Long-term safety of human retinal progenitor cell transplantation in retinitis pigmentosa patients
Liu et al. Stem Cell Research & Therapy
Long-term safety of human retinal progenitor cell transplantation in retinitis pigmentosa patients
Yong Liu 0
Shao Jun Chen 0
Shi Ying Li 0
Ling Hui Qu 0
Xiao Hong Meng 0
Yi Wang 0
Hai Wei Xu 0
Zhi Qing Liang 1
Zheng Qin Yin 0
0 Key Laboratory of Visual Damage, Regeneration and Repair, Southwest Eye Hospital, Third Military Medical University , Chongqing 400038 , China
1 Department of Gynecology and Obstetrics, Southwest Hospital, Third Military Medical University , Chongqing 400038 , China
Background: Retinitis pigmentosa is a common genetic disease that causes retinal degeneration and blindness for which there is currently no curable treatment available. Vision preservation was observed in retinitis pigmentosa animal models after retinal stem cell transplantation. However, long-term safety studies and visual assessment have not been thoroughly tested in retinitis pigmentosa patients. Methods: In our pre-clinical study, purified human fetal-derived retinal progenitor cells (RPCs) were transplanted into the diseased retina of Royal College of Surgeons (RCS) rats, a model of retinal degeneration. Based on these results, we conducted a phase I clinical trial to establish the safety and tolerability of transplantation of RPCs in eight patients with advanced retinitis pigmentosa. Patients were studied for 24 months. Results: After RPC transplantation in RCS rats, we observed moderate recovery of vision and maintenance of the outer nuclear layer thickness. Most importantly, we did not find tumor formation or immune rejection. In the retinis pigmentosa patients given RPC injections, we also did not observe immunological rejection or tumorigenesis when immunosuppressive agents were not administered. We observed a significant improvement in visual acuity (P < 0.05) in five patients and an increase in retinal sensitivity of pupillary responses in three of the eight patients between 2 and 6 months after the transplant, but this improvement did not appear by 12 months. Conclusion: Our study for the first time confirmed the long-term safety and feasibility of vision repair by stem cell therapy in patients blinded by retinitis pigmentosa. Trial registration: WHO Trial Registration, ChiCTR-TNRC-08000193. Retrospectively registered on 5 December 2008.
Progenitor cell; Visual improvement; Retinitis pigmentosa; Cell transplantation; Retina
Retinitis pigmentosa is an inherited retinal dystrophy
that is characterized by the onset of night blindness,
the early loss of peripheral vision, and later the loss
of central vision [
]. Retinitis pigmentosa is related to
different genetic etiologies, all of which induce the
death of photoreceptors. Therefore, the identification
of causative genes must be a prerequisite if gene
therapies are to be applied to treat retinitis pigmentosa
]. Compared to gene therapies, cell
transplantation might have good potential to rescue
or replace dysfunctional photoreceptors. Schwartz et
al. reported on mid-term and long-term outcomes
when using human embryonic stem cell
(hESC)-derived retinal pigment epithelial (RPE) cells to treat
dry atrophic age-related macular degeneration and
Stargardt's macular dystrophy [
]. Their studies
provided the first evidence that hESC-derived cells
can be used as a new form of therapy to treat retinal
Studies using retinitis pigmentosa animal models
indicated that the transplanted cells were able to
differentiate into photoreceptors, integrate into the
host retinas, and rescue vision [
]. These findings
suggested that it is possible to transplant human
RPCs to treat retinitis pigmentosa patients. Currently,
retinal progenitor cells (RPCs) are usually derived
from fetal retinas, embryonic stem cells (ESCs), and
induced pluripotent stem cells (iPSCs). With regard
to the source of RPCs, fetal-derived RPCs may have
an advantage for cell therapy. Firstly, RPCs derived
from fetal neural retinas have low immunogenicity
and can stably maintain their characteristics over
many passages [
]. RPCs derived from ESCs or
iPSCs require a longer time and more steps to induce
in vitro. Secondly, transplanting ESCs or iPSCs carries
the potential risk of tumor formation and gene
The most relevant previous clinical studies to treat
retinal degeneration used fetal retinal sheets and
immature neural retinal cells, and there were no obvious
signs of immune rejection; however, parallel animal
studies suggested there was only limited progenitor
cell integration into the neural retina [
Subsequent reports suggested that only transplanted
cells isolated from fetuses at a specific gestational
stage and already committed to a photoreceptor cell
fate could facilitate visual recovery [
]. This means
that the transplanted cells should be photoreceptor
precursors before being delivered to patients.
