Corneal Surface Reconstruction Research Articles

MO-0958 corneal surface reconstruction and pupil tracking for eye localization in ocular proton therapy

MO-0958 corneal surface reconstruction and pupil tracking for eye localization in ocular proton therapy

Combined umbilical cord patching with amniotic membrane graft for corneal surface reconstruction.

Umbilical cord patch (UCP) grafts have been successfully used for glaucoma shunt tube coverage and conjunctival surface reconstruction. In recent years, the technique has emerged as a novel alternative for the reconstruction of corneal perforation and descemetocele. This study aimed to evaluate the effectiveness of combined UCP grafting and human amniotic membrane (HAM) transplantation for the management of corneal perforation or descemetocele. This prospective, non-comparative, interventional case series included nine eyes of nine patients with corneal descemetoceles and 28 eyes of 28 patients with corneal perforations, all in a clinically quiescent state. UCP grafting and HAM transplantation were combined to treat all patients. We re-examined the patients daily throughout the first week, weekly for 1 month, and then monthly for the first 6 months using slit-lamp examination and anterior segment optical coherence tomography. We included 37 eyes with descemetocele or corneal perforation in a clinically quiescent state. The mean (standard deviation) ages of patients with corneal descemetocele and corneal perforation were 56.3 (18.8) years and 54.3 (18.1) years, respectively. The male-to-female ratios in patients with corneal descemetocele and corneal perforation were 56% to 44% and 61% to 39%, respectively. Postoperative corneal thickness increased significantly in eyes with descemetocele compared to preoperative values (P < 0.001). Postoperative best-corrected distance visual acuity improved significantly compared to preoperative values in eyes with descemetocele or corneal perforation (both P < 0.001), with relief of accompanying ocular symptoms. We did not observe any recurrence or complications such as rejection, infection, suture-related problems, or severe inflammation and all had a formed anterior chamber up to the final follow-up visit. Combined UCP grafting and HAM transplantation could be a promising alternative treatment for corneal perforation or descemetocele in clinically quiescent eyes, providing satisfactory reconstruction and functional outcomes. Further studies with robust designs, larger sample sizes, and longer follow-up are needed to verify the efficacy and safety of this modified surgical technique in enhancing vision and restoring anterior segment anatomical integrity in compromised corneas.

Amniotic membrane: Basic concepts, different kinds

AbstractAmniotic membrane (AM) use in ophthalmic surgery has provided an alternative for corneal and conjunctival reconstruction in many clinically challenging situations and it is currently part of our armamentarium to deal with ocular surface disease and corneal surgery. In 1910, AM was first utilized surgically as a skin graft substitute material. The first ophthalmic usage was in the 1940 s as a conjunctival substitute after removal of fibrotic tissue.The AM is a thin, avascular membrane. It is the innermost layer of the foetal membranes and is composed of three layers: the epithelium, a monolayer of cuboidal cells attached to the basal lamina by numerous hemidesmosomes; the basement membrane (BM), composed of collagen, fibronectin and laminin. and the stroma. Finally, the loose connective tissue of the stroma confers tensile strength.AMT is useful clinically due to its unique structure, biocompatible composition and subsequent biological functions. Studies on human AM have shown important biological properties: enhanced epithelialization by facilitating the migration of epithelial cells, reinforcing adhesion of basal epithelial cells, promoting epithelial differentiation maintaining the epithelial phenotype, and preventing apoptosis; inhibition of inflammation, neovascularization and scarring; and antimicrobial properties.Diverse Growth factors have been found, that may have an epithelial origin. The epithelium also provides cytokines that maintain the microenvironment of the stem cells (SCs) of the corneal epithelium. BM facilitates migration of epithelial cells, reinforces adhesion of basal epithelial cells, and prevents apoptosis. Finally, a component of the stroma suppresses signalling via TGF‐beta, which modulates the proliferation of normal corneal, conjunctival, and limbal fibroblasts, and reduces fibrosis. The stroma also contains antiinflammatory and antiangiogenic proteins and protease inhibitors that reduce stromal inflammation and ulceration.Different methods of AM preservation have been described, including Cryopreservation (the most common technique of preservation), lyophilisation and dehydration and low‐temperature vacuum evaporation.Preserved AM may be used as a graft, as a patch, or a combination of both. As a graft (epithelial‐side up) AM is used in cases of both epithelial and stromal defects. The AM replaces the missing stromal matrix and provides a BM for cells to grow on. As a patch (epithelial‐side down), the AM covers an epithelial defect without stromal loss, and is sutured to the episclera near the limbus and protects the ocular surface from external insults and provides substances that reduce inflammation and promote epithelialization. Combined transplantation utilizes both the inlay and onlay methodologies of transplantation, where the graft provides structural integrity, whilst the patch allows for the protection of the graft.Indications for AMT in Ocular Surface Diseases include (1) conjunctival reconstruction in areas with conjunctival tissue defects, (2) corneal surface reconstruction: AM as a graft provides a stroma and a BM on which normal corneal epithelium grows. AM heals corneal ulcers with stromal thinning of different aetiologies, the most common being neurotrophic corneal ulcers. Additionally, AMT can be used in cicatrizing disease associated with acute or chronic SC loss and acts as a substrate for culturing epithelial stem cells of the sclerocorneal limbus.

