Generation of allogeneic CAR-NKT cells from hematopoietic stem and progenitor cells using a clinically guided culture method.

Cancer immunotherapy with autologous chimeric antigen receptor (CAR) T cells faces challenges in manufacturing and patient selection that could be avoided by using 'off-the-shelf' products, such as allogeneic CAR natural killer T (AlloCAR-NKT) cells. Previously, we reported a system for differentiating human hematopoietic stem and progenitor cells into AlloCAR-NKT cells, but the use of three-dimensional culture and xenogeneic feeders precluded its clinical application. Here we describe a clinically guided method to differentiate and expand IL-15-enhanced AlloCAR-NKT cells with high yield and purity. We generated AlloCAR-NKT cells targeting seven cancers and, in a multiple myeloma model, demonstrated their antitumor efficacy, expansion and persistence. The cells also selectively depleted immunosuppressive cells in the tumor microenviroment and antagonized tumor immune evasion via triple targeting of CAR, TCR and NK receptors. They exhibited a stable hypoimmunogenic phenotype associated with epigenetic and signaling regulation and did not induce detectable graft versus host disease or cytokine release syndrome. These properties of AlloCAR-NKT cells support their potential for clinical translation.

Engineering allorejection-resistant CAR-NKT cells from hematopoietic stem cells for off-the-shelf cancer immunotherapy.

The clinical potential of current FDA-approved chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapy is encumbered by its autologous nature, which presents notable challenges related to manufacturing complexities, heightened costs, and limitations in patient selection. Therefore, there is a growing demand for off-the-shelf universal cell therapies. In this study, we have generated universal CAR-engineered NKT (UCAR-NKT) cells by integrating iNKT TCR engineering and HLA gene editing on hematopoietic stem cells (HSCs), along with an ex vivo, feeder-free HSC differentiation culture. The UCAR-NKT cells are produced with high yield, purity, and robustness, and they display a stable HLA-ablated phenotype that enables resistance to host cell-mediated allorejection. These UCAR-NKT cells exhibit potent antitumor efficacy to blood cancers and solid tumors, both in vitro and in vivo, employing a multifaceted array of tumor-targeting mechanisms. These cells are further capable of altering the tumor microenvironment by selectively depleting immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells. In addition, UCAR-NKT cells demonstrate a favorable safety profile with low risks of graft-versus-host disease and cytokine release syndrome. Collectively, these preclinical studies underscore the feasibility and significant therapeutic potential of UCAR-NKT cell products and lay a foundation for their translational and clinical development.

Comparison of Incidence or Recurrence of Anterior Uveitis in Patients with Ankylosing Spondylitis Treated with Tumor Necrosis Factor Inhibitors.

Background: Anterior uveitis (AU) is a significant concern in patients with ankylosing spondylitis (AS), and the choice of tumor necrosis factor inhibitors (TNFi) as a treatment modality raises questions regarding its effects on AU. We compared the effects of TNFi on AU in patients with AS. Methods: Patients diagnosed with AS and treated with at least one TNFi, including anti-TNFα antibodies (adalimumab and infliximab) or a soluble TNF receptor molecule (etanercept), between January 2010 and December 2022, were retrospectively reviewed. We compared the recurrence rate of AU in patients with a history of uveitis and the incidence of new-onset AU in those without a history of uveitis among the three TNFi groups. We also compared the effects of two different TNFi agents in patients who underwent TNFi switching. Results: Within two years of treatment initiation, there was no significant difference in AU recurrence among the three TNFi groups. However, the incidence of new-onset AU was significantly higher in the etanercept group than in the adalimumab group (26.4% vs. 6.3%; p = 0.024). After two years, the AU recurrence rate was significantly lower in the adalimumab group than in the other groups (p < 0.001). Among patients who underwent anti-TNFi switching, adalimumab treatment was associated with a significantly lower incidence of uveitis than etanercept (p = 0.023). Conclusion: In the short-term period following TNFi therapy, etanercept induced new-onset AU more frequently than adalimumab in patients with AS. Adalimumab recipients experienced fewer AU recurrences during the subsequent long-term period compared to other TNFi recipients.

Clinical relevance of thyroid-stimulating immunoglobulin as a biomarker of the activity of thyroid eye disease.

