This is the first randomized controlled study to investigate safety and efficacy of administering allogeneic deceased‐donor ex vivo expanded corneal epithelial stem cells in severe bilateral OSD arising from LSCD. Our robust study design allowed us to explore the specific benefits arising from the use of IMP. In addition, we examined serum cytokine profiles in LSCD patients which have not been previously reported.

The use of allogeneic deceased‐donor limbal tissue introduces variance due to donor age, time to procurement, transport conditions, tissue processing and innate biological variability. We were able to achieve adequate ex vivo outgrowth containing corneal epithelial stem cells from all but one of the donations we received. We chose to use AM as a matrix on which to grow and transfer the cells, rendering this a composite tissue engineered product. QC of such products is not achievable without sacrificing some product. Therefore, we tested remnant material from the transplanted composite product by immunohistochemistry. The stem‐like p63+ phenotype was detected in the remnant material from 5 out of the 9 composite transplanted products. The absence of p63+ cells in 4 may be because of the limited tissue availability and/or peripheral nature of the area sampled. Where p63 positive cells were not detected in remnant material from the transplanted product, p63+ cells were detected in the duplicate culture, so we may have a degree of confidence that the transplanted product did contain LSCD. Kolli et al. 11 describe a gradation in the stem cell marker p63 in these cultures, with colony formation highest proximate to the limbal explants and declining in the more distal zones of the culture. Expression of CK3 (a marker of differentiated corneal epithelium) showed the opposite gradation. In the cultures used for the shelf‐life validation, around half of the products expressed The CK3 marker, whereas in the transplanted products CK3+ cells were detected in 7 out of 9 products (and in the duplicate cultures from the 2 clinical cultures where CK3 was not detected). This indicates our product was similar to that manufactured by Kolli et al. Variability from product to product is likely a function of the original donated cadaveric material, or the outgrowth region sampled for immunohistochemistry analysis, as the GMP‐compliant manufacturing process is highly standardized. The IMP shelf‐life of 24 hours at 18°C–24°C allowed transportation of the product to clinical centers such as Glasgow and Liverpool from the manufacturing center (Edinburgh) within the validated timeframe.

Clinical Trial Outcomes

The IMP was demonstrated to be safe within the limits of the study, with no serious adverse reactions or events attributed to the IMP itself. The primary endpoint was improvement in visual acuity, and there were indicators of transient improvement throughout the study in both groups. These were not statistically significant, but the majority of patients in both groups ended up with an improved score at 18 months. The IMP group demonstrated statistically significant and sustained improvement in OSS over the period of the study. The control group also demonstrated an initial improvement in OSS, but the effect was not sustained, and the scores returned to near start levels by 18 months. One patient with aniridia was particularly informative. They entered the control arm and the OSS improved from 10 to 8 over 18 months. The patient was then readmitted to the study in respect of his/her other eye and allocated to the IMP arm where OSS improved from 9 to 3. This patient's visual acuity also improved from LogMAR 3 to 2 with control product and from LogMAR 3 to 1.08 with IMP. It therefore may be asserted with a degree of confidence that patients receiving the IMP did benefit overall in terms of OSS improvement, meeting the principal secondary endpoint of the trial.

The clinical trial was designed as a randomized, controlled and partially blinded phase I/II trial and hypothesized that the use of AM, topical autologous serum eye drops and systemic immunosuppression, may have therapeutic benefit. This clearly is the case, as both IMP and control groups show initial improvements in OSS—indeed this is statistically significantly improved in both groups at 6 months. This is not sustained in the control group, returning to be nonsignificantly different from the starting scores, but is clearly sustained in the IMP group. We may conclude that this study demonstrates a pro‐regenerative role for transplanted LSC in maintaining improved OSS for a significantly longer time post‐transplant than control product. The mechanism for this role is not associated with long‐term donor cell engraftment, as there is no sign of donor DNA in eyes of the majority of patients at 6 months. This finding agrees with 2 earlier studies, one which found no evidence of donor DNA in 7 patients 28 weeks postallografting 13, and one on small patient numbers (albeit after longer follow‐up) who found sustained benefit in eyes receiving LSC, but with no evidence of engrafted donor DNA 25. There may be scope to reduce the period of immune suppression in future trials if this is not required to support long‐term engrafted allogeneic cells.

In this study we were able to compare improvements in VA between the IMP treatment and control groups, which is a novel approach in this field. In other uncontrolled studies that included allogeneic donor products (although some were from related donors) successful improvement of any kind in VA at the assessment point was seen in, for example, 5/7 patients 26, 2/7 patients 27, 4/7 patients 28. We saw overall VA improvement in 5/8 IMP patients and 4/5 Control patients at the end of our study. Although generalizations must be drawn carefully between differing studies and protocols, the results of our study has at least comparable outcomes in VA changes to other studies, whether LSC are present or not. The magnitude of change is higher with the IMP but not significantly so, this may well be a factor of the small patient numbers and will require a larger trial to be investigated further.

It should also be noted that randomization led to a skew in the number of patients with different etiologies completing the IMP and control groups. Specifically, 4/8 patients in the IMP group had aniridia compared with 1/5 in the control group. It is possible that patients with aniridia respond better to the treatments. Larger studies with aniridia patients using our IMP are warranted. The majority of IMP group patients did not display sustained engraftment of donor DNA suggesting that engraftment is transient, as described previously 13. The benefits in OSS for the IMP group were due to early effects of the stem cells. How these observations relate to the mode of action is unclear. The IMP could induce repair of the limbal niche or stimulate small numbers of dormant residual host stem cells to multiply potentially through a paracrine or chemotactic mechanism, however this remains speculative and we believe that more research is required.