Candidate cells for this treatment exclusively express
the cone-rod homeobox-containing gene (CRX), but
no expression of Opsin (the main chromophore of
the mature photoreceptor) [
]. Human fetal neural
retinal cells from second-trimester fetuses can be
expanded into a large number of undifferentiated cells
in vitro and mature retinal cells [
]. These cells can
be ideal sources for transplantation when considering
a dose-response relationship in pre-clinical and
We previously reported on the technical feasibility
of human retinal transplantation using a mini-pig
model and a pars plana vitrectomy approach [
this study, we describe the preparation and isolation
of highly enriched human RPCs derived from early
developmental fetuses and investigated the potential
of these cells to integrate into the host retinas of
RCS rats and to restore a visual response. We then
initiated a clinical trial in patients with retinitis
pigmentosa to investigate the safety, immunological
response, and efficacy of human fetal-derived RPC
subretinal transplantation using pars plana vitrectomy
Study design and ethics
The animal study was approved by the Laboratory
Animal Welfare and Ethics Committee of the Third
Military Medical University, and the clinical trial was
approved by the Medical Ethics Committee of Southwest
Hospital, the Third Military Medical University. We
conducted a phase I clinical trial assessing the safety of RPC
transplantation into RP patients over a 24-month
followup period. This trial was conducted in the Southwest
Hospital, China. Recruitment of patients started in May
2008 and the study was completed in September 2013.
The research adhered to the principles of the
Declaration of Helsinki, and written informed consent
and surgical consent were obtained from all patients
(WHO Trial Registration, ChiCTR-TNRC-08000193).
The culture of RPCs was performed under Good
Manufacturing Practice (GMP) conditions in the Cell
Biology Therapy Center, Southwest Hospital, Third
Military Medical University. This center has been
awarded GMP certification and qualified for the
production of RPCs.
Ocular tissues of 12- to 16-week-old aborted fetuses
were collected from the embryonic tissue bank of the
Department of Obstetrics, Southwest Hospital, according
to the good tissue practice guidelines. Donors provided
informed consent and were not compensated for the use of
their terminated fetal tissue for research. Fetal neural
retinas were cut into pieces, rinsed, digested at 37 °C for
20–30 min with Tryple (CTS, Gibco) and diluted by the
addition of 3 ml medium (Ultraculture; Lonza)). The
tissue was dissociated by gentle agitation for 10 s and the
suspension was settled for 2 min. The supernatant
containing the precursors was carefully decanted into a new
tube with fresh medium, and the remaining pellet was
discarded. The collected supernatant was centrifuged for
5 min at 2400 g and re-suspended in Ultraculture
supplemented with 10 ng/ml human epithelial growth factor
(EGF; Peprotech) and 20 ng/ml human basic fibroblast
growth factor (bFGF; Peprotech) [
]. Cells were plated
onto matrigel-coated tissue culture surfaces (Cellstart CTS,
Invitrogen) and placed in an incubator for 120 min. The
majority of non-neuronal cells adhered to the bottom of
the plate, whereas neuronal progenitor cells remained in
suspension such that the non-adherent suspension can be
collected for primary culture and expansion [
purified RPCs were supplemented with Ultraculture, B27,
N-2, 20 ng/ml human EGF, and 20 ng/ml human bFGF,
placed on fibronectin-coated (10 μg/ml) plates and placed
in an incubator (37 °C, 5% CO2). RPCs at passage three
were used for the following study. The viability of RPCs
was determined using Trypan blue staining (0.4%; GIBCO).
The RPCs were plated onto glass cover slips. Primary
antibodies were used to characterize the cells (PAX6,
1:100, Santa Cruz; CRX, 1:50, Santa Cruz; Nestin, 1:500,
BD Bioscience; Sox2, 1:1000, Chemicon; GFAP, 1:1000,
Chemicon). Cy3 (1:1000, Santa Cruz) was used as the
secondary antibody and the slides subsequently imaged
using a confocal microscope (Leica TCS NT, Leica
In brief, RPCs were prepared in a cell suspension,
incubated with the primary antibodies (1:30 for Nestin,
PAX6, SOX2, and GFAP) or isotype control (1:30;
BioLegend), washed, and then incubated with
fluorophore-conjugated secondary antibodies (1:30).
