Transplantation of Human Fetal Retinal Pigment
Epithelium Rescues Photoreceptor Cells From
Degeneration in the Royal College of Surgeons Rat Retina
Caroline W. Little,* Bienvenido Castillo* David A. DiLoreto* Christopher Cox,\
Jeffrey Wyatt,% Coca del Cerro* and Manuel del Cerro*$\\
Purpose. The Royal College of Surgeons (RCS) rat suffers from a well-characterized, early-
onset, and relentless form of photoreceptor cell degeneration. It has been shown that allo-
grafts of retinal pigment epithelial cells from normal perinatal rats have rescue effects in this
condition. In preparation for human application, the authors determined whether human
fetal retinal pigment epithelium (RPE) grafts have a photoreceptor rescue effect in RCS
dystrophic rat retinas.
Methods. Sheets of RPE from human fetal eyes (10 to 16 weeks gestational age) were isolated
according to the authors' recently described method. Fragments of the RPE sheets were
transplanted to the subretinal space within the superior hemisphere. Transplants were per-
formed within the superior equatorial region of five dystrophic RCS rats, one eye per animal.
A similar volume of vehicle was injected into the subretinal space of five age-matched control
rats, again one eye per rat. All rats were immunosuppressed with daily injections of
cyclosporine. Using light microscopy, photoreceptor cell nuclear profiles of superior equato-
rial (SE) and inferior equatorial (IE) regions of transplanted and sham-injected control
animals were counted.
Results. Four weeks after transplantation, a dramatic rescue effect was observed. Microscopi-
cally, presumptive donor RPE cells were seen as single pigmented cells and as cell clusters in
the subretinal space. An outer nuclear layer three to four profiles thick was present in the
area of the RPE transplant but was nearly absent in the rest of the retina, as well as in the
retinas of control rats. The number of photoreceptor nuclear profiles per 100 //m was 34.7
± 2.2 (mean ± SEM) in the SE region of transplanted rats and 3.5 ± 1.4 in the same region
of sham-injected rats. There were 3.0 ±1.0 photoreceptor nuclear profiles in the IE region
of transplanted rats and 3.5 ± 1.2 in the IE region of sham-injected eyes. No evidence of
graft rejection was seen.
Conclusions. This study provides the first indication that transplanted human fetal RPE cells
are able to rescue photoreceptor cells in a model of hereditary retinal degeneration. Invest
Ophthalmol Vis Sci. 1996; 37:204-211.
X he Royal College of Surgeons (RCS) rat is an exten-
sively studied animal model of hereditary retinal de-
From the Departments of*Neurobiology, tBiostatistics, %Laboratory Animal
Medicine, ^Neurology, and ^Ophthalmology, University of Rochester School of
Medicine, Rochester, New York.
l*resented in part as a poster at the ARVO Annual Meeting, Sarasota, Florida, May
Supported by the Rochester Eye and Human Parts Bank and private gifts to the
University of Rochester Retinal Transplantation Team.
Submitted for publication April 14, 1995; revised August 23, 1995; accepted
September 25, 1995.
Proprietary interest category: N.
Reprint requests: Manuel del Cerro, Department of Neurobiology, University of
Rochester School of Medicine, P. O. Box 603, Rochester, NY 14642.
generation.1 6 The strain was initially maintained at
the Royal College of Surgeons (London, UK) and was
first described by Bourne, Campell, and Tansley in
1938.l The inbred, dystrophic strain is pink eyed and
is subject to an autosomal recessive disease that results
in progressive degeneration of the photoreceptors
after development is complete.7 By postnatal day 22,
there is a selective decrease in electroretinographic
(ERG) a-wave amplitude and increased rhodopsin
content.3 As the degeneration continues, the ERG b-
wave amplitude and rhodopsin content decrease. Loss
Investigative Ophthalmology & Visual Science, January 1996, Vol. 37, No. 1
Copyright © Association for Research in Vision and Ophthalmology
Transplanted Human Fetal RPE Rescues RCS Photoreceptors
F I G U R E 1. Isolation of human fetal retinal pigment epithe-
lium (RPE) under dark-field illumination (magnification,
X25). The sheet of RPE (arrows) dissects freely from the
choroid (CH) after 2% dispase treatment.