BACKGROUND/OBJECTIVES: Although it has been reported that thyroid-stimulating immunoglobulin (TSI) is associated with the clinical characteristics of thyroid eye disease (TED), there is a paucity of literature regarding the role of TSI in diagnosing active TED. This study investigated the relationship between the level of TSI and the activity of TED and assessed the cut-off value of TSI discriminating active TED from inactive TED. METHODS: This cross-sectional study included 101 patients with TED. TSI was quantitatively measured with a cell-based bioassay using a chimeric TSH receptor and a cyclic adenosine monophosphate response element-dependent luciferase. The association between TSI and a variety of demographic and clinical features of TED was analysed. Multivariate regression analysis was performed to determine possible independent factors affecting the level of TSI. RESULTS: TSI level was higher in males than in females (p = 0.023) and smokers than in nonsmokers (p = 0.004). TSI level was inversely correlated with the duration of ocular symptoms (r = -0.295, p = 0.003). The level of TSI was also significantly different when compared to the thyroid function (p = 0.003), TED activity (p < 0.001), and TED severity (p = 0.001). Multivariate regression analysis revealed a significant relationship between TED activity and thyroid function jointly and the TSI level. The cut-off level of TSI for predicting active TED was a specimen-to-reference ratio of 406.7 (p < 0.001, area under the curve = 0.847, sensitivity 77.4%, specificity 81.3%). CONCLUSIONS: TSI was a functional biomarker strongly associated with TED activity even after being adjusted by other clinical characteristics. Serum TSI level may help identify patients with active TED in clinics.

Nanoformulation improves antitumor efficacy of MAOI immune checkpoint blockade therapy without causing aggression-related side effects.

MAOIs, a well-established class of antidepressant that operate through the inhibition of monoamine oxidase to increase available serotonin, have recently been identified as a surprisingly effective candidate for the circumvention of tumor-induced immune suppression due to their abilities to enhance antitumor T cell activity through autocrine serotonin signaling and depolarize alternatively activated tumor-associated macrophages through a reduction in reactive oxygen species production. However, this impressive class of antidepressants-turned-cancer-drugs can induce aggressive behavioral side effects when administered in immunotherapeutic doses. In this study, we investigated the possibility of avoiding these neurological side effects while simultaneously improving antitumor activity by establishing crosslinked multilamellar liposomal vesicles (cMLVs) containing the MAOI phenelzine (PLZ). Our results showed that cMLV-PLZ treatment increases antitumor efficacy in a B16-OVA mouse melanoma model compared to treatment with free phenelzine. We also found that nanoformulation resulted in the complete elimination of MAOI-related aggression. These findings suggest a promising direction for the future of MAOIs repurposed for cancer immunotherapies.

The Link module of human TSG-6 (Link_TSG6) promotes wound healing, suppresses inflammation and improves glandular function in mouse models of Dry Eye Disease.

PURPOSE: To investigate the potential of the Link_TSG6 polypeptide comprising the Link module of human TSG-6 (TNF-stimulated gene/protein-6) as a novel treatment for dry eye disease (DED). METHODS: We analyzed the therapeutic effects of topical application of Link_TSG6 in two murine models of DED, the NOD.B10.H2b mouse model and the desiccating stress model. The effects of Link_TSG6 on the ocular surface and DED were compared with those of full-length TSG-6 (FL_TSG6) and of 0.05% cyclosporine (Restasis®). Additionally, the direct effect of Link_TSG6 on wound healing of the corneal epithelium was evaluated in a mouse model of corneal epithelial debridement. RESULTS: Topical Link_TSG6 administration dose-dependently reduced corneal epithelial defects in DED mice while increasing tear production and conjunctival goblet cell density. At the highest dose, no corneal lesions remained in ∼50% of eyes treated. Also, Link_TSG6 significantly suppressed the levels of inflammatory cytokines at the ocular surface and inhibited the infiltration of T cells in the lacrimal glands and draining lymph nodes. Link_TSG6 was more effective in decreasing corneal epithelial defects than an equimolar concentration of FL_TSG6. Link_TSG6 was significantly more potent than Restasis® at ameliorating clinical signs and reducing inflammation. Link_TSG6 markedly and rapidly facilitated epithelial healing in mice with corneal epithelial debridement wounds. CONCLUSION: Link_TSG6 holds promise as a novel therapeutic agent for DED through its effects on the promotion of corneal epithelial healing and tear secretion, the preservation of conjunctival goblet cells and the suppression of inflammation.