Cells were analyzed on a fluorescence-activated cell
sorting Calibur system (FACS, BD Biosciences, San
Jose, CA, USA). The ratio of positive cells within the
gated population was estimated based on comparison
with species-specific isotype control. Ten thousand
events/sample were collected, stored for analysis, and
the experiments were repeated three times.
Real-time quantitative polymerase chain reaction (RT-qPCR) analysis
Total RNA was extracted from the RPCs by the
RNeasy Mini Kit (Qiagen) and cDNA generated using
the iScript cDNA Synthesis Kit (Bio-Rad) according
to the manufacturer’s instructions. RT-qPCR) was
carried out using a Power SYBR Green PCR Master
Mix on the 7500 Real-time PCR System (Applied
Biosystems). hESC line H1, which was a gift from
Shanghai Institute of Biochemistry and Cell Biology,
was used for comparison with RPCs. RT-qPCR
assayed for the hESC markers Nanog and OCT4, and
for the RPCs markers PAX-6, Six6, Crx, and
recoverin. Relative gene expression was assayed in
triplicate replicates normalized to the GAPDH signal
present in each sample. The expression levels of cell
markers detected in RPCs were normalized to that of
an hESC sample which served as the zero set point.
Differentiation of RPCs into photoreceptors
Retinoic acid (10 μM; Sigma) was added into serum-free
conditioned medium and the cells were cultured for
2 weeks in order to induce the RPCs to differentiate into
mature photoreceptors [
]. Cells were then identified by
the specific markers recoverin (1:1000, Chemicon) and
rhodopsin (1:250, Chemicon). The cellular proliferating
properties were examined by anti-Ki67and Ki67 (1:200,
Abcam). Cy3-conjugated IgG was used as a secondary
Cell transplantation into RCS rats
RPCs were pre-labeled with the fluorescent marker CM-DiI
(2 mg/ml; Invitrogen) prior to transplantation. For the
efficacy study, RCS rats at 30 days old received an injection
with RPCs (n = 12); 0.01 M phosphate-buffered saline (PBS)
injections were used as a vehicle control (n = 6). The right
eyes served as the treatment eyes, whereas the left eyes
Rats were anesthetized by an intraperitoneal injection
of a solution of ketamine (120 mg/kg) and xylazine
(20 mg/kg). A scleral hole was created using a 30G
needle allowing access to the space between the neural
retinal layer and the retinal pigment epithelium layer. A
glass micropipette carrying 5 μl of a RPC suspension
(1 × 105 cells) was inserted tangentially into the space
beneath the degenerating photoreceptor layer at the
superior retinal hemisphere. Fundus examination was
performed immediately after surgery, and a successful
injection was confirmed by a small subretinal fluid bleb.
Cyclosporine A (200 mg/L) was given orally from day 1
Functional test after cell transplantation
Electroretinography (ERG) analysis was used to evaluate
the improvement in retinal function after cell injections.
Three and six weeks following transplantation, animals
were dark adapted for at least 12 h before the ERG test.
Anesthesia was performed as above. Pupils were dilated
using 1% tropicamide. The active gold lens electrode
was placed on each cornea, and the reference and
ground electrodes was respectively placed
subcutaneously in the mid-frontal area of the head and the base of
the tail. Light stimulation was delivered at –5 dB for the
dark-adapted test, and all recordings were processed by
software supplied by the manufacturer (Diagnosys LLC,
MA). The amplitudes of a-waves were measured from
the baseline to the cornea-negative peaks, and the
amplitudes of b-waves were measured from the
corneanegative peak to the major cornea-positive peak.
Rats were sacrificed at 6 weeks post-transplantation. The
eyes were fixed in paraformaldehyde in PBS, infiltrated with
sucrose, and then sectioned using a cryostat. The injected
cells were preliminary identified by the fluorescent marker
CM-DiI with fluorescence microscopy. Sections were
washed in PBS three times to remove CM-DiI. Mouse
antihuman mitochondria (1:200, Abcam) and rabbit
antihuman recoverin (1:1000) or rhodopsin (1:250, Chemicon)
were used as primary antibodies to detect the transplanted
RPCs, and then sections were incubated in the secondary
antibodies, Cy3-conjugated AffiniPure goat anti-mouse IgG
(1:300) and FITC-conjugated AffiniPure goat anti-rabbit
We chose three rats to quantify the percentage
recoverin/rhodopsin-positive cells among RPCs. From each
rat, three random sections that containing the typical
transplant areas were selected. The ratio of
doublestained cells among the human mitochondrial-positive
cells was considered as the photoreceptor cells
differentiated from the grafted RPCs.
