Corneal Stem Cells Research Articles

Comparison of two kinds of conjunctival transposition for the treatment of pterygium

Objective To compare the effects of migrating the corneal limbal stem cells of 12:00o'clock and simple lateral comjunctiva gliding for treatment of pterygium.Methods A prospective randomized paired-eye trial was studied.There were 234 patients (266 eyes)with initial pterygium,and they were allocated to two groups:130 cases (143 eyes) in group A had umdergone limbal stem cell of 12:00 o'clock migration,104 cases ( 123 eyes) in group B accepted simple lateral conjunctiva gliding.They were followed up for 10 -24 months.Foreign body sensation,limbal epithelial growth and pterygium recurrence were observed.Results Four eyes of 143 eyes (2.8％) in group A and 10 eyes of 123 eyes (8.1％) in group B were recurred (P ＜ 0.001 ).Conclusion 12:00 o'clock limbal stem cell transposition has lower rate of recurrence on treatment of pterygium. Key words: Ptergium; Corneal stem cells; Transposition; Therapy

Comparison of clinical efficacy of three surgical methods in the treatment of pterygium.

To investigate and compare the efficacy of amniotic membrane transplantation, corneal limbus stem cell conjunctival transplantation and pedicle conjunctival flap transposition in the treatment of pterygium. A total of 155 patients with pterygium were retrospectively analysed: 53 cases who received amniotic membrane transplantation, 41 who underwent corneal limbus stem cells conjunctival transplantation, and 63 patients who had pedicle conjunctival flap transposition. Patients in the three groups had no significant differences in terms of age, times since surgery, or extent of pterygium extension onto the cornea (F:1.194, 0.639, 0.140 respectively; all P>0.05). A total of 137 out of 155 patients (88.4%) completed 6 months of follow up. The recurrence rates of pterygium for the three surgeries (amniotic membrane transplantation, corneal limbus stem cells conjunctival transplantation and pedicle conjunctival flap transposition) were 14.6%, 13.9% and 7.7%, respectively. No significant difference was identified when comparing the recurrence rate between any two groups (X2: 0.008, 1.211 and 0.890; P: 0.593, 0.218 and 0.276). Five patients presented with postoperative complications, including 3 cases of sub-conjunctival hemorrhage, 1 case of amniotic membrane dissolution, and 1 case of conjunctival cyst, accounting for 3.64% of all participants. These three surgical methods are equally effective in preventing the recurrence of pterygium after excision.

A Review of Surgical Techniques for Harvesting Corneal Stem Cells From Allograft Donor Tissue

A Review of Surgical Techniques for Harvesting Corneal Stem Cells From Allograft Donor Tissue

Scotblood 2011 features advances in translational medicine, haemopoietic progenitor cells and milestones of blood banking systems

Amongst the presentations featured at Scotblood 2011 were advances in diagnostic tests for antibodies to red blood cells, the establishment of an islet isolation laboratory, and the development of a clinical product for corneal stem cell transplantation. In addition, the conference comprised presentations on state-of-the-art in collection, storage, and clinical utility of haemopoietic progenitor cells. It also included a session on blood banking systems dedicated to an SNBTS colleague, the late Russell Graham. Finally, the keynote lecture was delivered by Prof. John Forsythe on behalf of the Safety of Blood, Tissues and Organs (SaBTO) members, while the Iain Cook Memorial Lecture was delivered by the recently retired SNBTS R&D director, Prof. Chris Prowse.

Laser scanning confocal microscopy-assisted obtain of limbal tissue for the ex vivo culture of human limbal epithelial stem cells and identification