An Analysis of the Progression of Conjunctivalisation after Transplantation of Cultivated Corneal Epithelium.

Purpose To analyse the recurrence of superficial neovascularisation after previous corneal surface reconstruction with cultivated corneal epithelial cells. Materials and Methods Forty-eight eyes underwent autologous transplantation of cultivated corneal epithelium to treat partial or total limbal stem cell deficiency caused by chemical or thermal injury. The carrier for the epithelial sheets was a denuded amniotic membrane. Follow-up was conducted for up to 120 months. Recurrent revascularisation (measured in terms of clock hours affected) was evaluated with slit-lamp examination and the support of confocal microscopy. Results During the long-term observation, only 7 eyes had stable epithelia with no neovascularisation from the conjunctiva. Nineteen eyes developed pathologic vessels in 1 quadrant, with additional 4 eyes developing them in 2 quadrants. Twelve patients developed subtotal or total conjunctivalisation of the corneal surface. They were referred for second cultivated epithelium transplantation (3 patients), allogenic keratolimbal transplantation (7 patients), or keratoprosthesis (2 patients). Six patients withdrew consent. The use of confocal scans of up to 100 µm in resolution enabled the detection of pathologic microvasculature originating from the conjunctiva and the exclusion of stromal vascular ingrowth. Conclusions Local ingrowth of the conjunctiva is a common complication after the transplantation of cultivated epithelial cells. Severe and progressive vascularisation inevitably leads to graft failure. However, if local ingrowth stops before reaching the central cornea, the treatment even with this complication can be considered a success.

Efficacy of Modified Amnion-Assisted Conjunctival Epithelial Redirection (ACER) for Partial Limbal Stem Cell Deficiency.

Background and objectives: the aim of this study was to analyze the efficacy of a modified “amnion-assisted conjunctival epithelial redirection (ACER)” technique for the treatment of partial limbal stem cell deficiency (LSCD). Materials and methods: the medical records of three patients with partial LSCD who underwent corneal surface reconstruction with modified ACER following superficial keratectomy were retrospectively studied. Briefly, in this technique, an inner amniotic membrane (AM) layer was applied on the corneal surface to promote corneal re-epithelialization. The outer AM layer was applied as a barrier to prevent the invasion of conjunctival epithelial cells into the cornea before the corneal surface was completely covered by corneal epithelial cells derived from the remaining intact limbal stem cells. Results: in all three cases, the outer AM layer successfully kept the conjunctival epithelium away from the corneal surface and prevented an admixture of conjunctival epithelial cells with corneal epithelial cells. In all three patients, the cornea was completely re-epithelized with epithelial cells derived from the remaining healthy limbal stem cells, and a clear visual axis was maintained without recurrence for a mean follow-up period of 37.3 ± 8.6 months. Conclusions: the preliminary results suggest that modified ACER appears to be a viable option for patients with partial LSCD.

Peripheral Blood As a Source of Stem Cells for Regenerative Medicine: Emphasis Towards Corneal Epithelial Reconstruction-An In Vitro Study.

