Characterization of bone marrow-derived mesenchymal stem cells
BM-MSCs from all participants exhibited spindle-shaped-like cells. The stem cell phenotypes were in accordance with the international society for cellular therapy (ISCT), such as adherence to the plastic culture vessel; expression of more than 95% of CD73, CD90, and CD105; and negative (less than 2%) for CD34, CD45, and HLA-DR (Fig. 2). The trilineage differentiation ability to be adipocyte, osteocyte, and chondrocyte was confirmed in all samples. There was no microorganism or endotoxin contamination. The average cell viability was 92.12 ± 3.5% (Table 1).
Baseline characteristics of study participants
We recruited 14 participants with advanced RP, consisting of nine males and five females, with ages ranging from 31 to 61 (mean ± SD, 46.2 ± 9.3) years. Four participants had the intraocular lens (IOL) in both eyes and one had IOL in the study eye (Table 1), all of which underwent the surgeries longer than 6 months prior to enrollment. The average baseline BCVA was 2.00 ± 0.14 logMAR in the study eye. At baseline, the mean number of the cells and the values of flare in the anterior chamber of the study eye were 1.55 ± 0.88 cells per 0.5 mm3 and 6.8 ± 3.05 photons per millisecond (ph/ms), respectively, with an average IOP of 13.11 ± 1.71 mmHg. The ERG was nonrecordable in all participants. Seven out of 14 participants had VF less than 20° in the study eye, and one participant presented with a central scotoma of 20° with normal peripheral VF. In the remaining six participants, we could not evaluate the VF due to poor fixation. Seven participants were categorized based on the number of MSCs injected to group 1 (1 × 106 cells), three participants to group 2 (5 × 106 cells), and four participants to group 3 (1 × 107 cells) (Table 1).
Clinical safety evaluation
We did not observe any serious AEs after the intravitreal injection, such as central retinal artery occlusion, leakage, hemorrhage, retinal detachment, or endophthalmitis, in all participants. We noticed an increase in IOP in all groups (4.40 ± 2.07 mmHg in group 1; 6 ± 4.24 mmHg in group 2; and 9 ± 0 mmHg in group 3) 1 h post-intravitreal injection. However, the IOP returned to the baseline values on the first day (D1) and remained stable throughout the course of the study.
The Kowa FC-2000 was used to quantify the flare and cells. The intensity of flare and peaks of light scattered from the particles in the aqueous reflect the inflammation and cells in the anterior chamber, respectively. It is important to note that any large particles in the aqueous that can produce peaks, such as blood cells, pigment, debris, or other cells, cannot be differentiated from inflammatory cells [15, 16]. In the present study, the number of cells in the anterior chamber increased and peaked immediately in the study eye after the injection in groups 1 and 2 (Fig. 3a, c, e). In group 3, the cells gradually increased and reached the peak at D5 (Fig. 3a, g). Compared among the three groups, the highest increase in cells from the baseline was observed in group 2. In group 1, which received the lowest number of BM-MSCs, the cells returned to the baseline after D1, while in groups 2 and 3 they returned to the baseline after D14. The protein flare values in group 1 did not show a remarkable increase after the injection (Fig. 3d), while those in groups 2 and 3 peaked at D4–5 (Fig. 3f, g). Similar to the cells, the highest increase in flare values from the baseline was found in group 2. This was due to the acute inflammation in one participant in group 2. After given eye drops containing tobramycin and dexamethasone, and 1% atropine eye drops, the inflammation subsided within 1 week. In groups 2 and 3, the flare values returned to the baseline after 14 days and remained stable throughout the course of the study. The cells and flare in the fellow eyes remained stable, except in group 2 (Fig. 3e), since two participants displayed fluctuating high numbers of cells in the fellow eye throughout the study.
During the first week post-injection, two participants (14.28%) complained of mild pain in the study eye, one (7.14%) reported feeling pressure, two (14.28%) with redness, and four with mild irritation in the study eye. All symptoms subsided spontaneously within 1 week without treatment. Five participants (35.71%) described seeing flashing or moving lights or dots occasionally. No participant reported extreme discomfort in the study eye (Fig. 4).
In the course of 12 months, no participant experienced a sudden decline in the BCVA or VF. The fundus photographs, fundus autofluorescence, FFA, ICG (data not shown), and the OCT showed no significant changes in pigment accumulation, areas of hypo- and hyper-autofluorescence, the appearances of retinal and choroidal vasculature, and central subfield thickness, respectively, in the study eye and the fellow eye. This indicated the stable condition of RP, with no remarkable deterioration of the disease (Fig. 5).
