence, Alcon Nordic) immediately after surgery, one animal received systemic
cyclosporine-A (a descending dose of 20-5 mg/kg/d sc) (Teva Sweden AB), one animal
received both triamcinolone and cyclosporine-A, and one animal was maintained without
immunosuppression. On post-operative examination, none of the eyes showed signs of
extra- or intra-ocular infection or inflammation except for one animal (one eye) that had a
uveitis one week after surgery and was sacrificed. A second animal was sacrificed 6 weeks
post-transplantation after signs of cyclosporine-A toxicity (hematuria). Analysis of cohort one
showed no beneficial effect of cyclosporine-A on donor cell integration (defined as
appearance of pigmented areas within 4 weeks post-transplantation) whereas several
animals treated with only triamcinolone had successful integration. The second cohort (6
animals receiving 521-derived hESC-RPE) was therefore treated with intravitreal
triamcinolone alone without any post-operative complications. The third cohort received
either undifferentiated hESC (2 animals) or fibroblasts (2 animals) with post-operative
triamcinolone. In animals kept for long-term evaluation, intravitreal triamcinolone was readministered every 3 months.
Spectral domain-optical coherence tomography (SD-OCT)
Anesthetized rabbits were placed in an adjustable mount. A commercial Spectralis HRA +
OCT device (Heidelberg Engineering) with the Heidelberg Eye Explorer Software (version
1.9.10.0) was used to obtain horizontal cross-sectional b-scans of hESC-RPE treated
animals. The Spectralis has a real-time motion tracking system that minimizes eye motion
artifacts. At least 3 cross-sectional OCT scans were obtained with simultaneous infraredconfocal
scanning
laser
ophthalmoscope
(IR-cSLO)
reflectance
reference
images
representing the upper, central and lower portion of the transplanted area. The best overall
image quality was obtained when the OCT setting was on high-speed acquisition with at
least 50 averaged automatic real-time images. En-face fundus images were obtained by IRor multicolor cSLO (a composite of three simultaneously acquired color cSLO images).
These modalities have a higher contrast level compared to conventional fundus camera
photos. The total area of the pigmented lesions was estimated manually on IR-cSLO images
using the built-in measuring tool of the Spectralis software.
Photoreceptor rescue measurements
We previously defined ORT as the SD-OCT distance between the inner nuclear layer (INL)
and RPE using the ImageJ software (http://imagej.nih.gov/ij/) (Bartuma et al., 2015). ORT of
treated regions was obtained from a section overlying at least 500 μm of continuously
integrated cells (mean of 10 random measurements). ORT of non-treated control regions
was obtained from the same scan 500 μm outside the bleb (mean of 5 random
measurements). Relative ORT was then calculated as the ratio between treated and nontreated retina. Distances were normalized to the 200 μm scale bar of the original image. All
measurements were done one month post-transplantation, or at the nearest time-point after.
A 2-sided Student’s T-test was performed comparing the relative ORT between eyes with
integrated and with non-integrated rhLN-521-derived hESC-RPE (defined as a pigmented
area >2.5 mm2). Results are presented as mean ±SD (standard deviation).
Histology and tissue immunostaining
Immediately after sacrifice by intravenous injection of 100 mg/kg pentobarbital (Allfatal vet.
100 mg/ml, Omnidea), the eyes were enucleated and the bleb injection area marked with
green Tissue Marking Dye (TMD) (Histolab Products). An intravitreal injection of 100 μL
fixing solution (FS) consisting of 4% buffered formaldehyde (Solvenco AB) was made before
fixation in FS for 24-48 hours, and embedding in paraffin. 4 μm serial sections were made
through the TMD-labeled area and every 4 sections were stained with hematoxylin-eosin
(HE). Images were captured with an Iphone 4S mounted to a bright-field microscope (Zeiss
Axioskop 40, CarlZeiss).
For immunostaining, slides were deparaffinized in xylene, dehydrated in graded alcohols,
and rinsed with dH2O and Tris Buffered Saline (TBS, pH 7.6). Antigen retrieval was done in
10 mM citrate buffer (trisodium citrate dihydrate, Sigma-Aldrich, pH 6.0) with 1:2000 Tween20 (Sigma-Aldrich) at 96°C for 30 min, followed by 30 min cooling at room temperature.
Slides were washed with TBS and blocked for 30 min with 10% Normal Donkey Serum
(Abcam) diluted in TBS containing 5% (w/v) IgG and protease free bovine serum albumin
(Jackson Immunoresearch) in a humidified chamber. Primary antibodies diluted in blocking
buffer, were incubated overnight at 4°C: human nuclear mitotic apparatus protein (NuMA)
(1:200, Abcam ab84680), BEST1 (1:200, Millipore MAB5466), RPE65 (1:200, Abcam
ab78036, clone [401.8B11.3D9]) and Rhodopsin (1:2000, Millipore MAB5356). Secondary
antibodies (Alexa Fluor 555 donkey anti-rabbit IgG A31572 and Alexa Fluor 647 donkey antimouse IgG A31571, both from Life Technologies) diluted 1:200 in blocking buffer, were
incubated 1 hour at room temperature. Sections were mounted with vector vectashield with
DAPI mounting medium (Vector Laboratories) in a 24x50 mm coverslip. Images were taken
with Zeiss LSM710-NLO point scanning confocal microscope. Post-acquisition analysis of
the pictures was performed using ImageJ software.
In order to enhance the signal of the NuMA, TSA-Plus-Cyanine3 System was used (Perkin
Elmer Life Sciences). An extra blocking step of 30 min with 3% H2O2 in Methanol was added
prior to serum blocking followed by incubation with NuMA. Secondary antibody chicken antirabbit HRP (1:200, Santa Cruz sc-2963) diluted in the serum blocking buffer was incubated
for 30 min, followed by 8 min incubation with Tyramide-Cy3 according to manufacturer’s
instructions. Images were taken with an Olympus IX81 fluorescence microscope and postacquisition analysis of the pictures was performed using ImageJ software.
REFERENCES
Parinot, C., Rieu, Q., Chatagnon, J., Finnemann, S.C., and Nandrot, E.F. (2014). Large-scale
purification of porcine or bovine photoreceptor outer segments for phagocytosis assays on
retinal pigment epithelial cells. J Vis Exp 94, e52100, doi:10.3791/52100.
Kruse, P.F., and Patterson, M.K. (1973). Tissue culture: methods and applications; Section
IV: Replicate Culture Methods; Chapter 3, Leonard Hayflick (Academic Press, New York).