Development of allogeneic HSC-engineered iNKT cells for off-the-shelf cancer immunotherapy.

Cell-based immunotherapy has become the new-generation cancer medicine, and "off-the-shelf" cell products that can be manufactured at large scale and distributed readily to treat patients are necessary. Invariant natural killer T (iNKT) cells are ideal cell carriers for developing allogeneic cell therapy because they are powerful immune cells targeting cancers without graft-versus-host disease (GvHD) risk. However, healthy donor blood contains extremely low numbers of endogenous iNKT cells. Here, by combining hematopoietic stem cell (HSC) gene engineering and in vitro differentiation, we generate human allogeneic HSC-engineered iNKT (AlloHSC-iNKT) cells at high yield and purity; these cells closely resemble endogenous iNKT cells, effectively target tumor cells using multiple mechanisms, and exhibit high safety and low immunogenicity. These cells can be further engineered with chimeric antigen receptor (CAR) to enhance tumor targeting or/and gene edited to ablate surface human leukocyte antigen (HLA) molecules and further reduce immunogenicity. Collectively, these preclinical studies demonstrate the feasibility and cancer therapy potential of AlloHSC-iNKT cell products and lay a foundation for their translational and clinical development.

Targeting monoamine oxidase A for T cell-based cancer immunotherapy.

Monoamine oxidase A (MAO-A) is an enzyme best known for its function in the brain, where it breaks down neurotransmitters and thereby influences mood and behavior. Small-molecule MAO inhibitors (MAOIs) have been developed and are clinically used for treating depression and other neurological disorders. However, the involvement of MAO-A in antitumor immunity has not been reported. Here, we observed induction of the Maoa gene in tumor-infiltrating immune cells. Maoa knockout mice exhibited enhanced antitumor T cell immunity and suppressed tumor growth. MAOI treatment significantly suppressed tumor growth in preclinical mouse syngeneic and human xenograft tumor models in a T cell-dependent manner. Combining MAOI and anti-PD-1 treatments generated synergistic tumor suppression effects. Clinical data correlation studies associated intratumoral MAOA expression with T cell dysfunction and decreased patient survival in a broad range of cancers. We further demonstrated that MAO-A restrains antitumor T cell immunity through controlling intratumoral T cell autocrine serotonin signaling. Together, these data identify MAO-A as an immune checkpoint and support repurposing MAOI antidepressants for cancer immunotherapy.

Contrast, visibility, and color balance between the microscope versus intracameral illumination in cataract surgery using a 3D visualization system.

Purpose: To compare image resolution and depth between the microscope versus intracameral illumination images during 3D heads-up cataract surgery. Methods: We collected 25 consecutive patients who had cataract surgery using the 3D viewing system. Based on bright, contrast, visibility, and color balance, the digital images (RGB color and three monochromes) extracted at the same point of the procedures were compared between the two illuminations. Results: Contrast values of green and blue channels except for red channel and visibility values of all three channels were higher in the intracameral illumination images than in the microscope images (P < 0.001, t-test). Color balance values of both green/red and blue/red were higher in the intracameral illumination images than in the microscope images (P < 0.001, t-test). Conclusion: The digital images in the digitally assisted cataract surgery were enhanced by using the intracameral illumination. Considering the contrast and color balance in the 3D cataract surgery, the intracameral illumination may be better than the microscope illumination.

Creatine uptake regulates CD8 T cell antitumor immunity.

T cells demand massive energy to combat cancer; however, the metabolic regulators controlling antitumor T cell immunity have just begun to be unveiled. When studying nutrient usage of tumor-infiltrating immune cells in mice, we detected a sharp increase of the expression of a CrT (Slc6a8) gene, which encodes a surface transporter controlling the uptake of creatine into a cell. Using CrT knockout mice, we showed that creatine uptake deficiency severely impaired antitumor T cell immunity. Supplementing creatine to WT mice significantly suppressed tumor growth in multiple mouse tumor models, and the combination of creatine supplementation with a PD-1/PD-L1 blockade treatment showed synergistic tumor suppression efficacy. We further demonstrated that creatine acts as a "molecular battery" conserving bioenergy to power T cell activities. Therefore, our results have identified creatine as an important metabolic regulator controlling antitumor T cell immunity, underscoring the potential of creatine supplementation to improve T cell-based cancer immunotherapies.