Mesenchymal stem cell-based treatments are now emerging as a therapy for corneal epithelial damage. Although bone marrow, adipose tissue and umbilical cord blood are the main sources of mesenchymal stem cells (MSCs), other tissues like the peripheral blood also harbor mesenchymal-like stem cells called peripheral blood-derived mononuclear cells (PBMNCs). These blood derived stem cells gained a lot of attention due to its minimally invasive collection and ease of isolation. In this study, the feasibility of using PBMNCs as an alternative cell source to corneal limbal stem cells envisaging corneal epithelial regeneration was evaluated. Rabbit PBMNCs were isolated using density gradient centrifugation and was evaluated for mesenchymal cell properties including stemness. PBMNCs were differentiated to corneal epithelial lineage using rabbit limbal explant conditioned media and was evaluated by immuno-cytochemistry and gene expression analysis. Further, the differentiated PBMNCs were engineered into a cell sheet using an in-house developed thermo-responsive polymer. These blood derived cells were demonstrated to have similar properties to mesenchymal stem cells. Corneal epithelial lineage commitment of PBMNCs was confirmed by the positive expression of CK3/12 marker thereby demonstrating the aptness as an alternative to limbal stem cells. These differentiated cells effectively generated an in vitro cell sheet that was then demonstrated for cell sheet transfer on an ex vivo excised rabbit eye. PBMNCs as an alternative autologous cell source for limbal stem cells is envisaged as an effective therapeutic strategy for corneal surface reconstruction especially for patients with bilateral limbal stem cell deficiency.

A Decellularized Human Corneal Scaffold for Anterior Corneal Surface Reconstruction

A Decellularized Human Corneal Scaffold for Anterior Corneal Surface Reconstruction

Ex vivo expansion of human labial mucosal epithelium for corneal surface reconstruction

AbstractPurposeTo achieve ex vivo expansion of human labial mucosal epithelium using the explant method and simplified formulation of the culture media.MethodsLabial mucosa was obtained from six patients during conjunctival fornix reconstruction after signing informed contest. The substantia propria was severed, and the remaining tissue was into 1 × 1 mm explants. Explants were cultured in EpiLife (0.06 mM Ca) and DMEM/F12 (1:1) (1.05 mM Ca) media supplemented with 5% fetal bovine serum, hydrocortisone 5 µg/mL, human recombinant epidermal growth factor (hrEGF) 10 ng/ml, insulin 5 µg/ml, and standard antifungal‐antibiotic. Besides routine monitoring, primary cells were double stained with anti‐p75/p63, anti‐integrin b1/vimentin, and anti‐ZO‐1/CK10 antibodies.ResultsExplant outgrowths had cobblestone epithelial phenotype in all samples in both media; however, cell growths were delayed in the low‐Ca medium. In the high‐Ca medium, all the cultures had more unfastened cells and debris. Integrin b1, CK10, and p75 had no expression in all the primary cultures. p63 expression was registered in 39.2% and 34.7% (median, n = 3, ns) cells for EpiLife and DMEM/F12 media respectively. Expression of vimentin was found as very low in both media. ZO‐1 expression was calculated as 5.18 and 17.5 mkm (median, n = 3, ns) length per cells for EpiLife and media respectively.ConclusionsIt was confirmed that human labial mucosal epithelium could be cultured from a small biopsy without feeder cells in the culture media contained only three specific growth factors. Better cell morphology and lowered differentiation status were obtained in the 0.06Ca medium. Thus it was considered that the cell source, culture technique, and media formulations are suitable for autologous transplantation of cultured labial mucosal epithelium in patients with limbal stem cell deficiency.

Amniotic Membrane transplantation for corneal surface reconstruction

Amniotic Membrane transplantation for corneal surface reconstruction

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells.

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

Limbal epithelial stem cells in corneal surface reconstruction

Cornea serves as the partial front barrier and major light reflection organ of the eye. The integrity of corneal surface is essential for ocular function. Injuries or congenital diseases could significantly destruct the homeostasis of the ocular surface, especially the microenvironment of limbal epithelial stem cells (LESCs), and will eventually cause dysfunction of corneal regeneration and diminish of LESCs. The loss of LESCs by different reasons are named limbal stem cell deficiency (LSCD), which is one of the leading cause of vision loss worldwide. To restore the corneal surface, LESC transplantation in the form of tissue or cell cultures is currently a viable and promising method to treat LSCD. In this review, we aim to introduce the characters and niche of LESCs, and discuss different aspects of its application in cornea surface reconstruction.