To compare the degree of outer nuclear layer
(ONL) preservation between RPCs and vehicle groups,
the thickness of the ONL was measured on the areas
extending 100 μm either side of the injection site.
RPCs were also assessed for tumor formation in the
retina. RPCs were injected (as above) into the space
beneath the degenerating photoreceptor layer of P30
RCS rats (n = 36) and then examined 6 weeks
posttransplantation. Hematoxylin-eosin staining was used
to examine tumor formation in the injection area.
We enrolled eight patients diagnosed with rod-cone
dystrophy on the basis of eye examinations, visual
field testing, standard full-field fundus fluorescein
angiography (FA), and flash (f )ERG according to the
standards set by the International Society for Clinical
Electrophysiology of Vision (ISCEV) [
met the following inclusion criteria: (1) between 18
and 50 years of age; (2) best corrected visual acuity
(BCVA) ≤ 20/400 in the operated eye, or a visual field
of less than 20°, as assessed by Octopus 101
perimeter; and (3) the vision in the non-operated eye had
to be better than the operated eye. Exclusion criteria
included evidence of other eye disease such as a
cataract that could compromise the interpretation of
visual results; the inability to return for follow-up
according to pre-planned schedule during the study;
and history of intraocular surgery.
Surgical procedure for cell transplantation into retinitis pigmentosa patients
A standard three-port vitrectomy was performed and
the vitreous body was removed from the inner limiting
membrane of the retina. Using a 39G retinal
hydrodissection cannula (Storz, USA), a minimally invasive
retinotomy was performed temporally or
superatemporally to the macula and near the arcade vessels. A RPC
suspension (100 μl containing ~ 1 × 106 cells) was slowly
injected into the presumptive space thus creating a small
retinal bleb (Additional file 1: Video S1). Cells were
assessed prior to transplantation for microbial
contaminants and endotoxin. Post-surgical treatment followed
standard procedures for patients receiving three-port
Seven patients were followed for 24 months and one
patient for 12 months. BCVA was measured three
times at each visit using the Early Treatment Diabetic
Retinopathy Study (ETDRS) chart. Data were then
converted into logMAR (log of the minimum angle of
resolution) scores according to the formula 1.1 + log10
(designed distance/testing distance) – 0.02 × number
of letters [
]. A high logMAR score indicates poor
vision. Patients with only hand-motion vision were
assigned a score that was one line lower than the
largest printed line on the 4-m chart (<20/1600).
On each follow-up visit (1, 2, 3, 6, 9, 12, and 24 months
post-transplantation), photographs and autofluorescence/
fluorescein angiography of the fundus were performed
using a Heidelberg HRA II system (Heidelberg Engineering
GmbH, Germany). High-resolution optical coherence
tomography (OCT; OCT-1000 System, Topcon) and
Spectral Domain OCT (SD-OCT, Spectralis 3 Mode OCT,
Heidelberg Engineering) were used to evaluate retinal
structure. Bilateral full-field ERGs were recorded using a
Roland electrophysiology system (RETIscan, Roland
Consult, Germany) with ERG-jet contact lens electrodes.
For the ERG analyses, the pupils were dilated with 1%
tropicamide and the patient dark-adapted for 30 min. ISCEV
standard dark-adapted and light-adapted ERGs were
Pupillary light reflexes in the dark-adapted (40 min)
state were evaluated using a custom-built computerized
pupillometer and a modified commercial spherical
Ganzfeld according to the method of Aleman et al. [
Five continuous 200-ms blue stimuli with intensities
ranging from –2.8 to 0.85 log scot-cd/m2 and six white
stimuli ranging from –1.5 to 2 log scot-cd/m2 were
applied to elicit a transient light reflex; each stimulus (from
low to high) was followed by a 15-s dark recovery period
]. It was difficult to analyze the amplitude changes
for individual pupillary reflexes due to large amplitude
variations elicited by the same stimulus. A response
criterion of 0.3 mm was used to define a response
threshold. The threshold values were converted to ranked data.
A one-level improvement corresponded to a one-level
decrease in the threshold.
Data are given as the mean ± SD. Comparisons were
made using a two-tailed paired t test for visual acuity.
The treatment effect was compared to the baseline
condition and that in the follow-up time points.