Background Human limbal allograft transplantation or limbal autograft transplantation are the primary approaches to the severe corneal-blindness,but their application in clinic were limited because of the defects of donor material.With the development of tissue engineering technology,transplantation of in vitro cultured limbal epithelial stem cells is being an advanced management.Objective The aim of this work was to expand human limbal epithelial stem cells ex vivo under the guidance of confocal microscope and to lay the foundation for fabricating ex vivo cultured cell sheets.Methods Ten eyes of ten patients were examined with the Heidelberg Retina Tomography Ⅲ Rostock Cornea Module(HRT3-RCM)to elucidate the structure of the human corneoscleral limbus and to correlate limbal epithelial dimensions.According to the analysis of the images of limbal epithelia,the limbal tissues provided by Eye Bank of Henan Eye Institute were cut into suitable explants.Then,this study was conducted to expand limbal epithelial stem cells ex vivo on denuded amniotic membrane.The phenotypes of primary cultured cells were evaluated by morphology and immunofluorescent staining with antibodies for limbal epithelial stem cell markers (p63,cytokeratinl9)and differentiation markers(keratin 3,involucrin).This experimental procedure was approved by the Ethic Committee of Henan Provincial People's Hospital.The written informed consent was obtained from subjects before initiation of any examination.Results The palisade morphology of human limbus was imaged clearly on the laser scanning in vivo confocal microscopy and many hyperreflective cells were observed in palisade basal cells.The cell-island phenomenon was seen in the basement membrane under the laser scanning in vivo confocal microscopy.The oblique sections of limbus showed many papilla-like epithelial columns below the superficial limbal epithelia.Throughout the experiment duration,the epithelial cells grew well with the migration rates from limbal tissue (68.62± 16.94)％ and the migration time(5.83 ±2.04)days,which depended on the tissue freshness.Compared with the second and forth batch of tissue,the migration rates of the third and sixth batch of tissues were significantly higher(P＜0.05),and the migration time was evidently longer in the forth and sixth batch of tissue compared with the first,second,third and fifth batch(P＜0.05).The positively expressing rates in the cultured corneal stem cells were 4.05％ and 36.52％ for p63,26.07％ and 40.55％ for CK19,57.88％ and 40.81％ for K3,64.66％ and 59.19％ for involucrin.Conclusion Human limbal epithelial stem cells can be successfully and purposefully obtained from the limbal tissue based on the guidance confocal miscroscope.The cultured corneal stem cells can grow well on the denuded amniotic membrane Key words: Cornea/limbus ; stem cells ; Laser scanning confocal microscopy; Cell culture; Markers

Corneal stem cells in the eye clinic

Corneal opacity is a common cause of blindness. The majority of cases result from ulceration and scarring following infection or trauma, but in a proportion corneal epithelial stem cell (SC) deficiency leads to an inability to maintain a healthy corneal surface. This review includes systematic reviews and individual case series of treatments for corneal epithelial SC deficiency. Two techniques such as transplantation of large segments of cornea from a healthy eye and ex vivo expansion of corneal SCs in the laboratory were compared. Both have merits and their clinical outcomes are similar. The smaller biopsy in the cell expansion approach has less risk for the donor eye, which is a significant advantage. Treatment algorithms for different aetiologies of SC failure are evolving. The proportion of true corneal epithelial SCs in ex vivo culture is unclear and it is unknown whether these cells survive long term. In this study, the optimum method of cell culture and transplantation is being intensively investigated. Development of tissues using multiple cell types, genetic modification to treat hereditary corneal disorders and development of cell therapy for other eye diseases are future possibilities.

A comparative study on rabbit bone mesenchymal stem cells and human amniotic epithelial cells transplantation for rabbit limbal stem cell deficiency