Light exposure from microscope versus intracameral illumination during cataract surgery.

Purpose: To evaluate light exposure from microscope versus intracameral illuminations to patient's and surgeon's retina during cataract surgery. Methods: Thirty consecutive patients who had cataract surgery using microscope and intracameral illuminations. At the point of the ocular of an operating microscope, optical illuminance and irradiance from the microscope illumination (60, 40, 20% intensity) and the intracameral illumination (60% intensity) were measured using a light meter and a spectrometer at a pause after lens capsule polishing in cataract surgery. Results: Average illuminance (lux) was 1.46, 0.66, 0.27, and 0.1 from 60%, 40%, 20% intensity microscope illuminations and 60% intracameral illumination. Average total spectral irradiance (µW/cm2) was 1.25, 0.65, 0.26, and 0.03 from 60%, 40%, 20% intensity microscope illuminations and 60% intracameral illumination. Conclusion: Microscope ocular illuminance and irradiance during cataract surgery were higher in the microscope illumination than in the intracameral illumination. It suggests that light exposure reaching patient's and surgeon's retina during cataract surgery is lower in the intracameral illumination than in the microscope illumination.

The effects of Engelhardtia chrysolepis Hance on long-term memory and potential dopamine involvement in mice.

Engelhardtia chrysolepis Hance (ECH) is a perennial plant used in traditional medicine. A major active ingredient of ECH is astilbin (ASB), which has recently been shown to have neuroprotective effects as well as to affect catecholamine neurotransmissions in brain areas such as the prefrontal cortex. In this study, we investigated the effects of ECH and ASB on long-term memory in mice using a battery of behavioral tests. Acute ECH treatments dose-dependently facilitated nonspatial, but not spatial, memory. ECH treatments also upregulated expression of tyrosine hydroxylase, the enzyme mediating catecholamine synthesis, in neuroblastoma cell culture. Acute ASB treatments similarly improved nonspatial memory, whereas chronic ASB treatments improved both nonspatial and spatial memory. In accordance with such behavioral effects, the increased ratio of tissue concentrations of dopamine metabolites over dopamine in striatal regions was observed in mice with chronic ASB treatments. These results suggest that ECH and its active ingredient ASB may facilitate long-term memory by modulating catecholamine transmission.

Efficacy of 4-Haptic Bitoric Intraocular Lens Implantation in Asian Patients with Cataract and Astigmatism.

PURPOSE: To determine the efficacy of 4-haptic bitoric intraocular lens (IOL) implantation in Asian patients with cataract and astigmatism. METHODS: A total of 19 eyes with ≤25.0 mm axial length and ≥0.75 diopters (D) corneal astigmatism were included in this prospective non-comparative study. All subjects underwent phacoemulsification with implantation of an AT Torbi 709M IOL. Visual and refractive outcomes as well as toric IOL axis were evaluated during a 3-month follow-up. Errors in predicted residual spherical equivalent were calculated by subtracting predicted residual spherical equivalent from postoperative refraction. RESULTS: Uncorrected and corrected distance visual acuity improved significantly 3 months after surgery, from 0.43 to 0.05 and from 0.24 to -0.05, respectively. Mean refractive cylinders also decreased significantly, from -1.91 preoperatively to -0.54 D 3 months after surgery. Mean J0 and J45 decreased 3 months postoperatively, from 0.26 to 0.03 D and from 0.24 to -0.06 D, respectively. After 3 months, mean absolute IOL rotation was 1.81°. Errors in predicted residual spherical equivalent showed a hyperopic shift of 0.35 D. CONCLUSIONS: Implantation of 4-haptic bitoric IOL proved to be effective for correcting astigmatism in Asian eyes during cataract surgery.

Intraoperative Complications of Cataract Surgery Using Intracameral Illumination in the Elderly over 75 Years.