Novel implantable composite biomaterial by fibrin glue and amniotic membrane for ocular surface reconstruction.

Amniotic membrane transplantation (AMT) is considered a substantial treatment option in the management of ocular surface disorders. However, several inherent drawbacks still remain. The present study devised a novel implantable composite biomaterial of fibrin glue-double layer Amniotic membrane (AM) and evaluated the biomechanical properties and effects on corneal surface reconstruction in alkali-burned rabbit model. Biomechanic parameters were calculated by an electronic universal testing machine. Corneal alkali burning was done in the right eyes of thirty rabbits, which were randomized into three groups of ten animals each. The eyes in group 1 underwent fibrin glue-double layer AMT, the eyes in group 2 underwent ordinary single layer AMT, and the eyes in group 3 (control group) did not undergo any surgical procedure. Healing of corneal epithelial defect, extent of corneal vascularization and corneal clarity were assessed and compared at two time points. One month after surgery, animals were killed and the eyes were processed for histopathology. The fibrin glue-double layer AM composites had more ideal biomechanical properties. In fibrin glue-double layer AM group, the rate of epithelial healing, vascularization inhibition and corneal clarity was significantly better than the other two groups. Novel fibrin glue-double layer AMT with corneal alkali burns is more effective and useful for ocular surface reconstruction and has great potential applications.

Cultivation and Characterization of Limbal Epithelial Stem Cells on Contact Lenses With a Feeder Layer

Limbal epithelial sheets are used to promote corneal surface reconstruction after the detection of limbal epithelial stem cell deficiency. The aim of this study was to evaluate a novel combination of limbal stem cells (LSCs) maintained on contact lenses (CLs) in the presence of a 3T3 feeder cell layer regarding preservation of stem cell phenotype and the potential use for future in vivo transplantation. Limbal epithelial cells were isolated from rabbit cornea and cultured with 3T3 cells on CLs. The preservation of LSC phenotype was determined using p63α and ABCG2 immunostaining, whereas epithelial differentiation was evaluated using CK3 and CK19. The colony-forming assay was used to determine the percentage of LSCs in cultures. Finally, CLs seeded with PKH26-labeled LSCs were transferred to rabbit eyes after performing a surgical keratectomy, and the transition and phenotype of labeled cells on the corneal surface were evaluated in whole-mount corneas. Proliferation of individual limbal cells was observed on CLs with a 3T3 feeder cell layer, showing holoclone formation and retention of viable stem or progenitor cell phenotype. Finally, a higher transition of cultivated cells after a dual sequential CL transplantation to the ocular surface was observed, showing the preservation of the LSC phenotype in the corneal surface. Limbal cells cultivated on a CL carrier overlaying a 3T3 feeder layer are mitotically active and retain the LSC phenotype. This novel technique of using CLs as a carrier offers an easily manipulable and nonimmunogenic method for transferring LSCs for ocular surface reconstruction in patients with limbal epithelial stem cell deficiency.

Reconstruction of limbal stem cell deficient corneal surface with induced human bone marrow mesenchymal stem cells on amniotic membrane

The cornea can be damaged by a variety of clinical disorders or chemical, mechanical, and thermal injuries. The objectives of this study were to induce bone marrow mesenchymal stem cells (BMSCs) to corneal lineage, to form a tissue engineered corneal substitute (TEC) using BMSCs, and to treat corneal surface defects in a limbal stem cell deficiency model. BMSCs were induced to corneal lineage using limbal medium for 10days. Induced BMSCs demonstrated upregulation of corneal stem cell markers; β1-integrin, C/EBPδ, ABCG2, and p63, increased protein expression of CK3 and p63 significantly compared with the uninduced ones. For TEC formation, passage 1 BMSCs were trypsinized and seeded on amniotic membrane in a transwell co-culture system and were grown in limbal medium. Limbal stem cell deficiency models were induced by alkaline injury, and the TEC was implanted for 8weeks. Serial slit lamp evaluation revealed remarkable improvement in corneal regeneration in terms of corneal clarity and reduced vascularization. Histologic and optical coherence tomography analyses demonstrated comparable corneal thickness and achieved stratified epithelium with a compact stromal layer resembling that of normal cornea. CK3 and p63 were expressed in the newly regenerated cornea. In conclusion, BMSCs can be induced into corneal epithelial lineage, and these cells are viable for the formation of TEC, to be used for the reconstruction of the corneal surface in the limbal stem cell deficient model.

