Indumathi Mariappan

In vitro culture and expansion of human limbal epithelial cells

Limbal stem cells (LSCs) have an important role in the maintenance of the corneal surface epithelium, and autologous cultured limbal epithelial cell transplantations have contributed substantially to the treatment of the visually disabling condition known as LSC deficiency. In this protocol, we describe a method of establishing human limbal epithelial cell cultures by a feeder-free explant culture technique using a small limbal biopsy specimen and human amniotic membrane (hAM) as the culture substrate. This protocol is free of animal-derived products and involves the use of human recombinant growth factors. In addition, the recombinant cell dissociation enzyme TrypLE is used to replace trypsin and autologous serum replaces FBS. It takes ∼2 weeks to establish a confluent monolayer from which ∼3 × 106 cells can be harvested. This procedure can be adopted for both basic research purposes and clinical applications.

Simplifying corneal surface regeneration using a biodegradable synthetic membrane and limbal tissue explants

Currently, damage to the ocular surface can be repaired by transferring laboratory cultured limbal epithelial cells (LECs) to the cornea using donor human amniotic membrane as the cell carrier. We describe the development of a synthetic biodegradable membrane of Poly D,L-lactide-co-glycolide (PLGA) with a 50:50 ratio of lactide and glycolide for the delivery of both isolated LECs and of cells grown out from limbal tissue explants. Both isolated LECs and limbal explants produced confluent limbal cultures within 2 weeks of culture on the membranes without the need for fibroblast feeder layers. Outgrowth of cells from explants was promoted by the inclusion of fibrin. Membranes with cells on them broke down predictably within 4-6 weeks in vitro and the breakdown was faster for a lower molecular weight (MW) (44 kg/mol) rather than a higher MW (153 kg/mol) PLGA. Membranes could be reproducibly produced, sterilised with gamma irradiation and stored dry at -20 °C for at least 12 months, and the ability to support cell outgrowth from explants was retained. We demonstrate transfer of cells (both isolated LECs and of cells grown out from limbal explants) from the membranes to an ex vivo rabbit cornea model. Characterisations of the cells by immunohistochemistry showed both differentiated and stem cell populations. A synthetic membrane combined with limbal explants in theatre would avoid the need for tissue banked human amniotic membrane and also avoid the need for specialist laboratory facilities for LEC expansion making this more accessible to many more surgeons and patients.

Mitomycin C in dacryocystorhinostomy: the search for the right concentration and duration--a fundamental study on human nasal mucosa fibroblasts.

Purpose: To establish primary cultures of human nasal mucosal fibroblasts (HNMFs) and to test the effect of varying concentrations of mitomycin C (MMC) and treatment durations on cellular proliferation and viability of the fibroblasts. Design: Laboratory investigation. Methods: Nasal mucosa harvested from patients undergoing a dacryocystorhinostomy was used to establish primary cultures by explant culture method. Cells were expanded and frozen at every passage, and passage 3 cells were used for further experiments. The cells were then treated with different concentrations of mitomycin C (0.1–0.5 mg/ml) for different time periods (3, 5, and 10 minutes). Cell viability was checked by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cellular proliferation index was determined with bromodeoxyuridine immunostaining. Apoptotic index was measured using annexin A5 affinity assay, propidium iodide staining, and 4′,6-diamidino-2-phenylindole counterstaining. The actin cytoskeletons of fibroblasts were studied using phalloidin staining. Results: The doubling time of cultured HNMFs is approximately 24 hours. Similarly, 0.4 mg/ml beyond 5 minutes and 0.5 mg/ml concentration at all time points were lethal and caused extensive cell death when compared with controls. A concentration of 0.2 mg/ml for 3 minutes of exposure prevented cell proliferation of HNMF cells by inducing cell cycle arrest, without causing extensive apoptosis. Conclusions: The minimum effective concentration appears to be 0.2 mg/ml for 3 minutes. This in vitro study could be the starting point for further clinical and histopathologic studies to validate its clinical usefulness.

Oral epithelial cells transplanted on to corneal surface tend to adapt to the ocular phenotype.

