Culture of Corneal Limbal Epithelial Stem Cells: Experience From Benchtop to Bedside in a Tertiary Care Hospital in India (original) (raw)
Related papers
PLoS ONE, 2012
A simple, reproducible, animal-material free method for cultivating and characterizing cornea limbal epithelial stem cells (LESCs) on human lens capsule (LC) was developed for future clinical transplantation. The limbal tissue explants (26260.25 mm) were harvested from 77 cadavers and expanded ex vivo on either cell culture plates or LC in medium containing human serum as the only growth supplement. Cell outgrowth at the edge of the explants was observed within 24 hours of cultivation and achieved viable outgrowth (.97% viability as measured by MTT assay and flow cytometry) within two weeks. The outgrowing cells were examined by genome-wide microarray including markers of stemness (p63a, ABCG2, CK19, Vimentin and Integrin a9), proliferation (Ki-67), limbal epithelial cells (CK 8/18 and 14) and differentiated cornea epithelial cells (CK 3 and 12). Immunostaining revealed the non-hematopoietic, -endothelial and -mesenchymal stem cell phenotype of the LESCs and the localization of specific markers in situ. Cell adhesion molecules, integrins and lectinbased surface carbohydrate profiling showed a specific pattern on these cells, while colony-formation assay confirmed their clonal potency. The LESCs expressed a specific surface marker fingerprint (CD117/c-kit, CXCR4, CD144/VE-Cadherin, CD146/ MCAM, CD166/ALCAM, and surface carbohydrates: WGA, ConA, RCA, PNA and AIL) which can be used for better localization of the limbal stem cell niche. In summary, we report a novel method combining the use of a medium with human serum as the only growth supplement with LC for cultivating, characterizing and expanding cornea LESCs from cadavers or alternatively from autologous donors for possible treatment of LESC deficiency.
Chapter 5 Limbal Stem Cells: Application in Ocular Biomedicine
International review of cell and molecular biology
Corneal opacification due to limbal stem cell deficiency (LSCD) is an important cause for ocular morbidity, resulting from a number of intrinsic and extrinsic factors. While the extrinsic factors include conditions such as chemical or thermal injuries, intrinsic include dysfunction, or reduction in the number of stem cells either due to pathological changes in autoimmune diseases or secondary to certain clinical conditions such as diabetes, dry eye disorders, or multiple previous eye surgeries. LSCD is characterized by a classic triad of signs—conjunctivalization, neovascularization and decrease in vision. With the increasing knowledge of limbal stem cells, the treatment of this condition has evolved from simple debridement to use of biological materials, direct transplantation of the healthy limbal tissue from the contralateral eye, or allogenic source to the use of cultivated limbal epithelial sheets. This chapter provides an update on the disease pathology, various treatment meth...
STEM CELLS, 2000
The corneal epithelium is maintained by a population of stem cells known as limbal stem cells [LSCs] due to their location in the basal layer of the outer border of the cornea known as the limbus. Treatment of limbal stem cell deficiency [LSCD] has been achieved with transplantation of ex vivo expanded LSCs taken from a small biopsy of limbus. This is a relatively new technique and as such, specific national or international guidance has yet to be established. Due to the lack of such specific guidance, our group has sought to minimise any risk to the patient by adopting certain modifications to the research methodologies in use at present. These include the replacement of all non-human animal products from the culture system and the production of all reagents and cultures under Good Manufacturing Practice [GMP] conditions. In addition, for the first time, a strictly defined uniform group of patients with total unilateral LSCD and no other significant ocular conditions has been used to allow the success or failure of treating LSCD to be attributable directly to the proposed stem cell therapy.
Biomedicines
Damage to limbal epithelial stem cells can lead to limbal stem cell deficiency (LSCD). Current autologous treatment procedures for unilateral LSCD bear a significant risk of inducing LSCD in the donor eye. This complication can be avoided by grafting a stem cell containing cultured autologous corneal epithelium (CACE). The primary objective of this study was to demonstrate the safety of CACE grafted on eyes with LSCD. The secondary objective was to assess the efficacy of a CACE graft in restoring a self-renewing corneal surface with adequate anatomic structures, as well as improving the best corrected visual acuity (BCVA). Fifteen patients were grafted with a CACE on a fibrin gel produced from a 3 mm2 limbal biopsy harvested from the donor eye. Data were collected at baseline and after grafting. Follow-ups from 1 to 5 years were conducted. No major adverse events related to the CACE graft were observed. For every visit, an anatomic score based on corneal opacity as well as central v...
Cultivated limbal stem cell transplantation for ocular surface reconstruction
Clinical ophthalmology (Auckland, N.Z.), 2008
Severe damage to cell repair mechanisms of the limbal region can lead to many disorders such as vascularized conjunctivalization, keratinization, corneal scarring, and corneal opacification, collectively described as limbal stem cell deficiency (LSCD). Limbal stem cell deficiency may occur as a result of depletion of stem cells or destruction of their stromal niche. In such cases, apart from conventional corneal transplantation, limbal stem cell transplantation would be needed to restore vision. Limbal stem cells may be replenished by autologous limbal transplants from the healthy fellow eye in unilateral cases, and allografts from living related donors or cadaveric donors in bilateral cases. The induction of iatrogenic LSCD and its sequelae in donor eyes have motivated researchers to cultivate sheets of limbal epithelium ex vivo, from small fragments of donor tissue for the purpose of ocular surface reconstruction.
IP innovative publication pvt. ltd, 2019
Purpose: Evolution of techniques of limbal stem cell transplantation for restoration of ocular surface in ocular surface disorder with limbal stem cell deficiency. Materials and Methods: It is an observational study in which 32 eyes of LSCD were observed with different surgical techniques. Outcome parameters were evaluated in terms of visual acuity, corneal clarity, symblepharon score, vascularisation. Statistical analysis was done using Annova t-test with significance level P (<0.05). Patients were followed up at the day 7, month 1, and 1-month interval thereafter. Result: The mean preoperative logMAR best-corrected visual acuity (BCVA) in CLAU, SLET, KLAU, and KLAL were 2.150.70, 2.700.20, 2.750.22 and 2.800.11 respectively which improved to 1.250.50, 2.000.42, 2.010.15 and 2.300.18 respectively after 6 months of follow up. Improvement in structural parameter were also seen in different surgical techniques. Conclusion: Patients in which CLAU and SLET technique were used, had shown better improvement in Visual and structural outcome as compared to those in which KLAU and KLAL technique were used
The Indian journal of medical research, 2008
The limbus is enriched with the stem cells of corneal epithelium. Auto- and allograft limbal transplantations are effective in restoring the corneal epithelium and inhibiting inflammation and neovascularization. Preserved human amniotic membrane (AM) is now widely used as a substrate for ocular surface reconstruction. The combination of limbal and AM transplantation has been shown to improve the surgical outcome in patients with total limbal stem cell deficiency (LSCD). The purpose of this study was to compare the expression of putative stem cell markers ATP binding cassette protein (ABCG2) and keratinocyte stem cell marker: p63 and differentiation markers. (connexin 43 and keratin 3 / keratin 12) on the limbal epithelial cells cultured over the denuded AM with and without the 3T3 murine fibroblast cells as feeder layer. Human limbal tissues obtained from the cadaveric donor eyes were cultured over the denuded human amniotic membrane in the presence of mitomycin C treated 3T3 fibrob...