Evaluation of transplanted oral epithelium by confocal microscopy corelates with success rate in aniridia patients

AbstractPurpose To evaluate success of transplanted oral epithelium in corneal surface reconstruction in aniridia patientsMethods 25 patients suffered from congenital aniridia accompanied by aniridial keratopathy underwent COMET procedure. Confocal microscopy examination was performed 7, 14 and 28 days after transplantation. Number of cell layers, cell density and stability of epithelium was evaluatedResults The survival of transplanted oral epithelium is corelated with early postoperative evaluation of epithelial graftConclusion Confocal microscopy examination could be useful to predict survival time of oral epithelial graft

Dynamic Corneal Surface Mapping with Electronic Speckle Pattern Interferometry

In view of the fast advancement in ophthalmic technology and corneal surgery, there is a strong need for the comprehensive mapping and characterization techniques for corneal surface. Optical methods with precision non-contact approaches have been found to be very useful for such bio measurements. Along with the normal mapping approaches, elasticity of corneal surface has an important role in its characterization and needs to be appropriately measured or estimated for broader diagnostics and better prospective surgical results, as it has important role in the post-op corneal surface reconstruction process. Use of normal corneal topographic devices is insufficient for any intricate analysis since these devices operate at relatively moderate resolution. In the given experiment, Pulsed Electronic Speckle Pattern Interferometry has been utilized along with an excitation mechanism to measure the dynamic response of the sample cornea. A Pulsed ESPI device has been chosen for the study because of its micron-level resolution and other advantages in real-time deformation analysis. A bovine cornea has been used as a sample in the subject experiment. The dynamic response has been taken on a chart recorder and it is observed that it does show a marked deformation at a specific excitation frequency, which may be taken as a characteristic elasticity parameter for the surface of that corneal sample. It was seen that outside resonance conditions the bovine cornea was not that much deformed. Through this study, the resonance frequency and the corresponding corneal deformations are mapped and plotted in real time. In these experiments, data was acquired and processed by FRAMES plus computer analysis system. With some analysis of the results, this technique can help us to refine a more detailed corneal surface mathematical model and some preliminary work was done on this. Such modelling enhancements may be useful for finer ablative surgery planning. After further experimentation, this technique can possibly be developed for in-vivo experiments on animals and humans and then may prospectively be matured for future clinical usage.

Transplantation of Conjunctival Epithelial Cells Cultivated Ex Vivo in Patients With Total Limbal Stem Cell Deficiency

To report the outcomes of transplantation of autologous conjunctival epithelial cells cultivated ex vivo (EVCAU) in patients with total limbal stem cell deficiency (LSCD). EVCAU were cultivated on denuded human amniotic membrane and transplanted in 12 eyes of 10 patients with total LSCD. We evaluated the improvement in the defined clinical parameters of LSCD (loss of corneal epithelial transparency, superficial corneal neovascularization and epithelial irregularity/recurrent epithelial breakdown), vision acuity, impression cytology, immunocytochemical analysis (CK3/CK19), and the appearance of a regular hexagonal basal layer of cells on corneal confocal microscopy. Histologic and immunohistochemical features were studied in 3 corneal buttons of patients submitted to penetrating keratoplasty after EVCAU. Cultivated conjunctival epithelium formed 4 to 5 layers with the formation of basement membrane-like structures. Immunocytochemical analysis showed positivity for CK3, CK19, MUC5AC, Ki-67, P63, and ABCG2. The improvement of the clinical parameters for this treatment in our cohort was 10 of 12 (83.3%) in a mean follow-up time of 18.5 months (range, 15-26 months), and these eyes showed an improvement in impression cytology, immunocytochemistry, and in vivo confocal analysis. Corneal buttons showed a well-formed epithelium with 5 to 6 layers, with rare cells periodic acid-Schiff+, and positivity for CK3, CK19, P63, connexin 43, and MUC5AC. We demonstrated the preliminary results of transplantation of EVCAU for corneal surface reconstruction in cases with total LSCD. Future studies are needed to further assess the long-term efficacy of this procedure.