Background Limbal stem cell deficiency usually leads to blindness, and traditional therapy is limited. Recent research demonstrated that bone mesenchymal stem cells ( BMSCs ) and human amniotic epithelial cells(AECs) could differentiate into many kinds of cells including corneal epithelial cells, but the outcome and effect of these cells on corneal stem cell deficiency are still unclear. Objective This study aimed to observe and compare the effects of rabbit BMSCs and human AECs transplantation for rabbit limbal stem cell deficiency. Methods Eighteen clean New Zealand rabbits were randomly divided into the amniotic stroma(AS) group, rabbit BMSCs group and human AECs group with 6 rabbits for each group. Limbal stem cell deficiency models were established by putting a piece of filter paper that had been soaked in a NaOH solution at the corneal center. Rabbit BMSCs were isolated and purified by density gradient centrifugation combined with the attachment culture method, and human AECs were collected by a sequential trypsin digestion technique,and the third generation rabbit BMSCs and the first generation human AECs were identified with RT-PCR. After that,cells were inoculated onto the denuded AS and grafted to the corneal surface of the experimental animals. Twenty-eight days after cell transplantation, the therapeutic effects were evaluated based on the corneal neovascularization and opacity scores. Corneal histopathological examination and immunohistochemistry were performed to evaluate and compare the effectiveness among AS,rabbit BMSCs and human AECs on corneal stem cell deficiency. The procedure complied with the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. Results The third generation of rabbit BMSCs grew well after 12 hours, and the first generation of human AECs formed a membrane-like monolayer after 48 hours of incubation on AS. Immunohistochemistry staining showed that, 28 days after transplantation, the surface cells of the cornea were positive for cytokeratin 3 in both the rabbit BMSCs group and human AECs group.Compared with the AS group,the corneal neovascularization and opacity grades were significantly decreased in the rabbit BMSCs group( Z=-2. 983, P =0. 003 ; Z =-2. 844, P =0. 004 ) and human AECs group ( Z =-2. 817, P =0. 005 ; Z =-2.041, P =0. 041 ). Histopathological analysis exhibited that stratified corneal epithelial-like cells formed on the corneal stroms 28 days after grafting and no signs of goblet cells and neovascularization were found. Less inflammatory cells and regular collagen fiber could be seen in the rabbit BMSCs group and human AECs group. In addition,clinical observation also revealed that the corneas were much clearer in the rabbit BMSCs group than the human A ECs group( Z =-2. 091 , P=0. 037 ), but the corneal neovascularization score was similar between them (Z = -0. 267,P=-0. 789). Conclusions Rabbit BMSCs and human AECs can differentiate into corneal epithelial-like cells on the damaged corneal surface and further demonstrate remarkable inhibitory effects on corneal neovascularization and inflammatory cells. The more dominant and prominent effect is the role played by rabbit BMSCs in the improvement of corneal transparency. Key words: Bone mesenchymal stem cell; Amniotic epithelial cell; Limbal stem cell; Corneal neovascularization; Stem cell transplantation

Stem cells of the skin and cornea: their clinical applications in regenerative medicine

The use of stem cells is of great interest for the treatment of various pathologies and ultimately for the restoration of organ function. Progress pointing towards future treatments of skin and corneal epithelial stem cell defects are reviewed, including the transplantation of living tissue-engineered substitutes. This article focuses on substitutes optimized for permanent replacement of skin and cornea. New skin substitutes for burn care are currently under development. More complex tissue-engineered skin substitutes in which stroma, adipose tissue, capillaries, and neurons are combined with the epithelium are being developed. Some dermal/epidermal substitutes have been applied to the treatment of patients. Cultured corneal epithelial cells have been characterized and more complete corneal substitutes are being designed. Long-term clinical results on the transplantation of cultured corneal stem cells for the treatment of limbal stem cell deficiency have been reported. Advances in tissue engineering for the development of substitutes that will benefit patients suffering from skin or corneal stem cell deficiencies are reviewed. These products are often a combination of cells, scaffolds and other factors. Key considerations in the development of corneal and skin substitutes for clinical applications are discussed.

ΔNp63 is an ectodermal gatekeeper of epidermal morphogenesis

p63, a member of p53 family, has a significant role in the development and maintenance of stratified epithelia. However, a persistent dispute remained over the last decade concerning the interpretation of the severe failure of p63-null embryos to develop stratified epithelia. In this study, by investigating both p63-deficient strains, we demonstrated that p63-deficient epithelia failed to develop beyond ectodermal stage as they remained a monolayer of non-proliferating cells expressing K8/K18. Importantly, in the absence of p63, corneal-epithelial commitment (which occurs at embryonic day 12.5 of mouse embryogenesis) was hampered 3 weeks before corneal stem cell renewal (that begins at P14). Taken together, these data illustrate the significant role of p63 in epithelial embryogenesis, before and independently of other functions of p63 in adult stem cells regulation. Transcriptome analysis of laser captured-embryonic tissues confirmed the latter hypothesis, demonstrating that a battery of epidermal genes that were activated in wild-type epidermis remained silent in p63-null tissues. Furthermore, we defined a subset of novel bona fide p63-induced genes orchestrating first epidermal stratification and a subset of p63-repressed mesodermal-specific genes. These data highlight the earliest recognized action of ΔNp63 in the induction epidermal morphogenesis at E11.5. In the absence of p63, a mesodermal program is activated while epidermal morphogenesis does not initiate.