of photoreceptor nuclei begins by postnatal days 22
to 2 * 7 and is complete by postnatal day 60.3
The primary abnormality in this disease is in the
retinal pigment epithelial (RPE) cells, which are de-
fective in phagocytizing the shed rod outer seg-
ments.8'9 This defect results in the accumulation of
photoreceptor debris and the subsequent degenera-
tion of photoreceptors.10 The debris zone, adjacent to
the RPE, consists of extracellular lamellar material3
and shed rod outer segments.11
Grafting of normal rat RPE cells has been shown
to rescue photoreceptor cells in this animal model.12"14
Transplanted rat RPE and rescued photoreceptors ap-
pear to have normal structural and functional charac-
teristics.15'16 Given the growing interest in RPE trans-
plantation in humans, we decided to investigate
whether transplantation of human RPE cells has a res-
cue effect in this animal model of hereditary retinal
degeneration. Results described in this report provide
a positive answer to this question.
MATERIALS AND METHODS
Retinal Pigment Epithelium Isolation
Human fetal eyes (10 to 16 weeks gestational age)
were collected in Optisol-GS medium at 4°C (Chiron
Vision, Irvine, CA.).17 In Optisol, the anterior segment
of the eye was dissected away, lens, vitreous, and retina
were removed, and the RPE-choroid was isolated.
Within 24 hours of collection, sheets of RPE-choroid
were treated with 2% dispase in Dulbecco's minimal
essential medium (DMEM) for 25 minutes at 37°C.
Sheets of RPE were isolated by microdissection under
dark-field illumination as described (Fig. I).18
Hosts were 10 dystrophic RCS male rats, 24 days of age
at the time of transplantation. The animals originated
from our colony of RCS rats, which was derived from
breeding pairs (kindly provided by Dr. Matthew M.
La Vail) and were maintained following US Depart-
ment of Health and Human Services guidelines. They
were kept in climate-controlled quarters, room tem-
perature 22°C ± 2°C, under a 12-hour light-12-hour
dark cycle with an average light intensity of 11 footcan-
dles inside each cage, average room humidity of 40%
to 50%, and 19 room air changes per hour. Food and
sterile water were provided ad libitum. The RCS rat
colony is barrier maintained and seronegative for all
common murine pathogens as determined by enzyme-
linked immunoadsorbent assay and immunofluores-
Animals were anesthetized with intramuscular keta-
mine (Ketalar 40 mg/kg; Parke-Davis, Morris Plains,
NJ) and intramuscular diazepam (Valium 3 mg/kg;
Schein Pharmaceutical, Port Washington, NY) and
topical anesthetic (0.5% Proparacaine HC1; Allergan
America, Hormigueros, PR). Eyes were dilated with
1.0% mydriacyl (Akon, Humacao, PR) and 2.5%
phenylephrine HC1 (Bausch & Lomb, Tampa, FL).
Using a 29-gauge needle shaft connected by polyethyl-
ene tubing to a Kloehn (Brea, CA) microsyringe,19 3
fi\ of DMEM containing fragments of RPE sheets were
injected into the subretinal space within the superior
hemisphere of dystrophic RCS rat eyes (n = 5, one
eye per animal). A similar volume of DMEM was in-
jected into the superior equatorial subretinal space
of age-matched control dystrophic RCS rats (n = 5).
Injection sites were labeled by applying drops of a
dialyzed colloidal carbon suspension (Pelikan special
ink, batch C11/1431A; Pelikan AG, Hannover, Ger-
All rats were immunosuppressed with 10 mg/kg per
day of intramuscular cyclosporine (Sandimmune; San-
doz Pharma, East Hanover, NJ) to maintain a
cyclosporine blood level above 1500 ng/ml. Body
weight and general physical condition of each animal
were closely monitored every other day. Cyclosporine
blood levels were determined at the time of sacrifice
by means of a fluorescent polarization monoclonal
immunoassay using an Abbott TDX instrument (Ab-
bott Laboratories, Chicago, IL).