Differences in BCVA (logMAR) were obtained for
each patient at 0, 1, 2, 3, 6, 9, 12, and 24 months
post-transplantation. The differences following
treatment at each time point were normalized to the
baseline measurement obtained at month 0 in order to
perform comparisons across patients. A chi-square
test was used to assess differences in the pupillary
light reflexes between the operated and non-operated
eye. All statistical tests were considered significant if
P ≤ 0.05.
Human fetal-derived RPC transplants in RCS rats
RPCs after three passages were characterized by
checking the expression of Nestin (98%), Pax6 (96.6%), and
Sox2 (78%) using immunocytochemistry or flow
cytometry (Fig. 1a and b). There were limited glial cells in the
population of PRCs (0.1% GFAP+ cells). Gene expression
analysis confirmed that the typical early eyecup
transcription factor genes Pax6 and Six6 (Fig. 1c) increased
5–10 fold compared to that seen in hESC cultures; the
expression of photoreceptor precursor markers, such as
recoverin and CRX, was much higher in the RPCs. In
contrast, the expression of the pluripotency markers
NANOG and OCT4 clearly decreased by 5–10 times
compared to hESCs. RPCs were also able to differentiate
and express photoreceptor phenotypes (recoverin and
rhodopsin) in vitro following treatment with retinoic
acid, and lost proliferative properties without Ki67
staining (Fig. 1e). Since RPCs would be transplanted into
degenerated retina in retinitis pigmentosa patients, these
cells were extensively tested for animal and human
pathogens. The final RPC product had normal female
(46 XX) karyotype, confirming that the cells were free of
microbial contaminants (Fig. 1d).
Six week following human RPCs transplantation,
DiI-labeled cells could be readily identified within
RCS retinal sections and, in Fig. 2a, it is clear that
transplanted cells had spread across a broad area of
the region previously occupied by the photoreceptors.
Moreover, immunofluorescence staining confirmed
that these cells exclusively expressed human
mitochondria, a specific marker of the human species.
Most RPCs integrated in the host ONL. Some cells
co-expressed recoverin or rhodopsin (Fig. 2b and c).
We observed approximately 20.7 ± 3.1%
recoverinlabeled and 9.7 ± 1.5% rhodopsin-labeled RPCs in the
recipient ONL in each randomized section, indicating
the expression of a photoreceptor phenotype and
possible photoreceptor differentiation.
Statistical analysis of ONL thickness indicated that
transplants were associated with a significantly thicker ONL
compared with that in the PBS group (37.2 ± 2.8 μm vs
18.4 ± 2.0 μm, P < 0.05; Fig. 2d). Analysis of the recorded
ERGs at 3 and 6 weeks post-transplant indicated that the
bwave amplitudes were higher compared to those of the PBS
group (P < 0.05; Fig. 2e and f ). These findings showed that
retinal function was profoundly improved after RPC
transplantation, which corresponds to the morphological
results by ONL thickness measurements.
We then tested the risk of tumor formation by
injecting RPCs into the degenerating retinas. Cellular
survival was observed in 32 eyes from 36 RCS rats;
tumors were not observed in any retinal sections,
suggesting the safety of human RPC transplantation
in retinitis pigmentosa patients.
Clinical study of fetal-derived RPC transplantation in retinitis pigmentosa patients
Preoperatively, the visual acuity of the prospective
transplanted eye was 1.37 ± 0.34 logMAR in the eight
patients and is equivalent to ~ 20/500 on a Snellen’s
vision chart. Vitrectomy and subretinal
transplantation proceeded without complications, such as
iatrogenic retinal tears, cataracts, or endophthalmitis
(Table 1 and Additional file 9: Video S1). All
postoperative retinal examinations on day 1 were
unremarkable, apart from the formation of the retinal bleb
containing the transplanted cells. Clinical examination
on day 7 revealed that retinal detachments had not
occurred and the OCT analysis clearly showed a thick
region of transplanted cells beneath the neural retinal
layer (a typical example is shown in Fig. 3b).
