• Anti- VEGF agents
• Diabetic retinopathy
• Laser photocoagulation
• Polyol pathway
• Visual impairment

• Humans
• Diabetic Retinopathy/therapy
• Diabetic Retinopathy/physiopathology
• Renin-Angiotensin System
• Animals
• Vascular Endothelial Growth Factor A/antagonists & inhibitors
• Vascular Endothelial Growth Factor A/metabolism

• SG20230005 VIT SEED GRANT(RGEMS)
• SG20230005 VIT SEED GRANT(RGEMS)
• SG20230005 VIT SEED GRANT(RGEMS)

• differentiation
• ectoderm
• induced pluripotent stem cells
• limbal deficiency
• limbal stem cells
• neuronal cell lineage
• ocular surface damage
• pluripotency

• Humans
• Induced Pluripotent Stem Cells/cytology
• Induced Pluripotent Stem Cells/metabolism
• Induced Pluripotent Stem Cells/transplantation
• Limbus Corneae/cytology
• Cell Differentiation
• Animals
• Mesenchymal Stem Cells/cytology
• Mesenchymal Stem Cells/metabolism
• Amnion/cytology
• Cell Culture Techniques/methods
• Cells, Cultured
• Fibrin
• Corneal Diseases/therapy
• Limbal Stem Cells

• Immunotherapies
• Induced pluripotent stem cells
• Personalized medicine
• Regenerative medicine
• Therapy
• Tumorigenesis

• Humans
• Induced Pluripotent Stem Cells/metabolism
• Cell Differentiation
• Carcinogenesis/metabolism
• Cell Transformation, Neoplastic/genetics
• Cell Transformation, Neoplastic/metabolism
• Neoplasms/genetics
• Neoplasms/therapy
• Neoplasms/metabolism

• Humans
• Animals
• Swine
• Corneal Diseases/surgery
• Corneal Transplantation
• Cornea
• Biomedical Engineering
• Corneal Ulcer
• Epithelium, Corneal

• cell differentiation
• cellular reprograming
• epithelial cell
• extracellular matrix
• laminin
• ocular surface
• transdifferentiation

Corneal epithelium maintains visual acuity and is regenerated by the proliferation and differentiation of limbal progenitor cells. Transplantation of human limbal progenitor cells could restore the integrity and functionality of the corneal surface in patients with limbal stem cell deficiency. However, multiple protocols are employed to differentiate human induced pluripotent stem (iPS) cells into corneal epithelium or limbal progenitor cells. The aim of this study was to optimize a protocol that uses bone morphogenetic protein 4 (BMP4) and limbal cell-specific medium. Human dermal fibroblast-derived iPS cells were differentiated into limbal progenitor cells using limbal cell-specific (PI) medium and varying doses (1, 10, and 50 ng/mL) and durations (1, 3, and 10 days) of BMP4 treatment. Differentiated human iPS cells were analyzed by real-time polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemical studies at 2 or 4 weeks after BMP4 treatment. Culturing human dermal fibroblast-derived iPS cells in limbal cell-specific medium and BMP4 gave rise to limbal progenitor and corneal epithelial-like cells. The optimal protocol of 10 ng/mL and three days of BMP4 treatment elicited significantly higher limbal progenitor marker (ABCG2, ∆Np63α) expression and less corneal epithelial cell marker (CK3, CK12) expression than the other combinations of BMP4 dose and duration. In conclusion, this study identified a successful reprogramming strategy to induce limbal progenitor cells from human iPS cells using limbal cell-specific medium and BMP4. Additionally, our experiments indicate that the optimal BMP4 dose and duration favor limbal progenitor cell differentiation over corneal epithelial cells and maintain the phenotype of limbal stem cells. These findings contribute to the development of therapies for limbal stem cell deficiency disorders.

• bone morphogenetic protein 4 (BMP4)
• cornea
• induced pluripotent stem (iPS) cells
• limbal progenitor cells
• limbal stem cell deficiency (LSCD)

• Antioxidant response
• Cigarette smoke
• Corneal epithelium
• Ocular surface
• RNA-Seq
• The main finding

• Animals
• Epithelial Cells
• Epithelium, Corneal
• Mice
• Phenotype
• Smoke/adverse effects
• Smoking
• Transcriptome

• Corneal epithelium
• Ocular surface
• Stem cell
• Tissue engineering

Persistent epithelial defects (PED), associated with limbal stem cell deficiency (LSCD), require ocular surface reconstruction with a stable corneal epithelium (CE). This study investigated CE reformation using human adipose mesenchymal stem cells (ADSC), which derived epithelial progenitors via mesenchymal-epithelial transition (MET).