In vivo observation of the grafts was accomplished by
means of a Topcon (Paramus, NJ) fundus camera,
modified as recently described for optimal examina-
tion of the fundi of small animals.n General anesthe-
sia used during this procedure was the same one used
for the subretinal injections.
Investigative Ophthalmology 8c Visual Science, January 1996, Vol. 37, No. 1
FIGURE 2. Fundus photograph of the superior hemisphere of a transplanted eye. Trans-
planted cells form a localized band of pigmentation (arrows).
Four weeks after transplantation, rats were anesthe-
tized with intramuscular ketamine (Ketalar, 90 mg/
kg) and intramuscular xylazine (Rompun, 8 mg/kg;
Miles, Shawnee Mission, KS). When the animals
reached a deep plane of anesthesia, the eyes were
marked on the limbus with a small-tipped surgical cau-
tery, and the eyes were enucleated and placed in 6%
glutaraldehyde in 0.1 M sodium cacodylate at 40°C. A
midsagittal slit was made through the cautery mark to
allow penetration of the fixative into the eye. Eyes
were left at room temperature for 1 hour, kept for 48
hours at 4°C, and hemisected along a sagittal plane
through the injection site and the optic nerve. Both
hemispheres from each eye were postfixed in 1% os-
mium tetroxide (Electron Microscopy Sciences, Fort
Washington, PA) and 2% uranyl acetate, then dehy-
drated and embedded in Durcupan (Fluka Chemie,
Switzerland).22"" One-micrometer thick midsagittal
cross-sections of the eyes were cut using an ultramicro-
The numbers of photoreceptor cell nuclear profiles
at the superior equatorial (SE) and inferior equatorial
(IE) regions of transplanted and control animals were
counted using our previously published protocol.21
One-micrometer thick midsagittal sections were exam-
ined under an Olympus (Lake Success, NY) Vanox-S
microscope, with a calibrated reticle fit into a 10X
wide-field ocular. The equatorial region was desig-
nated as the midpoint between the ora serrata and
the optic nerve. The SE and IE points were identified
with a 4x objective. Once these points were deter-
mined, a 60X objective was substituted to count photo-
receptor nuclear profiles along a 100-^m segment of
retina that bisected the designated point. Mean num-
bers of surviving photoreceptor cell nuclei at the SE
and IE regions were calculated. Photoreceptor debris
was quantified using a method similar to the one de-
scribed to locate the regions of interest. Once die SE
or IE regions of the grafted or control animal were
identified, an ocular reticle was used to measure the
thickness of the debris zone within the subretinal
space. Macrophages in the peripheral vitreous were
counted for each eye on 1-fim midsagittal sections
within a region extending 50 fxm into the vitreous
from the inner limiting membrane along the entire
length of the retinal surface.
All research was carried out in strict accordance with
institutional, federal, and ARVO guidelines regulating
the use of human fetal tissue and the welfare of human
subjects and laboratory animals. Tissue was obtained
with written, informed consent of the patient
Transplanted Human Fetal RPE Rescues RCS Photoreceptors
• ^ 8
FIGURE 3. A scleral macrophage loaded with colloidal carbon
is dark brown under the microscope (arrow). Within the
retina, there are presumptive retinal pigment epithelial cells
with granules that take a greenish hue with Stevenel's blue
Only one eye per animal was used in this experiment
to minimize confounding variables. Measurements
were made in the equatorial regions of the superior
and inferior hemispheres of each eye. Data on num-
bers of photoreceptor cell nuclear profiles and thick-
ness of debris layer in each region were analyzed by
two-way, repeated measures analysis of variance (AN-
OVA) .2<1 The two factors included in the ANOVA, each
at two levels, were injection (graft versus sham) and
region of the eye (SE versus IE). Each ANOVA in-
cluded an analysis of residuals as a check on the re-
quired assumptions of normally distributed errors
with constant variance. The ANOVA provided overall
tests for a difference between treated and control eyes
and for a difference between SE and IE regions within
the same eye. The test of interest in our study is the
test for interaction, which examines whether the dif-
ference between two regions depends on whether the
eye was treated. A significant result of this test would
confirm an effect of transplantation. In addition to
this test, specific comparisons were made using paired
Wests between transplanted and sham-injected SE re-
gions and between the SE and IE regions of the trans-
planted eyes. Finally, results from the grafted SE re-
gion were compared with the average of the other
three regions (IE of grafted eye, SE and IE of sham-
injected eye). The results of /-tests are more conserva-
tive than the corresponding tests paired on the AN-
All research was carried out in strict accordance
with institutional, federal, ARVO, and Declaration of
Helsinki guidelines regulating the use of human fetal
tissue and the welfare of human subjects and labora-
tory animals. Tissue was obtained with the written,
informed consent of the pregnant women.
