Shimmura Shigeto

INTRODUCTION: Patients with limbal dysfunction, which occurs when corneal epithelial stem cells are depleted, require the transplantation of donor corneal epithelial stem cells or donor-independent cell sources. This study aimed to establish organoids with limbal epithelial progenitor cell function from the central cornea, where stem cells do not reside in vivo. We confirmed the regenerative capacity of organoids in a rabbit limbal deficiency model. METHODS: After treatment with collagenase, central corneal epithelial cells were scraped from corneal tissue and seeded onto Matrigel. For comparison, cells were collected from the limbus. The cells were cultured in Limbal Phenotype Maintenance Medium (LPMM). After 1 month, the organoids were observed in terms of number and size, immunohistochemistry, cell cycle, and colony-forming efficiency. Organoids were also transplanted into a rabbit model of limbal deficiency. RESULTS: Although we were able to form organoids from the central cornea, the number of organoids from the cornea was small (approximately one tenth compared to the limbus) after 1-month culture. Cornea-derived organoids were similar in shape and size to limbal-derived organoids, and expressed keratin 15 and p63, which are characteristics of the limbal epithelium, as well as collagen type IV, laminin, and tenascin-C, which are limbal basement membrane components. Cornea-derived organoids also showed colony forming efficiency, slow-cycling cells, and label-retaining cells. Transplanted corneal organoids were observed in the limbus of a rabbit limbal deficiency model, and the presence of organoid-derived cells extending into the host cornea was confirmed by immunohistochemistry using anti-human nuclei, -K12, -collagen type IV, and -laminin antibodies. CONCLUSIONS: Our data suggest that corneal organoids de-differentiated to gain a limbal phenotype and functionally supplied corneal epithelium in a rabbit limbal deficiency model for up to 1 month.

A first-in-human investigator-initiated clinical study of a corneal endothelial cell substitute (CLS001) derived from a clinical-grade induced pluripotent stem cell (iPSC) line shows improvement of visual acuity and corneal stromal edema, with no adverse events for up to 1 year after surgery for the treatment of bullous keratopathy. While preclinical tests, including multiple whole-genome analysis and tumorigenicity tests adhering to the Food and Drug Administration (FDA) draft guidelines, are negative, an additional whole-genome analysis conducted on transplanted CLS001 cells reveals a de novo in-frame deletion of exon22 in the EP300 gene. No adverse events related to the mutation are observed. Our study demonstrates the feasibility of using iPSC-derived cells to replace donor transplant for bullous keratopathy, while shedding light on risk management of gene mutation in cell products. Further follow-up is required for long-term analysis of clinical safety and efficacy.

PURPOSE: This study explores the application of adipose-derived mesenchymal stromal cells (adMSCs) as a therapy for ocular inflammatory diseases utilizing a chronic GVHD model. METHODS: Human adMSCs were administered via subconjunctival injection into mice with chronic ocular GVHD. Clinical scores and changes in T cell populations were analyzed. RESULTS: The study showed significant improvement in corneal integrity, including epithelial damage, opacity, thickness, and structure, after subconjunctival adMSC transplantation. Additionally, adMSC transplantation increased CD45+ and Foxp3+ Tregs while decreasing CD4+ T cells, 1IL17A+ Th17 cells, and IFNγ+ Th1 cells in local cervical lymph nodes. Moreover, adMSC-conditioned media enhanced wound closure and cell migration toward the wound bed in vitro. The cells disappeared within a week suggesting that trophic factors were involved. CONCLUSION: The dual benefit of adMSCs in immune-related ocular disorders underscores their potential for clinical application. This study focuses on subconjunctival delivery, effects of adMSCs and migration post-injection, with implications for optimizing cellular therapy application. The observed dual action, combining immunomodulation and tissue repair enhancement, underscores holistic approach of adMSC therapy in regenerative medicine, making it a potent treatment for diseases involving inflammation and tissue damage in the ocular surface.

Rationale: Patients with atopic dermatitis undergoing penetrating keratoplasty (PKP) face a high risk of postoperative complications. Endothelial keratoplasty may be a safer alternative for such patients, including those with abnormal anterior chamber anatomy. Patient concerns: 3 male patients, aged 33 to 44, presented with blurred vision at Keio University Hospital. Diagnosis: Bullous keratopathy was diagnosed through slit-lamp examination and specular microscopy. Two patients had well-controlled systemic atopic dermatitis, while 1 had blepharitis associated with atopic dermatitis. Two patients had peripheral anterior synechia, and 2 had undergone glaucoma surgery before keratoplasty. Interventions: Non-Descemet stripping endothelial keratoplasty (nDSAEK) was performed by a single surgeon. Outcomes: The best-corrected visual acuity ranged from 0.7 to 1.5 logMAR before surgery and from 0.2 to 2.3 logMAR after surgery. One year post-surgery, the graft remained clear in 2 cases; however, in the case of repeated glaucoma surgeries after nDSAEK, the graft became edematous. Corneal endothelial cell density was 1586 and 1988 cells/mm² in 2 cases and undetectable in the failed case. The follow-up period ranged from 2.5 to 9 years. Lessons: Despite the presence of peripheral anterior synechia or prior glaucoma surgery, some patients experienced a favorable long-term postoperative course following nDSAEK. This procedure may offer a safer alternative for treating patients with atopic dermatitis who have ocular complications that present a high risk for PKP.