Ethics committee approval for the umbilical cord Wharton’s jelly derived mesenchymal stem cell study was obtained from the Ankara University Faculty of Medicine Clinical Research Ethics Committee (19–1293-18) and was also approved by the Review Board of the Cell, Organ and Tissue Transplantation Department within the Turkish Ministry of Health (56,733,164/203 E.507). The study was performed in accordance with the tenets of the 1964 Declaration of Helsinki. Written informed consent was obtained from the patients prior to enrollment.
This prospective, open-label clinical trial was conducted between April and October of 2019 at Ankara University Faculty of Medicine, Department of Ophthalmology. 32 RP patients (34 eyes) were included in the study. The preliminary diagnosis was based on clinical history, patients’ complaints, and fundus appearance. All patients enrolled in this study underwent a complete routine ophthalmic examination, including the best-corrected visual acuity (BCVA) measurement with the early treatment of diabetic retinopathy study (ETDRS) chart (Topcon CC 100 XP, Japan). The patients were further evaluated with optical coherence tomography angiography (OCTA) (RTVue XR “Avanti”, Optovue, Fremont, CA, USA) to confirm the diagnosis that provided a typical multimodal imaging platform. Retinal and macular functions were evaluated using the Compass 24/2 visual field test (VF) (Compass, CenterVue, Padova, Italy). Photoreceptor functions were evaluated using multifocal electroretinography (mfERG) (Retiscan, Roland Germany) and with a full-field flicker ERG device (RETeval, LKC Tech. Inc., Gaithersburg, MD, USA).
Dietary supplements were suspended in RP patients 1 month before enrolling in the study because these may interfere with visual functions.
Subjects
The study included 34 eyes from 32 RP patients and in these patients, phase-3 clinical stem cell research was conducted.
RP patients were included in this study if they met the following criteria:
18 years of age or older;
Diagnosis of any phenotypic or genotypic variation of RP, confirmed by clinical history, fundus appearance, visual field (VF), electroretinogram (ERG) and genetic mutation analysis;
Having experienced various degrees of VF loss;
BCVA from 50 letters to 110 letters in the ETDRS chart testing (Topcon CC-100 XP, Japan);
Mean deviation (MD) values ranging between − 33.0 and − 5.0 dB with Compass visual field analysis (threshold 24–2, Sita Standard, Stimulus 3-white);
Intraocular pressure (IOP) of < 22 mmHg.
RP patients were excluded from the study if any of the following were found:
The presence of cataracts or other media opacity that might affect the VF, MD, or ERG recordings;
The presence of glaucoma, which causes visual field and optic disc changes;
The presence of any systemic disorder (e.g.,diabetes, neurological disease, or uncontrolled systemic hypertension) that may affect visual function;
The habit of smoking.
Umbilical cord Wharton’s jelly derived mesenchymal stem cell preparation
The mesenchymal cells that were used in this study were isolated from Wharton’s jelly of the umbilical cord that was collected allogenicly from a single donor with the mother’s consent. The umbilical cord sample was treated as follows: briefly, cord tissue was washed twice with PBS (Lonza, Switzerland) and the Wharton’s jelly part was minced using forceps and a scalpel. Minced pieces were cultivated in a cell culture dish (Greiner Bio-One, Germany) with Dulbecco’s modified Eagle’s medium F12 (DMEM)-low glucose no L-Glutamine (Bilogical Industries, Israil) with 10% human AB serum (Capricorn, Germany), 1% 10.000 U/mL penicillin and 10.000 μg/mL streptomycin (Gibco,USA). All cell preparation and cultivation procedures were conducted in a current Good Manufacturing Practice (cGMP) accredited laboratory (Onkim Stem Cell Technologies, Turkey). The culture-expanded cells were cryopreserved at P3 using standard cryopreservation protocols until their use in the following experiment. The cells were characterized at the time of cryopreservation with flow cytometric analysis to determine the expression of positive surface markers CD90, CD105, CD73, CD44, CD29, and negative for CD34, CD45 and CD11b; also, using real-time polymerase chain reaction (PCR), the expression of LDHA, HLA-DR, HLA-G, BMP2, BMP4, BMP6, JAG1, ZPF42, NANOG, POU5F1, ENG, CD44, TNF, ICAM1, VIM, THY1, VCAM1, VEGFA NES, RUNX2, SMURF1 and COL1A1 genes were analyzed. Additionally, quality control analyses like mycoplasma analysis (using PCR), endotoxin analysis (using the LAL test and sterility analysis) were also completed. Cells were solubilized from cryopreservation before being made ready for injection. Average cell viability for each treatment was over 90.0% and each patient received cell numbers between 2-6 × 106 in a 1.5 ml saline solution (Fig. 1a, b).