Clinically, there were no signs of pathologic responses
to the grafts. Funduscopic examination showed the
grafts as distinct, heavily pigmented patches primarily
located in the SE region of the retina (Fig. 2). The
carbon injection site was visible to die naked eye. His-
tologically, it was observed that the label was taken up
by scleral macrophages (Fig. 3).
Histologic examination of the sham-injected eyes
showed no obvious rescue effect of photoreceptor
cells. Within the SE and IE of these animals, only a
discontinuous layer of degenerating photoreceptors
remained 4 weeks after injection (Fig. 4). There was
a prominent debris layer between the remaining pho-
toreceptors and the RPE (see Results below). The in-
travitreal macrophage population remained very
low—4.0 ± 2.7 (mean ± SD).
Histologic examination of the grafted eyes dem-
onstrated a clear rescue effect of photoreceptor cells
in the superior equatorial region of the grafted retina
4 weeks after transplantation, when the animals were
52 days of age. The outer nuclear layer in this region
consisted of three to four layers of photoreceptor cells
(Figs. 5, 6), with a gradual decrease in the number of
photoreceptor cells in regions extending beyond the
FIGURE 4. Superior equatorial region of a sham-injected eye.
Notice the single layer of degenerating photoreceptors. RPE
= retinal pigment epithelium; DB = debris zone; INL =
inner nuclear layer.
Investigative Ophthalmology 8e Visual Science, January 1996, Vol. 37, No. 1
F I G U R E 5. Superior equatorial region of a transplanted eye.
This micrograph of the transplanted area shows a rescue
effect of the outer nuclear layer (ONL) and the soma of two
grafted cells (arrows). RPE = retinal pigment epithelium; DB
= debris zone; INL = inner nuclear layer.
graft site. Presumptive pigmented RPE cells* were
seen as single cells and as small cell clusters within the
subretinai space (Fig. 5). Occasionally, a few of these
cells were seen in the inner layers of the host retina.
Pigment granules also were seen within the cytoplasm
of some RPE cells attached to the host Bruch's mem-
brane in the transplant area, whereas the host RPE
cells beyond the transplant area retained their nonpig-
mented characteristics (Fig. 7). Regions distant to
transplantation maintained a single discontinuous
layer of photoreceptors, similar to that seen in the
Mean numbers of photoreceptor cell nuclear pro-
files of the four regions are shown in Figure 8. These
measurements revealed that the number of photore-
ceptor nuclear profiles was greatest in the SE region
of RPE transplantation. Results of the ANOVA for the
number of nuclear profiles indicated a highly signifi-
cant interaction (P < 0.0001). In addition, the differ-
ence between the SE region of transplanted and SE
sham eyes was highly significant (P < 0.0002). Also,
there was a highly significant difference between SE
and IE regions in transplanted eyes (P< 0.0001). The
difference between the SE region for transplanted
eyes and the average of the other three regions was
highly significant (P < 0.0001). Even more interest-
ing, essentially all the variation in treatment, region,
and their interaction was accounted for by this last
comparison—that is, this effect was the only one pres-
ent in the data, and it was limited to the general area
of the transplant. This can be seen clearly in the means
for the four regions (Fig. 8). The value of the coeffi-
* Pigmented cells in the subretinai space were not present in
control animals. They, however, will be termed "presumptive" reti-
nal pigment epithelial cells at the request of a reviewer.
cient of determination was very high (/? - 97.8% of
the total variation explained by the effects of treat-
ment, region, and their interaction). Thus, there was
relatively little variation among the 10 eyes.