To understand the response of oral epithelial cells, transplanted on corneal surface to the ocular cues in vivo. The corneal button obtained after penetrating keratoplasty (PK) of an eye of a patient with total limbal stem cell deficiency (LSCD), previously treated with cultured oral mucosal epithelial transplantation (COMET) was examined by immunohistochemistry for the expression of keratins, p63, p75, PAX6, Ki-67, CD31, and CD34. COMET followed by optical-PK has improved visual acuity to 20/40 and rendered a stable ocular surface. The excised corneal tissue showed the presence of stratified epithelium with vasculatures. The epithelial cells of the corneal button expressed K3, K19, Ki-67, p63, p75 and the cornea-specific PAX6 and K12. This study confirms that the oral cells, transplanted to corneal surface, survive and stably reconstruct the ocular surface. They maintain their stemness at the ectopic site and acquire some of the corneal epithelial-like characters.

Unilateral partial limbal stem cell deficiency: contralateral versus ipsilateral autologous cultivated limbal epithelial transplantation.

PURPOSE To report the outcomes of autologous cultivated limbal epithelial transplantation using the healthy part of the affected eye or the fellow eye as a source of limbal stem cells in patients with unilateral, partial limbal stem cell deficiency (LSCD). DESIGN Retrospective, nonrandomized, interventional case series. METHODS setting: L. V. Prasad Eye Institute, Hyderabad, India. study population: Patients with unilateral, partial LSCD who underwent autologous cultivated limbal epithelial transplantation between 2001 and 2011. intervention: The limbal biopsy was taken either from the healthy part of the limbus of the same eye (ipsilateral group) or from the healthy fellow eye (contralateral group). Cells were cultivated using a xeno-free explant culture technique, and cultivated cells were transplanted onto the affected surface. primary outcome measure: Success of cultivated limbal epithelial transplantation, defined as a completely epithelialized, avascular, and clinically stable corneal surface. RESULTS Seventy eyes of 70 patients were studied. The mean follow up was 17.5 ± 7 months. In 34 eyes the limbal biopsy was taken from the ipsilateral eye and in the remaining 36 eyes from the contralateral eye. Clinical success was achieved in 70.59% of eyes in the ipsilateral group and 75% of eyes in the contralateral group (P = .79). Limbal transplant survival rates at the final follow-up visit were 65.1% ± 0.09% in the ipsilateral group and 53.6% ± 0.12% in the contralateral group (P = .74). CONCLUSIONS Ocular surface restoration in partial LSCD is possible with cell-based therapy. Outcomes are similar irrespective of whether the limbal biopsy is taken from the healthy part of the ipsilateral eye or the contralateral eye.

Spatial Distribution of Niche and Stem Cells in Ex Vivo Human Limbal Cultures

Stem cells at the limbus mediate corneal epithelial regeneration and regulate normal tissue homeostasis. Ex vivo cultured limbal epithelial transplantations are being widely practiced in the treatment of limbal stem cell deficiency. In this report, we examined whether the limbal niche cells that nurture and regulate epithelial stem cells coexist in ex vivo limbal cultures. We also compared the inherent differences between explant and suspension culture systems in terms of spatial distribution of niche cells and their effect on epithelial stem cell proliferation, migration, and differentiation in vitro. We report that the stem cell content of both culture systems was similar, explaining the comparable clinical outcomes reported using these two methods. We also showed that the niche cells get expanded in culture and the nestin‐positive cells migrate at the leading edges to direct epithelial cell migration in suspension cultures, whereas they are limited to the intact niche in explant cultures. We provide evidence that C/EBPδ‐positive, p15‐positive, and quiescent, label‐retaining, early activated stem cells migrate at the leading edges to regulate epithelial cell proliferation in explant cultures, and this position effect is lost in early suspension cultures. However, in confluent suspension cultures, the stem cells and niche cells interact with each another, migrate in spiraling patterns, and self‐organize to form three‐dimensional niche‐like compartments resembling the limbal crypts and thereby reestablish the position effect. These 3D‐sphere clusters are enriched with nestin‐, vimentin‐, S100‐, and p27‐positive niche cells and p15‐, p21‐, p63α‐, C/EBPδ‐, ABCG2‐, and Pax6‐positive quiescent epithelial stem cells.

Transforming ocular surface stem cell research into successful clinical practice

It has only been a quarter of a century since the discovery of adult stem cells at the human corneo-scleral limbus. These limbal stem cells are responsible for generating a constant and unending supply of corneal epithelial cells throughout life, thus maintaining a stable and uniformly refractive corneal surface. Establishing this hitherto unknown association between ocular surface disease and limbal dysfunction helped usher in therapeutic approaches that successfully addressed blinding conditions such as ocular burns, which were previously considered incurable. Subsequent advances in ocular surface biology through basic science research have translated into innovations that have made the surgical technique of limbal stem cell transplantation simpler and more predictable. This review recapitulates the basic biology of the limbus and the rationale and principles of limbal stem cell transplantation in ocular surface disease. An evidence-based algorithm is presented, which is tailored to clinical considerations such as laterality of affliction, severity of limbal damage and concurrent need for other procedures. Additionally, novel findings in the form of factors influencing the survival and function of limbal stem cells after transplantation and the possibility of substituting limbal cells with epithelial stem cells of other lineages is also discussed. Finally this review focuses on the future directions in which both basic science and clinical research in this field is headed.

