Limbal Stem Cell Therapy Research Articles

Limbal stem cell therapy.

To highlight the progress and future direction of limbal stem cell (LSC) therapies for the treatment of limbal stem cell deficiency (LSCD). Direct LSC transplantation have demonstrated good long-term outcomes. Cultivated limbal epithelial transplantation (CLET) has been an alternative to treat severe to total LSCD aiming to improve the safety and efficacy of the LSC transplant. A prospective early-stage uncontrolled clinical trial shows the feasibility and safety of CLET manufactured under xenobiotic free conditions. Other cell sources for repopulating of the corneal epithelium such as mesenchymal stem cells (MSCs) and induced pluripotent stem cells are being investigated. The first clinical trials of using MSCs showed short-term results, but long-term efficacy seems to be disappointing. A better understanding of the niche function and regulation of LSC survival and proliferation will lead to the development of medical therapies to rejuvenate the residual LSCs found in a majority of eyes with LSCD in vivo. Prior efforts have been largely focused on improving LSC transplantation. Additional effort should be placed on improving the accuracy of diagnosis and staging of LSCD, and implementing standardized outcome measures which enable comparison of efficacy of different LSCD treatments for different severity of LSCD. The choice of LSCD treatment will be customized based on the severity of LSCD in the future. New approaches for managing different stages of LSCD are being developed. This concise review summarizes the progresses in LSC therapies for LSCD, underlying mechanisms, limitations, and future areas of development.

A Hyaluronan Hydrogel Scaffold for Culture of Human Oral Mucosal Epithelial Cells in Limbal Stem-Cell Therapy.

Hyaluronan (HA), a major component of the extracellular matrix, plays a key role in cell proliferation, growth, survival, polarization and differentiation. We investigated the optimization of a HA hydrogel scaffold for culture of human oral mucosal epithelial cells (OMECs) for potential application in limbal stem cell therapy. The effect of the optimized scaffold on OMEC cell sheet morphology, cell metabolic activity and expression of genes associated with stemness, adherence and cell damage was studied. The results indicate that HA hydrogels crosslinked with polyethylene glycol diacrylate (PEGDA) failed to support OMEC attachment and growth. However, HA hydrogel scaffolds dried for three days and coated with 1 mg/mL collagen IV produced a full OMEC sheet. Cell morphology was comparable to control after three weeks culture, maintaining 76% metabolic activity. Of apoptosis-related genes, the pro-apoptotic markers CASP3 and BAX2 were upregulated and downregulated, respectively, compared to control whereas the anti-apoptotic marker BCL2 was downregulated. The expression level of stemness genes ΔNp63α and ABCG2 was significantly higher than control. Genes associated with improved scar-less wound healing (integrin-αV) and protection of the ocular surface (cadherin-1) had ~3-fold increased expression. These data suggest that our optimized HA-hydrogel scaffold could enhance culture of OMEC cell sheets for use in ocular reconstruction.

"Basal Cell Migration" in Regeneration of the Corneal Wound-Bed.

In this issue of Stem Cell Reports, Park et al. (2019) describe real-time in vivo visual monitoring of keratin-14+, Confetti-labeled limbal epithelial stem cells and their progeny as they contribute to central corneal wound-healing. The authors show that corneal wounds initially heal by “basal epithelial cell migration” into the wound-bed.

Ocular Stem Cells to Treat Retinal and Corneal Disorders

Background:According to WHO, 285 million people are visually impaired out of which, 39 million are classified as blind and the remaining 246 million people have low vision which comprises of moderate vision impairment and severe vision impairment. Therapies to treat major disorders leading to visual impairment like Age-related Macular Degeneration (AMD), Stargardt’s Disease (STGD), Retinitis Pigmentosa (RP) and corneal scarring are required.In the last decade, many advances have been made to treat these disorders using stem cell therapy. For corneal damage by accidental burns, scarring or limbal stem cell deficiencies which can lead to partial or total blindness, are treated with a risky intervention like keratoplasty. To overcome issues like graft rejection caused by keratoplasty as well as have a better outcome, limbal stem cell therapy has been introduced. Similarly, Retinal Pigment Epithelium (RPE) is a supporting tissue essential in nutrient transport, production of growth factors, phagocytosis of the photoreceptors and retinol cycling.Discussion and Conlusion:Degeneration of this monolayer causes many diseases that have no prevailing treatment; however, research is being carried out to replace this simple epithelial monolayer primarily with an autologous source of cells and currently using stem cells. This review discusses the advances made in the field of ocular stem cell therapy with regards to development, cultivation and novel methods used to deliver these cells to replace the corneal and retinal epithelium as a new standard for treatment.