Corneal Stem Cells and Their Origins: Significance in Developmental Biology

Adult corneal stem cells (SCs) have been the subject of substantial research over the past 2 decades, with promising clinical applications being devised, refined, and tried. However, there have been few studies on the early development of these cells in humans, perhaps due to ethical and practical constraints. This review highlights work that has yielded significant insights from developmental studies in the cornea and other SC repositories. This field merits further research to improve our current knowledge of the origin of SCs, their location, phenotype, function, and niche structure, as well as providing fresh insight into the pathogenesis of congenital diseases and new therapeutic avenues for treating a range of blinding corneal diseases.

Cultured human corneal epithelial stem/progenitor cells derived from the corneal limbus

Stem/progenitor cells of the human corneal epithelium are present in the human corneal limbus, and several corneal epithelial stem/progenitor cell markers have been reported. Recently, the neurotrophin family receptors were reported to be useful markers of corneal epithelial stem/progenitor cells. Therefore, we examined an enzymatic separation method for obtaining corneal epithelial stem/progenitor cells and measuring the change in the expression of low-affinity neurotrophin receptor p75 (p75(NTR)), a receptor belonging to the neurotrophin family. As a result, it was found that our separation method preserved cell viability. Furthermore, p75(NTR) was mainly observed in epithelial basal cells as were the corneal epithelial stem/progenitor markers p63 and integrin β1. p75(NTR) was also observed in the cultured cells, but its frequency decreased with passage. In conclusion, we propose that our culture method will enable the culture of corneal stem cells and that it is a useful tool for elucidating the molecular basis of the niche that is necessary for the maintenance of epithelial stem cells in the corneal limbus. Furthermore, we conclude that p75(NTR) is a useful cell marker for evaluating the characteristics of stem/progenitor cells in culture.

Differentiation Status of Limbal Epithelial Cells Cultured on Intact and Denuded Amniotic Membrane Before and After Air-Lifting

We have investigated differences in bovine limbal epithelial cell differentiation when expanded upon intact (amniotic epithelial cells and basement membrane remaining) and denuded human amniotic membrane (AM), a commonly used substrate in ophthalmic surgery for corneal stem cell transplantation. Ex vivo expansion of the epithelial cells, in supplemented media, continued for 2 weeks followed by 1 week under air-lifting conditions. Before and after air-lifting the differentiated (K3/K12 positive) and undifferentiated (K14 positive) cells were quantified by immunohistochemistry, Western blotting, and quantitative polymerase chain reaction. Limbal epithelial cells expanded upon AM formed 4-6 stratified layers, both on intact AM (iAM) and denuded AM (dAM). On dAM the proportion of differentiated cells remained unaltered after air-lifting. Within cells grown on iAM, however, the number of differentiated cells increased significantly after air-lifting. These results have important implications for both basic and clinical research. First, they show that bovine limbal epithelia can be used as an alternative source of cells for basic research investigating ex vivo limbal stem cells expansion. Second, these findings serve as a warning to clinicians that the effect of AM on transplantable cells is not fully understood; the use of iAM or dAM can produce different results in terms of the amount of differentiation, once cells are exposed to the air.