Surgical Management of Bilateral Limbal Stem Cell Deficiency

Limbal stem cell deficiency (LSCD) is now established as a distinct entity with a spectrum of clinical manifestations. Bilateral LSCD presents a unique set of challenges to the clinician dealing with ocular surface disease, due to the underlying causes, clinical presentation, and adnexal status, as well as lack of a source of autologous limbal stem cells. Various surgical modalities have been described to achieve visual rehabilitation in patients with bilateral LSCD. These can primarily be divided into cell-based therapies and implantation of keratoprostheses. In this review, the surgical options for management of bilateral LSCD, including autologous and allogeneic cell-based therapies and different types of keratoprostheses are described and classified. The indications, prerequisites, technique, results and complications of each modality are discussed. Based on the status of the ocular surface, an algorithm for choosing appropriate surgical management for vision restoration in bilateral LSCD has been proposed.

Generating minicorneal organoids from human induced pluripotent stem cells

Corneal epithelial stem cells residing within the annular limbal crypts regulate adult tissue homeostasis. Autologous limbal grafts and tissue-engineered corneal epithelial cell sheets have been widely used in the treatment of various ocular surface defects. In the case of bilateral limbal defects, pluripotent stem cell (PSC)-derived corneal epithelial cells are now being explored as an alternative to allogeneic limbal grafts. Here, we report an efficient method to generate complex three-dimensional corneal organoids from human PSCs. The eye field primordial clusters that emerged from differentiating PSCs developed into whole eyeball-like, self-organized, three-dimensional, miniature structures consisting of retinal primordia, corneal primordia, a primitive eyelid-like outer covering and ciliary margin zone-like adnexal tissues in a stepwise maturation process within 15 weeks. These minicorneal organoids recapitulate the early developmental events in vitro and display similar anatomical features and marker expression profiles to adult corneal tissues. They offer an alternative tissue source for regenerating different layers of the cornea and eliminate the need for complicated cell enrichment procedures. Summary: A new method for generating 3D corneal organoids from human pluripotent stem cells provides a detailed spatiotemporal analysis of marker expression and tissue formation during corneal development.

Enriched Cultures of Retinal Cells From BJNhem20 Human Embryonic Stem Cell Line of Indian Origin.

PURPOSE To test the retinal differentiation potential and to establish an optimized protocol for enriching retinal cells from an Indian origin, human embryonic stem cell (hESC) line, BJNhem20. METHODS The BJNhem20 cells were cultured and expanded under feeder-free culture conditions. Differentiation was initiated by embryoid body (EB) formation and were cultured on Matrigel in neural induction medium (NIM) for 1 week and further maintained in retinal differentiation medium (RDM). After 1 month, the neuro-retinal progenitor clusters located at the center of pigmented retinal patches were picked and cultured as suspended neurospheres in RDM for 3 days and subsequently on Matrigel in neuro-retinal medium. The mildly pigmented, immature retinal pigmented epithelial (RPE) cells were picked separately and cultured on Matrigel in RPE medium (RPEM). After 1 week, the confluent neuro-retinal and RPE cultures were maintained in RDM for 2 to 3 months and characterized by immunofluorescence and RT-PCR. RESULTS The BJNhem20 cells efficiently differentiated into both neuro-retinal and RPE cells. The early retinal progenitors expressed Nestin, GFAP, Pax6, Rx, MitfA, Chx10, and Otx2. Neuro-retinal cells expressed the neural markers, Map2, β-III tubulin, acetylated tubulin and photoreceptor-specific markers, Crx, rhodopsin, recoverin, calbindin, PKC, NeuroD1, RLBP1, rhodopsin kinase, PDE6A, and PDE6C. Mature RPE cells developed intense pigmentation within 3 months and showed ZO-1 and Phalloidin staining at cell-cell junctions and expressed RPE65, tyrosinase, bestrophin1, Mertk, and displayed phagocytic activity. CONCLUSIONS This study confirms the retinal differentiation potential of BJNhem20 cells and describes an optimized protocol to generate enriched populations of neuro-retinal and RPE cells.