Isolation and characterization of equine amniotic membrane-derived mesenchymal stem cells

Recent studies have shown that mesenchymal stem cells (MSCs) are able to differentiate into multi-lineage cells such as adipocytes, chondroblasts, and osteoblasts. Amniotic membrane from whole placenta is a good source of stem cells in humans. This membrane can potentially be used for wound healing and corneal surface reconstruction. Moreover, it can be easily obtained after delivery and is usually discarded as classified waste. In the present study, we successfully isolated and characterized equine amniotic membrane-derived mesenchymal stem cells (eAM-MSCs) that were cultured and maintained in low glucose Dulbecco's modified Eagle's medium. The proliferation of eAM-MSCs was measured based on the cumulative population doubling level (CPDL). Immunophenotyping of eAM-MSCs by flow cytometry showed that the major population was of mesenchymal origin. To confirm differentiation potential, a multi-lineage differentiation assay was conducted. We found that under appropriate conditions, eAM-MSCs are capable of multi-lineage differentiation. Our results indicated that eAM-MSCs may be a good source of stem cells, making them potentially useful for veterinary regenerative medicine and cell-based therapy.

Coculture of autologous limbal and conjunctival epithelial cells to treat severe ocular surface disorders: Long-term survival analysis

Background:Cultivated limbal epithelium for reconstruction of corneal surface is a well-established procedure; however, it is not adequate for damage which also extensively involves the conjunctiva. In severe cases of ocular surface damage that warrant additional conjunctival transplantation apart from cultivated limbal stem cell transplantation, we describe the long-term survival of a novel method of cocultivating autologous limbal and conjunctival epithelium on a single substrate.Materials and Methods:Forty eyes of 39 patients with severe limbal stem cell deficiency and conjunctival scarring or symblepharon underwent transplantation of autologous cocultivated epithelium on human amniotic membrane. A ring barrier was used to segregate the central limbal and peripheral conjunctival epithelia in vitro. Patients were followed up at regular intervals to assess stability of the ocular surface, defined by absence of conjunctivalization into the central 4 mm of the cornea and absence of diffuse fluorescein staining. Penetrating keratoplasty (PKP) was subsequently performed, where indicated, in patients with surface stability.Results:The cumulative survival probability was 60% at 1 year and 45% at 4 years by Kaplan–Meier analysis (mean follow-up duration: 33 ± 29 months, range: 1–87 months). Best-corrected visual acuity improved to greater than 20/200 in 38% eyes at the last follow-up, compared with 5% eyes before surgery. Immunohistochemistry in five of the corneal buttons excised for PKP showed an epithelial phenotype similar to cornea in all five.Conclusions:Synchronous use of cultured limbal and conjunctival epithelium offers a feasible alternative and a simpler one-step surgical approach to treat severe ocular surface disorders involving limbus and conjunctiva.

Isolation and Characterization of Canine Amniotic Membrane-Derived Multipotent Stem Cells

Recent studies have shown that amniotic membrane tissue is a rich source of stem cells in humans. In clinical applications, the amniotic membrane tissue had therapeutic effects on wound healing and corneal surface reconstruction. Here, we successfully isolated and identified multipotent stem cells (MSCs) from canine amniotic membrane tissue. We cultured the canine amniotic membrane-derived multipotent stem cells (cAM-MSCs) in low glucose DMEM medium. cAM-MSCs have a fibroblast-like shape and adhere to tissue culture plastic. We characterized the immunophenotype of cAM-MSCs by flow cytometry and measured cell proliferation by the cumulative population doubling level (CPDL). We performed differentiation studies for the detection of trilineage multipotent ability, under the appropriate culture conditions. Taken together, our results show that cAM-MSCs could be a rich source of stem cells in dogs. Furthermore, cAM-MSCs may be useful as a cell therapy application for veterinary regenerative medicine.