Injection of umbilical cord WJ-MSCs
The WJ-MSC suspension from the culture was delivered to the operating room by cold chain for use within 24 h. A total of 1.5 ml of the WJ-MSC suspension was withdrawn using a 2.5 cc syringe and was immediately injected into the subtenon space of each eye. The injection of the WJ-MSC suspensions were carried out by two ophthalmologist (EÖ - UA) using two distinct methods. Procedures were conducted under topical anesthesia with proparacaine hydrochloride drops (Alcaine, Alcon, USA) and sterile conditions. In the first method, the preplaced suture technique, a small cut was made through the conjunctiva and tenon capsule up to the sclera in the infero-nasal quadrant, 13 mm away from the limbus, for the insertion of a 20 G subtenon curved canulla (BD, Visitec, UK). Subsequently, a 7/0 vicryl suture was passed through the conjunctiva and tenon and tied down with a loop creation. A curved subtenon canulla attached to the 2.5 cc syringe filled with 1.5 ml fluid containing stem cells was inserted through the cut, and forwarded into the extraocular muscle conus until reaching the sclera. 1.5 ml of fluid was then injected. While the canulla was drawn back, a loop was tightened in order to prevent leakage. The second ophthalmologist performed a subtenon injection using a 25-gauge sharp-tip syringe without any incision into the supero-temporal region because the largest quadrant for the effective delivery of the 1.5 ml fluid containing stem cells. Both methods were used in an equal number of eyes (17 eyes for each method). In both methods, in order to expose the more sub-tenon space in the chosen region, a traction by a 5/0 atraumatic silk suture with a round needle was exerted into the limbus, pulling away from the cut / injection site. In both methods, it was confirmed using orbital ultrasound (Quantel, Cournon d’Auvergne, France) that the injection was delivered to the deep sub-tenon region near the sclera and within the extraocular muscle conus. Postoperatively, loteprednol + tobramycin combination eye drops were given 4 times per day for 1 week and oral amoxicillin clavulonate was given at 1 g, twice a day for 5 days.
The patients were followed for 6 months after the WJ-MSC injection and underwent 5 consecutive examinations to monitor the individuals closely and record any possible adverse/side effects. The quantitative results were obtained by comparing the pre-injection and final examination (6th month) values. The primary aim of this clinical study was to assess the effects of WJ-MSCs on BCVA, VF, outer retinal thickness (ORT), mfERG and full-field flicker ERG. The secondary aim of the study was to investigate whether both surgical techniques are safe and the amount of stem cells used is sufficient to elicit clinical responses.
For VF analysis, in order to avoid mistakes during the test, practice rounds were carried out three times before the WJ-MSC injection of each eye. These visual field practice tests were completed using the same parameters as the real test to exclude learning effects.
To evaluate retinal functions, mfERG could be performed on patients who had sufficient fixation according to the ISCEV standard protocol [31,32,33]. The mfERG measures neuroretinal function (postreceptoral responses, cone mediated ON and OFF bipolar cells, and inner retinal cell contributions) in localized retinal areas. The amplitude (nv/deg2) and implicit times (ms) of the first-order kernel mfERG responses (N1 and P1 waves) were obtained and grouped into five rings (ring 1, central 2°; ring 2, 2–5°; ring 3, 5–10°; ring 4, 10–15°; ring 5,> 15°). In all subjects, the mfERG testing protocol was began 20 min after preadaptation to an ambiently lit environment equivalent to the mean luminance of the stimulus at 100 cd/m2. Pupils were pharmacologically (with tropicamide 1%) dilated to 8–9 mm. The cornea was anesthetized with proparacaine hydrochloride drops. The mfERGs were recorded monocularly, patching the contralateral eye using a DTL electrode. A small gold skin ground electrode was placed at the center of the forehead after preparing the skin with abrasive gel. Meanwhile, a skin electrode was placed at the outer canthus to be used as a reference. mfERG was performed by correcting refraction errors. The multifocal stimulus, which consisted of 61 scaled hexagons, was displayed on a high resolution, black and white cathode ray tube (CRT) monitor with a frame rate of 75 Hz. The signal was amplified (gain 100,000) and filtered (band pass 3–300 Hz). After automatic rejection of artifacts, the first-order kernel response, K1, was examined. These parameters were obtained from five concentric annular retinal regions (rings) centered on the fovea.