The accumulation of outer segment debris in the
subretinai space decreased in the area of outer nuclear
layer rescue, and, in fact, it seemed to decrease
throughout the retina of transplanted eyes (Figs. 4 to
6). Debris thickness (mean ± SEM) in the grafted SE
region was 15.8 ± 2.7; in the IE region of the grafted
eye, it was 22.4 ± 1.0; in the sham-injected SE regions,
it was 25.8 ± 1.4; and in the IE region of the sham-
injected eye, it was 27.3 ± 1.5. For analysis of the
debris layer, the difference between the SE regions in
transplanted and sham eyes was significant (P =
0.019), whereas the difference between SE and IE re-
gions of the transplanted eyes was not significant (P
= 0.12). A one-sided test was nearly significant (P =
0.06). Also, the difference between the SE region for
the transplanted eyes and the average of the other
three regions was significant (P = 0.033); most of the
variation from treatment, region, and their interaction
was accounted for by this difference.
Graft rejection was not observed. Throughout the
survival time, there were no clinical signs of rejection
(redness, swelling, or exudates). In addition, the vi-
treal macrophage population, which in our experi-
ence is a sensitive indicator of posterior pole inflam-
mation,25 remained low. Grafted eyes had an average
of 3.0 ± 2.6 macrophages per retinal surface, and
sham-injected eyes had 4.0 ± 2.7 macrophages per
retinal surface. There was no significant difference
between the two populations of cells (P = 0.79, two
FIGURE 6. Superior equatorial region of another trans-
planted animal. Three to four rows of photoreceptor cells
are present, and a decreased layer of debris (DB) is present.
Aside from minor abnormalities, the other layers of the ret-
ina, including the retinal pigment epithelium (RPE), appear
normal. INL = inner nuclear layer; ONL = outer nuclear
Transplanted Human Fetal RPE Rescues RCS Photoreceptors
FIGURE 1. Inferior equatorial region of a transplanted eye.
The rescue effect does not extend to the opposite hemi-
sphere. Note the absence of an outer nuclear layer. The
inner nuclear layer (INL) directly contacts the retinal pig-
ment epithelium (RPE).
Blood levels for cyclosporine, as determined by
the TDX monoclonal antibody technique (Abbott
Laboratories), were high, above 1500 ng/ml. Host ani-
mals remained in good health as demonstrated by
the consistent increase in body weight during survival
time. Also, in spite of the fact that high levels of
cyclosporine in blood were achieved, detailed histo-
logic analysis failed to reveal signs of toxicity toward
the host retina.
The dystrophic RCS rat is affected by a well-character-
ized, hereditary retinal degeneration.1 Previous stud-
ies by other groups 12'l3l2b have shown that it is possible
to rescue photoreceptor cells in the dystrophic RCS
rat by means of subre final grafts of normal rat RPE
cells. These important observations have been con-
firmed and extended.Ml27 A profound and lasting res-
cue effect appears to be specific to RPE cell transplants
because sham injections in the subretinal space of the
RCS rats have been shown to have a modest and tran-
sient effect. 16>ah'1!8 Grafts of macrophages have failed
to produce long-term rescue.29 Our data indicate that
transplanted human fetal RPE can rescue RCS degen-
An important issue involves the mechanism (s)
that regulate the specific rescue effect of RPE cells.