Ex Vivo Expansion of Corneal Stem Cells on Amniotic Membrane and Their Outcome

To identify the stem-cell property of the ex vivo expansion of limbal stem cells (LSCs) on amniotic membrane (AM) in culture system and after clinical transplantation. Four key factors have to be performed in the defined culture system: (1) the label-retaining cells have to be identified; (2) the cells can be serially expanded and passaged in vitro; (3) the expanded cells can be labeled by tissue-specific keratin or markers, and (3) their stem cells cannot be labeled by those keratin or markers. The ex vivo-expanded LSCs on AM were positive for p63 and ABCG2 and BrdU label-retaining studies on flat mount preparation. When the ex vivo-expanded LSCs with AM were transplanted into a subcutaneous layer of nude mice, they formed multiple layers of cells. Only the basal layer of cells was positive for p63 and BrdU. The cells over the suprabasal layers were positive for K12/K3. The pathologic studies of corneal specimen of successful LSC transplantation after penetrating keratoplasty demonstrated that P63-positive cells were noticed all over the basal layer of central cornea and AM could be identified at 10 months after LSC transplantation. These results indicate that the AM provided the niche function for cultured LSCs and maintained the limbal-like environment for the transplanted area of cornea. The survival of cases depends on the severity of the disease entity, culture technique, and maintenance of the niche environment for LSCs in the culture and after clinical transplantation.

Long-Term Phenotypic Study after Allogeneic Cultivated Corneal Limbal Epithelial Transplantation for Severe Ocular Surface Diseases

To determine the long-term epithelial lineage of origin of surgically removed grafts after allogeneic cultivated corneal limbal epithelial transplantation (CLET). Interventional case reports. We studied 2 eyes from 2 patients with total corneal stem cell destruction; 1 eye was from a patient with Stevens-Johnson syndrome and 1 eye had sustained chemical injury. Allogeneic cultivated corneal limbal epithelial sheets on human amniotic membrane (AM) were transplanted onto the ocular surface. Regrafting (1 eye, 42 months later) or penetrating keratoplasty (1 eye, 75 months later) were performed after the initial transplantation procedure for further visual rehabilitation. The excised grafts were subjected to clinical evaluation and to light- and transmission electron microscopy (TEM) examination and to immunohistochemical analysis. In clinically conjunctival grafts, TEM and immunohistochemical analysis disclosed only small areas where the original cultivated corneal epithelial cells persisted. Neighboring conjunctival epithelial cells had apparently invaded a large portion of the corneal surface (keratin 3/12(-), Muc5ac(+)). In clinically corneal grafts, transplanted allogeneic cultivated corneal epithelial cells clearly survived for a long period of time (keratin 3/12(+), Muc5ac(-)); there was no infiltration by inflammatory cells, nor was there dissolution of the AM substrate. We theorize that the process of graft opacification after allogeneic CLET is responsible for the loss of transplanted cultivated corneal epithelial cells and that this is followed by conjunctival cell invasion onto the corneal surface. The results of this study confirmed that in the clinically evaluated corneal graft, transplanted cultivated corneal epithelial cells indeed survived for a long period of time on the corneal surface and maintained ocular surface integrity, even though the transplanted cells were allogeneic.

Isoforms of p63 in corneal stem cells cultured on human amniotic membrane

The p63 gene supports stem cell proliferation and regulation in epithelial cells. In this study, corneal epithelial cells were cultured on human amniotic membrane (HAM) and investigated for p63 and its isoform genes. Human limbal biopsies obtained from cadaveric donor eyes were cultivated on intact and denuded HAM. Transactivation (TA) specific domain was positive in the limbal cells cultured over denuded HAM and negative on others. TAp63α,β,γ isoforms are negative in all the limbal cells cultured on intact, denuded and limbal tissues but not in cornel epithelial tissue. p63α isoform is present in all except on denuded HAM. αβ sharing region is not expressed only in cornel epithelial tissue. γ isoform is expressed in all the samples. ΔNp63α region is present in cells cultured over the intact HAM whereas it is negative on the cells cultured over the denuded HAM. The other isoforms such as ΔNp63β and ΔNp63γ are negative in all samples. The limbal cells cultured over the intact HAM were able to maintain high proliferative potential when compared to denuded HAM. Thus, p63 isoforms plays a biological function to retain the proliferative capacity of corneal epithelial cells and maintains the stemness when cultured on intact HAM.