Although reduced in thickness, the layer of RPCs was
still clearly present in OCT scanning 1 month after
surgery (Fig. 3c). The reduced thickness at the
BCVA best corrected visual acuity, F female, M, male, MAR minimum angle of resolution, OD right eye without treatment, OS left eye with transplant
transplant site suggested that the grafted cells may
have migrated within the subretinal space or
integrated into other layers of the host retina, in a similar
fashion to that seen within the RCS rat retinas. One
and two years post-transplantation, it was no longer
possible to unequivocally define the region of injected
cells using the OCT methodology (Fig. 4 and
Additional files 2–7: Figures S2–S7). In some cases
the region of the injection site could be defined by the
presence of small retinal scarring, characterized by locally
thickened retina with OCT scanning. Autofluorescence provided
useful information on conditions where the health of the
RPE played a key role; areas of hypo-autofluorescence
indicated missing or dead RPE cells [
imaging showed a limited hypo-autofluorescence area beneath
the corresponding retinal injection site, a sign of local RPE
disruption (Fig. 4c’). The restricted area of these regions
indicated that our surgery was safe and minimally invasive.
Fluorescein angiography indicated that our procedure
did not lead to changes in vascular leakage in the region
of the macular in eight patients. Distribution of
autofluorescence also showed no absent autofluorescence in the
macular area compared to the baseline (Fig. 4c’ and c”),
indicating that our injected cells did not lead to further
retinal degeneration or oxidative injury to the remaining
functional RPE cells. OCT scanning throughout the
24month follow-up period did not detect the presence of
inflammation or cystoid macular edema (Fig. 4 and
Additional files 2–7: Figures S2–S7), and none of the
patients developed any sign of immunological rejection
in the fundus. However, one patient formed a macular
membrane (Additional file 1: Figure S1). Given these
results, medication for systemic or intraocular
immunosuppression was not administered.
Visual acuity in the RPC-treated eyes showed a
significant improvement for grouped data compared
to the baseline (P < 0.05) between 2 and 6 months
after surgery (Fig. 5, Table 2), but acuity then
declined so that overall no differences were seen by
24 months. When individual patients were examined,
BCVA improved in five eyes, but remained stable in three
eyes during the 12 month follow-up; at 24 months,
improved vision was only seen in one eye.
The recorded signal during global ERG
measurements was too small and not clearly distinguishable
either before or after RPC transplantation, indicating
the poor visual function of the patients prior to the
MAR minimum angle of resolution, OD right eye without treatment, OS left
eye with transplant
study. Similarly, visual field tests were also unreliable
for this reason. Therefore, we developed a computerized
pupillometry to assess photoreceptor dysfunction. The
thresholds for blue and white light-induced pupillary light
reflexes were recorded. The threshold to blue light
improved in four subjects (patients 1, 3, 4, and 7) between 3
and 6 months post-transplantation (Additional file 8:
Figure S8A, C, D, and G), remained stable in three others
(patients 2, 6, and 8; Additional file 8: Figure S8B, F, and
H), but declined in one patient (patient 5; Additional file 8:
Figure S8E). The threshold to white light improved in
patients 1, 3, and 4 (Additional file 8: Figure S8A,’ C,’ and
D’) and remained stable in the other five patients. These
findings indicated that, compared to the preoperative
baseline, the retina became more photosensitive during the
6-month post-transplantation period in patients 1, 3, and 4,
but this was not sustained and threshold levels declined to
the baseline at 12 months. In the other five patients, three
maintained preoperative baseline levels, while two patients
showed a decline in the light reflex.
Clinical trials using hESC-derived RPE cells have been
previously attempted [
], and human RPCs derived
from the fetus after pregnancy termination are currently
being conducted in NIH-approved clinic trials [
In contrast to previous clinical trials using fetal retinal
], our study shows marginal
beneficial effects on visual acuity and pupillary responses
during the 2- to 6-month follow-up periods, although
this efficacy is not maintained in the long term.
Our study was designed to test the safety and
tolerability of human RPCs in patients with advanced retinitis
pigmentosa; we did not set up cell-dose cohorts due to
the small sample sizes. To optimize the chances that the
cells would achieve potential efficacy, we selected 1 × 106
RPCs/eye preoperatively in the clinical trial. A previous
report suggested that the optimal dose of human RPCs
for preserving visual function/retinal structure in
dystrophic rats was 0.5 × 105 to 1.0 × 105 cells per
], and for this reason we used ~ 1 × 105 cells
per injection in the animal study. This dosage of cells
resulted in significant differences between the treated
and controlled RCS rats, in agreement with previous
authors. Given the size differences between the RCS rat
and human retina (80 mm2 versus 10 cm2) [
suggest that an appropriate number of cells needed to gain
an improvement of visual acuity would be in the range
of ~ 1 × 106 RPCs/eye.