STEMPRO human ADSC were cultured with specific inhibitors antagonizing glycogen synthase kinase-3 and transforming growth factor-β signaling, followed by culture under a defined progenitor cell targeted-epithelial differentiation condition to generate epithelial-like cells (MET-Epi), which were characterized for cell viability, mesenchymal, and epithelial phenotypes using immunofluorescence and flow cytometry. Tissue-engineered (TE) MET-Epi cells on fibrin gel were transplanted to corneal surface of the rat LSCD model caused by alkali injury. Epithelial healing, corneal edema, and haze grading, CE formation were assessed by fluorescein staining, slit lamp bio-microscopy, anterior segment optical coherence tomography, and immunohistochemistry.

CD73^high/CD90^high/CD105^high/CD166^high/CD14^negative/CD31^negative human ADSC underwent MET, giving viable epithelial-like progenitors expressing δNp63, CDH1 (E-cadherin), epidermal growth factor receptor, integrin-β4, and cytokeratin (CK)-5, 9. Under defined epithelial differentiation culture, these progenitors generated MET-Epi cells expressing cell junction proteins ZO1 and occludin. When transplanted onto rat corneal surface with LSCD-induced PED, TE-MET-Epi achieved more efficient epithelial healing, suppressed corneal edema, and opacities, when compared to corneas without treatment or transplanted with TE-ADSC. CE markers (CK3, 12, and CDH1) were expressed on TE-MET-Epi-transplanted corneas but not in other control groups.

Human ADSC-derived epithelial-like cells, via MET, recovered the CE from PED associated with LSCD. ADSC can be a viable adult stem cell source for potential autologous epithelial cell-based therapy for corneal surface disorders.

• Adipose mesenchymal stem cells
• Corneal epithelium
• Epithelial reconstruction
• Limbal stem cell deficiency
• Mesenchymal-epithelial transition

Keeping the integrity and transparency of the cornea is the most important issue to ensure normal vision. There are more than 10 million patients going blind due to the cornea diseases worldwide. One of the effective ways to cure corneal diseases is corneal transplantation. Currently, donations are the main source of corneas for transplantation, but immune rejection and a shortage of donor corneas are still serious problems. Graft rejection could cause transplanted cornea opacity to fail. Therefore, bioengineer-based corneas become a new source for corneal transplantation. Limbal stem cells (LSCs) are located at the basal layer in the epithelial palisades of Vogt, which serve a homeostatic function for the cornea epithelium and repair the damaged cornea. LSC-based transplantation is one of the hot topics currently. Clinical data showed that the ratio of LSCs to total candidate cells for a transplantation has a significant impact on the effectiveness of the transplantation. It indicates that it is very important to accurately identify the LSCs. To date, several putative biomarkers of LSCs have been widely reported, whereas their specificity is controversial. As reported, the identification of LSCs is based on the characteristics of stem cells, such as a nuclear-to-cytoplasm ratio (N/C) ≥ 0.7, label-retaining, and side population (SP) phenotype. Here, we review recently published data to provide an insight into the circumstances in the study of LSC biomarkers. The particularities of limbus anatomy and histochemistry, the limits of the current technology level for LSC isolation, the heterogeneity of LSCs and the influence of enzyme digestion are discussed. Practical approaches are proposed in order to overcome the difficulties in basic and applied research for LSC-specific biomarkers.

• LSC biomarker
• LSC niche
• heterogeneity
• limbal stem cell (LSC)

• Animals
• Biomarkers/metabolism
• Cell Separation
• Corneal Diseases/therapy
• Corneal Transplantation
• Epithelium, Corneal/chemistry
• Epithelium, Corneal/cytology
• Humans
• Limbus Corneae/chemistry
• Limbus Corneae/cytology
• Mice
• Models, Animal
• Regeneration
• Stem Cell Transplantation
• Stem Cells/cytology
• Stem Cells/metabolism