Two possibilities are immediately obvious. One is that
the normal grafted cells phagocytize the photorecep-
tor membranes, a function that is abnormal in the
RCS RPE. Another possibility is that grafted RPE cells
provide trophic support to photoreceptor cells,
thereby surmounting the detrimental effects of the
unfavorable milieu created by the accumulation of
membranous debris in the subretinal space of the RCS
rat. These possibilities are not mutually exclusive, and
both should be considered in attempting to explain
the rescue phenomenon. Further, it needs to be taken
into account that both perinatal12'30 and fetal31 RPE
cells are able to rescue photoreceptor cells in RCS
Although phagocytic and trophic effects may take
place simultaneously, it is our hypothesis that, at least
initially, trophic effects play a prominent role. This is
supported by our observation that whereas the main
rescue effect was localized to the region of the graft,
there was a less pronounced, but still evident, effect
extending beyond where presumptive grafted RPE
cells could be visualized. Remarkably, the extent of
debris was decreased throughout transplanted eyes;
such an effect did not occur in sham-injected eyes.
The rescue effect was sufficient to preserve a sig-
nificant portion of the original photoreceptor cell
population, but not all of it, because the RCS retina
has approximately eight cell rows in the outer nuclear
layer at 4 weeks of age32; at the time of sacrifice (nearly
2 months of age), the animals retained three to four
rows of photoreceptor cells. Nonetheless, the data un-
equivocally show that human fetal RPE grafts rescue
photoreceptor cells at 52 postnatal days, a time when
only a discontinuous layer of degenerating photore-
ceptor nuclear profiles remain in the control retinas
and in the opposite region of grafted eyes. Presump-
tive donor RPE cells were seen in the subretinal space
of host animals. We assume most, if not all, the RPE
cells attached to Bruch's membrane were of host ori-
gin and that some of them incorporated human mela-
nin released at the injection site.
There was no graft rejection as detected by
+ SEM, lOOum
FIGURE 8. This graph shows die mean number of photore-
ceptors in the superior and inferior equatorial regions of
transplanted and sham-injected eyes. The number of photo-
receptors in the superior equatorial region of transplanted
eyes was statistically greater dian in all other regions counted
(P < 0.0001). SEM = standard error of the mean; Trans
= transplantation; SE = superior equatorial; IE = inferior
Investigative Ophthalmology 8c Visual Science, January 1996, Vol. 37, No. 1
the continued presence of transplanted cells and by
the absence of vitreal macrophages. Although
cyclosporine treatment is not without potential side
effects,33"36 this finding, considered together with the
substantial body of literature that shows good survival
of intraocular allografts and xenografts,12'262937 pro-
vides a hopeful perspective for attempts at protecting
human intraocular grafts. Naturally, the reactivity of
every species is likely to be different, with some spe-
cies, such as the mouse, having a clear but limited
immunologic tolerance to subretinal allografts.38"40
This is in contrast with the situation in the rat. del
Cerro et al,41 as well as Li and Turner,12 have suc-
ceeded in transplanting RPE cells and neural retina
across rat strains, even in the absence of immunosup-
Convincing evidence has been provided by other
researchers that grafts of normal rat RPE could halt
the progress of the RCS-associated photoreceptor de-
generation. 12~14'26'42'43 We decided to test whether hu-
man fetal RPE is endowed as well with such protective
action, and the current study demonstrates that it is.
These data constitute the first experimental evidence,
to our knowledge, that grafted human fetal RPE cells
are able to rescue degenerating photoreceptor cells
associated with hereditary retinal degeneration. This
study provides an experimental basis for current clini-
cal trials involving transplantation of human fetal RPE.
human fetal tissue, photoreceptor rescue, retinal degenera-
tion, retinal pigment epithelium, transplantation
The authors thank Dr. Matthew M. LaVail for kindly provid-
ing the breeding pairs of Royal College of Surgeons dystro-
phic and congenic animals used to start our colony, Ms.
Nancy Lawson for providing us with useful information on
the husbandry of these animals, Karen Jensen for her techni-
cal assistance, Dr. Barbara McKenna and associates for per-
forming the cyclosporine blood tests, and Dr. Gustavo Agu-
irre for his valuable feedback. They also thank the personnel
at Chiron Vision (Irvine, CA) for providing Optisol.
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