Given the crucial role played by photoreceptors in
visual perception, current transplantation strategies aim to
replace degenerating photoreceptors as a way of
restoring some functional vision. The use of photoreceptor
precursors may play a major role in attaining this goal.
Unfortunately, a convincing fluorochrome-conjugated
antibody that recognizes cell surface antigens is not
available and is necessary if photoreceptor precursors
are to be efficiently sorted. Singh et al. [
Gonzalez-Cordero et al. [
] isolated photoreceptor
precursors: respectively a GFP reporter under the control of
a neural retina leucine zipper transcription factor or a
rhodopsin promoter from the retina in order to sort
suitable cells for transplantation. We modified the cell
culture method used by Schmitt et al. [
confirmed that our human RPCs can partially rescue some
visual function in RCS rats. Our results suggest that the
improvement in visual response was due to
photoreceptor replacement and possibly the secretion of trophic
factors from the transplanted cells. In our study, grafted
RPCs expressed both recoverin (a photoreceptor and
bipolar cell marker) and rhodopsin (a photoreceptor
marker). This finding suggests that the transplanted cells
are able to differentiate into retinal cells. In addition,
eyes with transplants maintained a significantly thicker
ONL compared to the control group, which indicates
that the preservation of visual function was partially
achieved by rescuing the host ONL.
The developmental stage of the donor RPC is
important in determining its ability to integrate; early postnatal
RPCs integrate into the host ONL with greater efficiency
than do late postnatal or adult mature photoreceptors
]. The RPCs used by Luo et al.  were isolated
from fetal neural retinas at a gestational age of 16 weeks;
however, little evidence of replacement of degenerated
photoreceptors with donor cells was confirmed [
Their study indicated that trophic factors played a
major role in rescuing endogenous photoreceptors via
RPCs, with a greater number of preserved ONL cells.
Our RPCs were collected from slightly younger
fetuses (12–16 weeks). These RPCs might be better
committed to cell fate compared to those used by
Luo et al. [
], and therefore are likely to follow a
more appropriate differentiation which would account
for differences in our results compared to the data of
Luo et al.
Although our animal study demonstrated that human
RPC transplantation preserved the vision of RCS rats, a
similar statistical improvement in vision between 6 to
24 months was not seen in the clinical trial. A major
difference between our human trial and animal studies is
that the human recipients were generally at an advanced
stage of retinal degeneration compared to the earlier
stage of the disease process in the RCS rats. Chronic
inflammatory reaction is present in the eyes of patients
with retinitis pigmentosa and plays an important role in
the pathogenesis of retinitis pigmentosa [
]. As the
retinal degeneration aggravates, more microglial cells in the
dystrophic retina are activated gradually, and
inflammatory factors secreted by microglial cells will increase
accordingly, which is actually detrimental to donor cell
]. The subretinal space of the host retina in
the earlier disease process of the rats may provide a
more suitable environment for donor cells, resulting in
better survival and functional improvement. In addition,
cortical changes occur following visual loss, including
retinitis pigmentosa, that also seriously affect visual
perception of phoshene [
]. These results indicate
that the baseline status of a recipient’s retinal function
may have a direct impact on postoperative recovery and
should be taken into consideration in devising future
It was difficult to compare the pupillary diameter
changes during the visual function analysis because the
pupillary light reflex (PLR) tests were variable and each
test lasted for over 40 min. However, the threshold in
our study was stable in three repeated tests. Therefore,
we adopted pupillary threshold changes to objectively
represent retinal function. We did not find an
association between improvements in the PLR and visual
acuity for each subject; however, the trend was identical
in showing improved visual function from 3 to 6 months
post-transplantation. Differences between visual acuity and
the PLR test no doubt arise because visual acuity mainly
reflects foveal function, particularly cone sensitivity. During
the PLR test, we most likely activated primarily rods under
scotopic conditions by using a dimmer blue or white light
In our study, clinical examination observed that
allogeneic transplantation of human fetal-derived RPCs into
the diseased retina did not induce any signs of apparent
rejection, such as local retinal inflammation, vascular
leakage, or neovascularization. It is known that the
subretinal space possesses relative immune privilege [
and animal experiments have shown that fetal neural
retinas have low immunogenicity . Immunological
rejection was not observed after transplantation of
retinal cells or retinal tissue together with retinal pigment
epithelium from human fetuses into the subretinal space
of retinitis pigmentosa patients [
]. In agreement
with previous studies, we did not observe any signs of
immunological rejection in response to RPC transplants,
confirming this strategy is a safe procedure.