Understanding transcription factor (TF) regulation of limbal epithelial stem/progenitor cells (LEPCs) may aid in using non-ocular cells to regenerate the corneal surface. This study aimed to identify and characterize TF genes expressed specifically in LEPCs isolated from human donor eyes by laser capture microdissection. Using a profiling approach, preferential limbal expression was found for SoxE and SoxF genes, particularly for Sox9, which showed predominantly cytoplasmic localization in basal LEPCs and nuclear localization in suprabasal and corneal epithelial cells, indicating nucleocytoplasmic translocation and activation during LEPC proliferation and differentiation. Increased nuclear localization of Sox9 was also observed in activated LEPCs following clonal expansion and corneal epithelial wound healing. Knockdown of SOX9 expression in cultured LEPCs by RNAi led to reduced expression of progenitor cell markers, e.g. keratin 15, and increased expression of differentiation markers, e.g. keratin 3. Furthermore, SOX9 silencing significantly suppressed the proliferative capacity of LEPCs and reduced levels of glycogen synthase kinase 3 beta (GSK-3ß), a negative regulator of Wnt/ß-catenin signaling. Sox9 expression, in turn, was significantly suppressed by treatment of LEPCs with exogenous GSK-3ß inhibitors and enhanced by small molecule inhibitors of Wnt signaling. Our results suggest that Sox9 and Wnt/ß-catenin signaling cooperate in mutually repressive interactions to achieve a balance between quiescence, proliferation and differentiation of LEPCs in the limbal niche. Future molecular dissection of Sox9-Wnt interaction and mechanisms of nucleocytoplasmic shuttling of Sox9 may aid in improving the regenerative potential of LEPCs and the reprogramming of non-ocular cells for corneal surface regeneration.

• Cell transplantation
• Corneal endothelium
• Corneal epithelium
• Gene therapy
• Keratocyte
• Pluripotent stem cell
• Stem cell
• Wound healing

• Humans
• Stem Cells/metabolism
• Wound Healing/physiology

• R01 EY013431 NEI NIH HHS
• R01 EY023429 NEI NIH HHS
• R01 EY025377 NEI NIH HHS

The development of cell-based therapies using stem cells represents a significant breakthrough in the treatment of limbal stem cell deficiency (LSCD). The aim of this study was to develop a novel protocol to differentiate human embryonic stem cells (hESCs) into corneal epithelial progenitor cells (CEPCs), with similar features to primary cultured human limbal stem cells (LSCs), using a medium composed of DMEM/F12 and defined keratinocyte serum-free medium (KSFM) (1:1) under different carbon dioxide (CO₂) levels in culture. The differentiated cells exhibited a similar morphology to limbal stem cells under 5%, 7%, and 9% CO₂ and expressed the LSC markers ABCG-2 and p63; however, CK14 was only expressed in the cells cultured under 7% and 9% CO₂. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis indicated that the ABCG2, p63, and CK14 levels in the 7% CO₂ and 9% CO₂ groups were higher than those in the 5% CO₂ group and in undifferentiated hESCs (p<0.05). The highest expression of ABCG2 and p63 was exhibited in the cells cultured under 7% CO₂ at day 6 of differentiation. Western blotting indicated that the ABCG2 and p63 levels were higher at day 6 than the other time points in the 7% CO₂ and 9% CO₂ groups. The highest protein expression of ABCG2 and p63 was identified in the 7% CO₂ group. The neural cell-specific marker tubulin β3 and the epidermal marker K1/10 were also detected in the differentiated cells via immunofluorescent staining; thus, cell sorting was performed via fluorescence-activated cell sorting (FACS), and ABCG2-positive cells were isolated as CEPCs. The sorted cells formed three to four layers of epithelioid cells by airlifting culture and expressed ABCG2, p63, CK14, and CK3. In conclusion, the novel induction system conditioned by 7% CO₂ in this study may be an effective and feasible method for CEPC differentiation.

• Blotting, Western
• Cell Differentiation
• Cell Line
• Cells, Cultured
• Cornea/cytology
• Epithelium, Corneal/cytology
• Flow Cytometry
• Human Embryonic Stem Cells/cytology
• Human Embryonic Stem Cells/metabolism
• Humans
• Reverse Transcriptase Polymerase Chain Reaction

• the National Natural Science Fund of China
• the Key Scientific and Technological Project of Shandong Province

• Age-related Macular Degeneration (AMD)
• Induced Pluripotent Stem Cells (iPSCs)
• Mesenchymal Stem Cells (MSCs)
• Photoreceptors
• Retinal ganglion cells (RGCs)
• Retinal pigment epithelium (RPE)

The on-demand harvesting of cell/nanosheet constructs and their subsequent transplantation in a minimally-invasive manner are described.