PURPOSE: To evaluate intraoperative complications and utilization of adjunctive devices between microscope and intracameral illuminations during cataract surgery in the elderly over 75 years. DESIGN: A retrospective, consecutive, interventional case series Participants. Two hundred eighty-six eyes of 184 patients older than 75 years who underwent cataract surgery using microscope and intracameral illuminations. METHODS: A chart review was performed on an advanced cataract surgery group of 141 consecutive cases in which the intracameral illumination was used and on a standard cataract surgery group of 145 consecutive cases in which the intracameral illumination was not used. MAIN OUTCOME MEASURES: Intraoperative complications (posterior capsule rupture, radial tear of the anterior capsule, dropped nucleus, or sulcus-implanted/sclera-fixated IOL) and utilization of adjunctive devices (pupil expansion device or anterior capsule staining). RESULTS: The frequency of use of the pupil expansion device was lower in the advanced cataract surgery group than that in the standard cataract surgery group (0.7% vs 6.9%; p=0.007). Furthermore, the rates of a posterior capsule rupture and at least one intraoperative complication were lower in the advanced cataract surgery group than those in the standard cataract surgery group (0.7% vs 4.8%; p=0.067) (0.7% vs 7.6%; p=0.004). CONCLUSIONS: In the current cohort of patients over 75 years, the rate of intraoperative complications was lower when using the intracameral illumination than that when using the conventional method. Cataract surgery using intracameral illumination would be good option for elderly people.

Air-Perfused 23-Gauge Sutureless Diabetic Vitrectomy for Control of Intraoperative Bleeding during Removal of Fibrovascular Membrane.

PURPOSE: To demonstrate the advantages and efficacy of an air-perfused membrane dissection to control intraoperative bleeding in 23-gauge sutureless vitrectomy for proliferative diabetic retinopathy with severe fibrovascular membranes. Meterials and Methods: A prospective, consecutive, interventional case series of 15 eyes that underwent air-perfused diabetic vitrectomy (air vitrectomy group) for removal of the membranes was compared with a retrospective, membrane-matched case series of 10 eyes that underwent conventional diabetic vitrectomy (conventional vitrectomy group). The main outcome measures were real vitrectomy time, intraoperative and postoperative complications, and anatomic and functional successes at the final examination. RESULTS: The incidence of intraoperative retinal tears was 30% (3/10 eyes) in the conventional vitrectomy group and 20% (3/15 eyes) in the air vitrectomy group (p > 0.05). The postoperative complications such as vitreous hemorrhage or tractional retinal detachment were not common in both groups during the 6-month follow-up (p > 0.05). In addition, the final anatomic and functional success rates did not differ significantly between the groups (p > 0.05). However, the vitrectomy time was significantly shorter in the air vitrectomy group (67.0 ± 21.8 min) than in the conventional group (84.6 ± 21.1 min) (p = 0.04). CONCLUSION: Air-perfused vitrectomy showed comparable anatomic and functional success rates and shorter surgical time, compared with conventional vitrectomy in diabetic eyes with severe fibrovascular membranes. We suppose that the shortened surgical time in the air vitrectomy group is related to less intraoperative bleeding and more efficient hemostasis.

Clinical features and outcome of corneal opacity associated with congenital glaucoma.

BACKGROUND: To investigate the clinical features of corneal opacity and the surgical outcome of penetrating keratoplasty (PK) in eyes with congenital glaucoma. METHODS: A retrospective review was made of the records from 320 eyes of 193 patients who were diagnosed with congenital glaucoma between January 1981 and January 2016. Anterior segment photographs at disease presentation were examined for the presence and severity of corneal opacity. Data on patient demographics, intraocular pressure (IOP), ocular and systemic comorbidities, ocular surgery and its outcome were collected. RESULTS: Overall, corneal opacification was observed in 248 of 320 eyes (77.5%). Out of 248 eyes with corneal opacification, 53 eyes had Haab striae alone, and 195 eyes presented with either nebulomacular corneal opacity (128 eyes, iris details visible through opacity) or leukomatous corneal opacity (67 eyes, iris details invisible through opacity). In 12 eyes with severe leukomatous corneal opacity, PK was performed at the mean age of 18.6 months (range 4-57 months). The grafts failed in 6 eyes (50%) due to endothelial rejection (4 eyes) or graft infection (2 eyes) during the mean 80.6 months of follow-up (range 15-228 months). The median survival time was 36 months. The graft failure was significantly associated with smaller corneal diameter at the time of surgery, but not with the age, IOP, combined aniridia, simultaneous glaucoma or lens surgery. CONCLUSION: Congenital glaucoma was combined with corneal opacity in 77.5%. The corneal transplant survival was 50% in eyes with congenital glaucoma and total corneal opacity.

CAR-T Cells Surface-Engineered with Drug-Encapsulated Nanoparticles Can Ameliorate Intratumoral T-cell Hypofunction.