Our major finding is that fetal RPCs can be safely
transplanted into the retinas of retinitis pigmentosa patients.
These results provide useful information for future
investigations related to cell-based therapies for the
treatment of retinitis pigmentosa and other inherited retinal
Additional file 1: Figure S1. Morphologic changes after RPC
transplantation into the retina of patient 1. Color fundus photographs,
fluorescein angiograms (FA), and OCT images are shown pre- and
postoperatively. OCT showed macular membrane formation at the
12-month follow-up. OCT ocular coherence tomography. (TIF 2469 kb)
Additional file 2: Figure S2. Morphologic changes in patient 2.
(TIF 2215 kb)
Additional file 3: Figure S3 Morphologic changes in patient 3.
(TIF 1489 kb)
Additional file 4: Figure S4 Morphologic changes in patient 4.
(TIF 2045 kb)
Additional file 5: Figure S5. Morphologic changes in patient 5.
(TIF 1318 kb)
Additional file 6: Figure S6. Morphologic changes in patient 7.
(TIF 2092 kb)
Additional file 7: Figure S7. Morphologic changes in patient 8.
(TIF 2646 kb)
Additional file 8: Figure S8. Pupil responses in all patients after RPC
transplantation. (A–H) Figures show pupillary light reflex (PLR) elicited by
blue stimuli, while (A’–H’) show PLR elicited by the white stimuli. (A, C, D,
G) thresholds decreased after 3 to 6 months indicating patients were
more photosensitive, but by 12 months thresholds had returned to
baseline. Using a white stimulus (A’, C’, D’, E’) produces similar results.
(TIF 1077 kb)
Additional file 9: Video S1. Injection of transplanted cells. After
vitrectomy, a 39G cannula was used to create a bleb space for the
injection of transplanted cells. (WMV 29837 kb)
BCVA: Best corrected visual acuity; bFGF: Basic fibroblast growth factor;
CRX: Cone-rod homeobox-containing gene; EGF: Epithelial growth factor;
ERG: Electroretinography; ESC: Embryonic stem cell; FA: Fluorescein
angiography; FACS: Fluorescence-activated cell sorting; fERG: Flash
electroretinography; GFAP: Glial fibrillary acidic protein; GMP: Good
Manufacturing Practice; hESC: Human embryonic stem cell; iPSC: Induced
pluripotent stem cell; ISCEV: International Society for Clinical
Electrophysiology of Vision; MAR: Minimum angle of resolution; OCT: Optical
coherence tomography; ONL: Outer nuclear layer; PAX6: Paired box protein
6; PBS: Phosphate-buffered saline; PLR: Pupillary light reflexes; RCS: Royal
College of Surgeons; RPC: Retinal progenitor cell; RPE: Retinal pigment
epithelial; RT-qPCR: Real-time quantitative polymerase chain reaction;
Sox2: SRY (sex determining region Y)-box 2
We thank Drs. T. FitzGibbon and Shikun He, as well as Prof. Kwok-Fai So, for
their comments regarding the manuscript. We also thank Dr. Herong Yang
for arranging the donor fetal tissues, Drs. Yanji Yu, Sha Li, Minfang Zhang,
and Bo Liu for help with retinal examinations, and Dr. Yuxiao Zeng for the
isolation of retinal progenitor cells.
The study was supported by the National Basic Research Program of China
(973 Program, 2013CB967002 and 2013CB967003 to ZQY), and National
Natural Science Foundation of China (No. 81470671 to YL)
Availability of data and materials
The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request.
YL and ZQY designed the research. LHQ, HWX, and YL performed the
preclinical study, SJC, SYL, XHM, YW, and ZQL performed the clinical study.
SYL, XHM, and YW analyzed the data. YL and ZQY wrote the paper. All
authors read and approved the final manuscript.
Ethics approval and consent to participate
The trial was conducted at the Southwest Hospital, Chongqing, China, and
was approved by the Medical Ethics Committee of Southwest Hospital, Third
Military Medical University. The research adhered to the principles of the
Declaration of Helsinki, and all participants provided their written informed
consent and surgical consent and approved the procedure for publishing
our studies (WHO Trial Registration, ChiCTR-TNRC-08000193).
Consent for publication
The authors declare that they have no competing interests.
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