We observed several mild AEs, none of which affected the BCVA in the study eye. One participant in group 3 developed localized posterior synechiae at the superotemporal quadrant, at D6 (Fig. 6a). After treatment with 1% atropine eye drops, the synechiae was released at D14 (Fig. 6b). Minimal cystoid macular edema (CME) occurred in one participant in group 1 at month (M) 3 and persisted until the last follow-up at year (Y) 2.5 (Fig. 6c, d). We noticed a slight displacement of the IOL toward the temporal side of the study eye, with the IOL optic still in the visual axis, in two out of five participants with IOL, from groups 2 and 3, which started at M8 and M6, respectively (photographs not shown). The last mild AE was observed in a participant in group 1 who demonstrated localized flat choroidal detachment at the superotemporal quadrant of the study eye at M9. The ultra-widefield retinal imaging displayed that the choroidal detachment was slightly elevated at Y5 (Fig. 6e). The ultrasound demonstrated double-peak reflectivity in the A-scan with anechoic suprachoroidal space in the B-scan, indicating fluid accumulation (Fig. 6f).
Although this phase I study was completed in the 12-month period, we constantly monitored 12 out of 14 study participants during hospital visits, with a duration ranging from 1.5 to 7 years (mean ± SD, 4 ± 1.8 years). We documented two AEs. The first one happened at Y3M4, when a participant from group 2 experienced sudden vision loss in the study eye after waking up in the morning. Upon immediate evaluation, we found diffuse vitreous hemorrhage, which obscured fundus details. Pars plana vitrectomy was performed to remove the vitreous hemorrhage (Fig. 7a). Intraoperatively, we observed thick fibrous membrane along the vitreous base at the superotemporal quadrant. The fibrous membrane wrapped around the capsular bag; thus, the IOL and the capsular bag had to be removed. This membrane also caused traction to the peripheral retina with retinal dialysis superotemporally. Histopathological evaluation of the membrane demonstrated a membranous sheet of bone surrounded by ciliary tissue. Higher magnification showed calcified matrix sheets with parallel alignment and regularly distributed osteocytes, indicating osseous metaplasia or osseous heterotopia in the ciliary body (Fig. 7b). After the operation, the retina was attached and the BCVA was restored to the same level as the time of enrollment (logMAR 1.22 and 1.20, at baseline and Y5, respectively). Another mild AE happened in one participant from group 1, who developed minimal IOL subluxation in both eyes at Y4 post BM-MSC injection.
Short-term efficacy evaluation
In this study, we evaluated the visual acuity, visual field, and central subfield thickness for the short-term efficacy of the BM-MSCs. The BCVA was evaluated individually for each group. Since the number of participants that could perform reliable VF and CST assessment was low in some groups, we analyzed the available data as a mean from all participants, comparing between the baseline and each time point.
There was a slight improvement of BCVA in all study groups; however, it slowly returned to the baseline within 12 months (Fig. 8a). Interestingly, when compared to the fellow eye, the highest improvement was observed in group 1 (Fig. 8b), which received the lowest number of BM-MSCs. Specifically, when compared to the baseline, the BCVA in group 1 improved at M2, 5, 7, and 8, reaching statistically significant improvements at M7 (P = 0.04) and M8 (P = 0.02), slowly regressed after M9, and reached the baseline at M12. In group 2, the BCVA improvement started after M5 and persisted up to M9, then slowly declined until it reached the baseline at M12 (Fig. 8c). Conversely, in group 3, although the BCVA significantly improved immediately at M1 (P = 0.04), it returned to the baseline at M6 (Fig. 8d). We noted that both the study and fellow eyes showed the similar trends of the BCVA changes, especially in group 1 and group 2, although the study eye showed higher improvements. A pair match with the fellow eye was also tested and we did not observe a statistically significant difference in the BCVA between the study and the fellow eye, although some participants reported subjective improvements of vision on activities of daily living. The details of logMAR values (mean ± SD) of each study group were provided in Supplementary Table S1.
Eight out of 14 participants showed unremarkable VF changes between the baseline and at M12, while it was not able to be evaluated in the remaining six participants due to severe reduction in the visual acuity. We evaluated the CST in 11 out of 14 participants (the CST was unmeasurable in the remaining three study participants). The average CST at the baseline was 172 ± 59.2 μm in the study eyes and remained stable throughout the study, i.e., M1 (178 ± 61.7 μm), M3 (178 ± 58.7 μm), M6 (176 ± 60.1 μm), and M12 (176 ± 59.8 μm). On the other hand, the fellow eyes at M12 displayed thinner CST than baseline (185 ± 66.8 vs 178 ± 65.4 μm). Interestingly, we noticed slight improvements in individual CST in the study eye in three out of 11 participants (27.27%), with baseline/M12 values of 142/150 μm, 150/177 μm, and 102/131 μm, respectively. The BCVA of two participants (with CST baseline/M12 of 142/150 μm and 150/177 μm) showed some improvement in the study eye (logMAR 1.34 at baseline to 1.02 at M12; and logMAR 2.30 at baseline to 1.66 at M12, respectively). However, this did not occur in the other participant with CST improvement.
Subjectively, most participants experienced improvements in the quality of life during the 12-month period after the BM-MSC injection. Seven out of 14 (50%) participants described a stable vision, five (35.7%) participants could see better in dim light, five (35.7%) could see light better than the fellow eye, three (21.4%) could see some colors, and four (28.6%) could perform daily activities better, such as walking to the bathroom at night, using the cell phone, watching TV, and riding the bicycle (Fig. 4).