Limbal stem cells (LSC) are the progenitor cells that maintain the transparency of the cornea. Limbal stem cell deficiency (LSCD) leads to corneal opacity, inflammation, scarring, and blindness. A clinical approach to treat this condition consists in LSC transplantation (LSCT) after ex vivo expansion of LSC. In unilateral LSCD, an autologous transplant is possible, but cases of bilateral LSCD require allogenic LSCT. Cadaveric donors represent the most important source of LSC allografts for treatment of bilateral LSCD when living relative donors are not available. To evaluate the suitability of aged cadaveric donors for LSCT, we compared three pools of LSC from donors of different ages (<60 years, 60–75 years, and >75 years). We evaluated graft quality in terms of percent of p63-positive (p63+) cells by immunofluorescence, colony forming efficiency, and mRNA and protein expression of p63, PAX6, Wnt7a, E-cadherin, and cytokeratin (CK) 12, CK3, and CK19. The results showed that LSC cultures from aged donors can express ≥3% of p63+ cells—considered as the minimum value for predicting favorable clinical outcomes after LSCT—suggesting that these cells could be a suitable source of LSC for transplantation. Our results also indicate the need to evaluate LSC graft quality criteria for each donor.

Regeneration of the corneal surface after an epithelial insult involves division, migration, and maturation of a specialized group of stem cells located in the limbus. Several insults, both intrinsic and extrinsic, can precipitate destruction of the delicate microenvironment of these cells, resulting in limbal stem cell deficiency (LSCD). In such cases, reepithelialization fails and conjunctival epithelium extends across the limbus, leading to vascularization, persistent epithelial defects, and chronic inflammation. In partial LSCD, conjunctival epitheliectomy, coupled with amniotic membrane transplantation, could be sufficient to restore a healthy surface. In more severe cases and in total LSCD, stem cell transplantation is currently the best curative option. Before any attempts are considered to perform a limbal stem cell transplantation procedure, the ocular surface must be optimized by controlling causative factors and comorbid conditions. These factors include adequate eyelid function or exposure, control of the ocular surface inflammatory status, and a well-lubricated ocular surface. In cases of unilateral LSCD, stem cells can be obtained from the contralateral eye. Newer techniques aim at expanding cells in vitro or in vivo in order to decrease the need for large limbal resection that may jeopardize the “healthy” eye. Patients with bilateral disease can be treated using allogeneic tissue in combination with systemic immunosuppressive therapy. Another emerging option for this subset of patients is the use of noncorneal cells such as mucosal grafts. Finally, the use of keratoprosthesis is reserved for patients who are not candidates for any of the aforementioned options, wherein the choice of the type of keratoprosthesis depends on the severity of the disease. In summary, limbal stem cell transplantation improves both vision and quality-of-life in patients with ocular surface disorders associated with LSCD, and overall, the use of autologous tissue offers the best results. Future studies aim at improving cellular expansion and finding different sources of stem cells.

• cultivated limbal epithelial transplantation (CLET)
• keratolimbal allograft (KLAL)
• limbal stem cell deficiency (LSCD)
• simple limbal epithelial transplantation (SLET)

Corneal transplantation is the most common surgical procedure amongst solid organ transplants with a high survival rate of 86% at 1-year post-grafting. This high success rate has been attributed to the immune privilege of the eye. However, mechanisms originally thought to promote immune privilege, such as the lack of antigen presenting cells and vessels in the cornea, are challenged by recent studies. Nevertheless, the immunological and physiological features of the cornea promoting a relatively weak alloimmune response is likely responsible for the high survival rate in “low-risk” settings. Furthermore, although corneal graft survival in “low-risk” recipients is favourable, the prognosis in “high-risk” recipients for corneal graft is poor. In “high-risk” grafts, the process of indirect allorecognition is accelerated by the enhanced innate and adaptive immune responses due to pre-existing inflammation and neovascularization of the host bed. This leads to the irreversible rejection of the allograft and ultimately graft failure. Many therapeutic measures are being tested in pre-clinical and clinical studies to counter the immunological challenge of “high-risk” recipients. Despite the prevailing dogma, recent data suggest that tissue matching together with use of systemic immunosuppression may increase the likelihood of graft acceptance in “high-risk” recipients. However, immunosuppressive drugs are accompanied with intolerance/side effects and toxicity, and therefore, novel cell-based therapies are in development which target host immune cells and restore immune homeostasis without significant side effect of treatment. In addition, developments in regenerative medicine may be able to solve both important short comings of allotransplantation: (1) graft rejection and ultimate graft failure; and (2) the lack of suitable donor corneas. The advances in technology and research indicate that wider therapeutic choices for patients may be available to address the worldwide problem of corneal blindness in both “low-risk” and “high-risk” hosts.

• “High-risk” grafts
• Graft rejection
• Systemic immunosuppression
• Cell-based immunomodulation
• Keratoprosthesis
• Collagen-based hydrogels

• Corneal endothelium
• Corneal epithelium
• Gene therapy
• Keratocyte
• Stem cell
• Wound healing