Scleral Ischemia in Acute Ocular Chemical Injury: Long-Term Impact on Rehabilitation With Limbal Stem Cell Therapy

This study describes the impact of scleral ischemia on globe survival in cases of acute chemical injury and the outcome of limbal stem cell transplant in these eyes. This is a single-center, retrospective case study that reviews outcomes in all patients with acute chemical injury of grades IV and above who presented within 6 weeks from injury. The period of the study is between May 2012 and May 2017. Eyes presenting without scleral ischemia underwent amniotic membrane transplantation (group A), whereas eyes presenting with scleral ischemia underwent tenonplasty with amniotic membrane transplantation (group B). Simple limbal epithelial transplantation (SLET) was performed at 6 months in all consenting patients from both these groups. The primary outcome was defined as globe preservation at 3 months, whereas the secondary outcome was defined as SLET success at 1 year. Twenty-three eyes of the 23 patients were included in our study. Of the 13 eyes in group B, 2 eyes developed phthisis and 2 eyes developed hypotony. None of the 10 eyes in group A developed hypotony or phthisis. The eyes that developed into hypotony and phthisis had presented with more extensive scleral ischemia (>180 degrees). In group B, 5 of 7 patients failed SLET. This was high compared with group A in which 2 of 8 patients failed SLET. The presence and extent of scleral ischemia are important prognostic factors when assessing recovery in eyes with acute chemical injury. The authors recommend that it be added as a poor prognostic indicator in the existing classifications of acute chemical injury.

Human Platelet Lysate as a Replacement for Fetal Bovine Serum in Limbal Stem Cell Therapy

ABSTRACTPurpose: To evaluate the use of human platelet lysate (HPL) as an alternative supplement for limbal explant culture.Methods: Culture media were prepared using either 10% pooled HPL (PHPL), single donor HPL, or fetal bovine serum (FBS). Limbal tissues, obtained from the Minnesota Lions Eye Bank, were cultured in each medium on plastic plates or on denuded amniotic membrane (AM). Immunofluorescence staining was performed for ABCG2, tumor protein p63α, and cytokeratin 3 (K3). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to evaluate the expression of ABCG2 and p63. Limbal explants grown in each medium were labeled with bromodeoxyuridine (BrdU) to assess the proliferative capacity in each medium. Concentration of growth factors including epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and platelet derived growth factor (PDGF) in HPL and PHPL was compared to that in human serum (HS).Results: Immunofluorescence staining on AM showed prominent expression of ABCG2, p63α but sparse expression of K3 in HPL and PHPL supplemented medium. Real time-PCR showed 1.7 fold higher expression of ABCG2 in PHPL supplemented medium (p = 0.03), and similar expression of p63 in HPL and PHPL supplemented medium compared to FBS medium. The proliferation assay showed that LSCs retained their proliferative potential in HPL supplemented medium. Higher concentration of growth factors were found in HPL, compared to HS.Conclusions: Human platelet lysate has higher concentration of grown factors and is effective in maintaining growth and stem cell phenotype of corneal limbal explant cultures.

Concise Review: Transplantation of Cultured Oral Mucosal Epithelial Cells for Treating Limbal Stem Cell Deficiency—Current Status and Future Perspectives

A number of diseases and external factors can deplete limbal stem cells, causing pain and visual loss. Ten years have passed since the first transplantation of cultured oral mucosal epithelial cells in humans, representing the first autologous cell-based therapy for severe bilateral limbal stem cell deficiency. Its steady increase in popularity since then can be attributed to the accumulating evidence of its efficacy in reverting limbal stem cell deficiency. In this review, the focus is on clinical, and to a lesser degree laboratory, features of cultured oral mucosal epithelial transplants over the past 10 years. Comparisons with other available technologies are made. Avenues for research to stimulate further improvements in clinical results and allow worldwide distribution of limbal stem cell therapy based on oral mucosal cells are discussed. These include storage and transportation of cultured oral mucosal epithelial sheets and in vivo culture of oral mucosal epithelial cells.

Lithium increases p63 levels in cultured human limbal epithelial stem cells

AbstractPurpose p63 (isoform ΔNp63α) is a putative stemness marker for limbal epithelial stem cells (LESCs), and is essential for the proliferative potential of stem cells in stratified epithelia. The niche for LESCs is the limbus, located between the clear cornea and bulbar conjunctiva. LESCs are responsible for and able to renew and repair the corneal epithelium throughout life. In a number of disorders and after trauma, limbal stem cell deficiency (LSCD) may occur, and this can be treated by isolation and cultivation of LESCs from a healthy eye followed by transplantation to the affected eye. It is known that the percentage of p63‐bright cells affect the success rate of the transplantation. Thus, we wanted to reveal whether lithium, a known Wnt signaling inducing agent, may increase the expression of p63 in human LESC cultures.Methods Small limbal biopsies (1x1‐2mm) were dissected from donor corneas and cultured in a standard medium at 37 oC and 5% CO2 under different concentrations of lithium (LiCl2). The cells were cultured for 3‐6 weeks and medium was changed every 2‐3 days.Results The epithelial cells migrated from the limbal biopsy to form an epithelial sheet. Lithium enhanced the expression of p63 compared to controls, and this was confirmed by qRTPCR. Further studies mapping epigenetic alterations of the lithium treatment are currently conducted.Conclusion Lithium induced upregulation of p63 in LESC could be clinically important by increasing the long term success of limbal stem cell therapy.

Autologous and Allogeneic Limbal Stem Cell Therapy ‐ Where are the Limits?

AbstractAbstract not provided