One limiting factor of CAR T-cell therapy for treatment of solid cancers is the suppressive tumor microenvironment (TME), which inactivates the function of tumor-infiltrating lymphocytes (TIL) through the production of immunosuppressive molecules, such as adenosine. Adenosine inhibits the function of CD4+ and CD8+ T cells by binding to and activating the A2a adenosine receptor (A2aR) expressed on their surface. This suppression pathway can be blocked using the A2aR-specific small molecule antagonist SCH-58261 (SCH), but its applications have been limited owing to difficulties delivering this drug to immune cells within the TME. To overcome this limitation, we used CAR-engineered T cells as active chaperones to deliver SCH-loaded cross-linked, multilamellar liposomal vesicles (cMLV) to tumor-infiltrating T cells deep within the immune suppressive TME. Through in vitro and in vivo studies, we have demonstrated that this system can be used to effectively deliver SCH to the TME. This treatment may prevent or rescue the emergence of hypofunctional CAR-T cells within the TME. Cancer Immunol Res; 6(7); 812-24. ©2018 AACR.

Macular photostress and visual experience between microscope and intracameral illumination during cataract surgery.

PURPOSE: To evaluate macular photostress and visual experience between coaxial microscope illumination versus oblique intracameral illumination during cataract surgery. SETTING: Gachon University Gil Hospital, Incheon, South Korea. DESIGN: Prospective case series. METHODS: Consecutive patients who had cataract surgery using microscope illumination and intracameral illumination were included. The patients were asked to complete a questionnaire (seeing strong lights, feeling photophobia, feeling startled (fright) when seeing lights, seeing any colors, seeing any instruments or surgical procedures, and estimating intraoperative visual function) designed to describe their cataract surgery experience. The images projected on the retina of the model eye (rear view) with artificial opaque fragments in the anterior chamber during simulating cataract surgery were compared between the 2 illumination types. RESULTS: Sixty patients completed the questionnaire. Scores for strong lights, photophobia, fright, and color perception were significantly higher with microscope illumination than with intracameral illumination (all P < .001). More patients preferred the intracameral illumination (45 [75.0%]) to the microscope illumination (13 [21.7%]). In the rear-view images created in a model eye, only the bright microscope light in the center was seen without any lens image in the microscope illumination. However, in the intracameral illumination, the less bright light from the light pipe in the periphery and the lens fragments were seen more clearly. CONCLUSIONS: In a view of the patients' visual experience, oblique intracameral illumination caused less subjective photostress and was preferred over coaxial microscope illumination. Objective findings from the model-eye experiment correlated to the result of visual experience.

Prospective Clinical Trial of Corneal Reconstruction With Biomaterial-Free Cultured Oral Mucosal Epithelial Cell Sheets.

PURPOSE: To investigate the efficacy and safety of transplantation with biomaterial-free cultured oral mucosal epithelial cell sheets (COMECs) for ocular reconstruction in subjects with total limbal stem cell deficiency. METHODS: A prospective clinical trial (NCT02149732) was conducted in 8 subjects with total limbal stem cell deficiency after approval from the institutional review board of Seoul National University Hospital (H-0707-043-213) and the Ministry of Food and Drug Safety of Korea. COMECs were prepared in a culture system without the use of any temperature-sensitive polymers or carriers. The COMECs were transplanted without suture fixation. Four subjects underwent penetrating keratoplasty after stabilization of the COMEC transplant. Stable epithelialization, changes in visual acuity, and postoperative complications were evaluated for 6 months. Corneal cytokeratins (K) of 4 subjects who underwent penetrating keratoplasty were stained with an immunofluorescent agent. RESULTS: The ocular surface was successfully reconstructed in 6 eyes. Complete stable epithelialization was achieved within a mean of 53.6 days. Visual improvement (≥2 lines) was achieved in 62.5% of the eyes. K12 (corneal phenotype), K4, and K13 (mucosal phenotype) were well expressed in grafts after keratoplasty, whereas K1, K8, and K19 were barely expressed. No ocular infections, local tumor formation, or remarkable systemic complications were observed. Ocular reconstruction using COMECs failed in 2 eyes, which had full symblepharon in 4 quadrants. CONCLUSIONS: Transplanting biomaterial-free COMECs seems to be an efficient and safe procedure to reconstruct the ocular surface in patients who are completely limbal stem cell deficient without a full symblepharon.