Full-field flicker ERG is a noninvasive objective test that measures the electrical activity of the retina in response to a light stimulus. The 30 Hz flicker ERG reveals a response from the cone bipolar cells. Flicker stimulation is valuable for studying the neurovascular coupling, which is a physiological process, that adjusts the microcirculation in response to neural activity [34, 35]. Full-field flicker ERGs were recorded without mydriasis using the RETeval system. The measurements were taken according to the instructions provided with the instrument for both eyes. We used the 16 and 32 Tds protocol, which combines implicit time and amplitude to create a numerical result.
Time frame
The patients were checked during the following time points:
Before application: a period of 3 months prior to the WJ-MSC application
0 (baseline): just before the WJ-MSC injection
1: 1st month after injection
2: 2nd month after injection
3: 3rd month after injection
4: 6th month after injection
Primary outcome measure
Visual acuity was measured at the 0,1,2,3 and 4 time points. The visual acuity scores obtained from the baseline testing and the final examination were analyzed and compared (using statistical tests) to determine effectiveness.
Secondary outcome measures
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Visual field sensitivity (time frame: before application, 0, 1, 2, 3 and 4)
A Compass visual field analyzer, threshold 24–2 modality, was used at the 0, 1, 2, 3 and 4 time-points. In addition, it was used three times before application during experimentation to exclude the learning effect. The MD values, which were obtained from the baseline test and the final examination, were analyzed and compared (using statistical tests) to determine the effectiveness of the treatment.
Outer retinal thickness (time frame: before application, 0, 1, 2, 3 and 4)
The structural parameters were measured on OCTA at the 0, 1, 2, 3 and 4 time points. Outer retinal thickness (ORT): This is the thickness from the outer plexiform layer to the Bruch membrane in the 3 × 3 mm area of the fovea measured (and recorded automatically) by the multimodal imaging OCTA device.
The retinal electrical responses from mfERG were measured in patients by correcting refraction errors at the 0 and 4 time points. The amplitudes of each ring obtained during baseline testing and in the final examination were analyzed and compared (using statistical tests) to determine the effectiveness of the treatment.
The implicit times of each ring obtained from the baseline testing and the final examination were analyzed and compared (using statistical tests) to determine the effectiveness of the treatment. Full-field flicker electroretinogram (time frame: 0, 1, 2, 3 and 4).
The amplitudes and implict times obtained from the baseline testing and the final examination were analyzed and compared (using statistical tests) to determine the effectiveness of the treatment. mfERG was started as soon as necessary permissions were obtained due to electrophysiology laboratory density. Some deviations in the time frame were found to not change the mfERG results.
Definition of safety outcome
Intraocular/intraorbital inflammation, proptosis, diplopia, afferent pupillary defect, corneal/lenticular haze, ocular allergic reactions, intravitreal/subretinal/macular hemorrhages, vitreoretinal interface alterations, retinal tear(s)/retinal detachment (exudative, rhegmatogenous), intraocular pressure change from baseline (≤5 mmHg) were considered to be serious adverse ocular events. Besides the routine ophthalmic examinations, OCTA multimodal imaging was also used to detect and confirm the presence of complications and anatomical changes during each examination for the study period. Systemic allergic reactions and anaphylaxis were considered to be systemic side effects.
Statistical methods
The statistical comparisons were made primarily between the baseline and final values from the same eye. The BCVA and parametric results for visual field, ORT, mfERG and full field flicker ERG were analyzed using a Student’s paired t-test. Results are presented as means and standard deviations. P values less than 0.05 are considered statistically significant. A 95% confidence interval for the difference in means was used for double confirmation. Analyses were carried out with SPSS for Windows (v22; IBM Corp.; Armonk, NY, USA).