Techniques for Culture and Assessment of Limbal stem Cell Grafts

The therapeutic use of limbal cultures for the permanent regeneration of corneal epithelium in patients with limbal stem cell deficiency (LSC D) has been reported in many studies. A ccording to the guidelines for good manufacturing practice (GMP), strictly regulated procedures and stringent quality control tests are now required to manipulate stem cells as “medicinal products” and make engraftment safer and eventually more successful. This paper describes techniques for optimal preparation of limbal stem cell grafts, including 1) a reliable impression cytology assay for the grading of LSCD, 2) culture methods that maintain high percentages of limbal stem cells, 3) the use of specific markers for the detection of corneal, conjunctival, and limbal stem cells, namely keratin 12, mucin 1, and ΔNp63α, and 4) assays to assess the presence of contaminants, such as murine fibroblasts, endotoxins, mycoplasmae, and viral particles, in the cultured graft. The use of some of these assays allowed us to obtain a regenerated normal corneal epithelium in approximately 80% of 166 LSCD patients who received transplants from 2004 to 2008.

Cornea and Ocular Surface Treatment

In addition to being a protective shield, the cornea represents two thirds of the eye's refractive power. Corneal pathology can affect one or all of the corneal layers, producing corneal opacity. Although full corneal thickness keratoplasty has been the standard procedure, the ideal strategy would be to replace only the damaged layer. Current difficulties in corneal transplantation, mainly immune rejection and shortage of organ supply, place more emphasis on the development of artificial corneas. Bioengineered corneas range from prosthetic devices that solely address the replacement of the corneal function, to tissue-engineered hydrogels that allow regeneration of the tissue. Recently, major advances in the biology of corneal stem cells have been achieved. However, the therapeutic use of these stem cell types has the disadvantage of needing an intact stem cell compartment, which is usually damaged. In addition, long ex vivo culture is needed to generate enough cell numbers for transplantation. In the near future, combination of advanced biomaterials with cells from abundant outer sources will allow advances in the field. For the former, magnetically aligned collagen is one of the most promising ones. For the latter, different cell types will be optimal: 1) for epithelial replacement: oral mucosal epithelium, ear epidermis, or bone marrow- mesenchymal stem cells, 2) for stromal regeneration: adipose-derived stem cells and 3) for endothelial replacement, the possibility of in vitro directed differentiation of adipose-derived stem cells towards endothelial cells provides an exciting new approach.

Human epithelial stem cells in corneal regeneration and epidermal gene therapy

Adult stem cells are cells with a high capacity for self‐renewal that can produce terminally differentiated progeny. Stem cells generate an intermediate population of committed progenitors, often referred to as transit amplifying (TA) cells, that terminally differentiate after a limited number of cell divisions. Human keratinocyte stem cells are clonogenic and are known as holoclones. Human corneal stem cells are segregated in the limbus while limbal‐derived TA cells form the corneal epithelium. Self‐renewal and proliferation and deifferentiation of limbal stem cells are regulated by the ΔNp63 (α,β and γ), C/EBPδ and Bmi1 transcription factors. Cultivated limbal stem cells generate sheets of corneal epithelium suitable for clinical application. We report long‐term (up to 10 years) clinical results obtained in an homogeneous group of 112 patients presenting with corneal opacification and visual loss due to chemical burn‐dependent limbal stem cell deficiency. The corneal epithelium and the visual acuity of these patients have been restored by grafts of autologous cultured limbal keratinocytes. Clinical results were assessed by Kaplan‐Meyer, Chi‐square, Mann Whitney, Mc‐Nemar and Kruskal‐Wallis statistical analyses.Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder. Epidermal stem cells from an adult patient affected by LAM5‐β3‐deficient JEB were transduced with a retroviral vector expressing the β3 cDNA and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient's legs. Engraftment was complete after 8 days. Synthesis and proper assembly of normal levels of functional LAM5 was observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow‐up (3.5 years) in the absence of blisters, infections, inflammation or immune response. Proviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease.

A Review of Surgical Techniques for Harvesting Corneal Stem Cells From Allograft Donor Tissue

A Review of Surgical Techniques for Harvesting Corneal Stem Cells From Allograft Donor Tissue

The therapeutie effects of corneal limbus stem cells autograft and with amntotic membrane autograft for treating stage pterygium

Objective To compare the therapeutie.effects of corneal limbus stem cells autograft and with amntotic membrane autograft for treating stage pterygium.Methods One hundrand twenty-six cases of 132 eyes were.Randomly divided into Aand B group,In sixty three cases of 68 eyes were stage pterygium resection followed by corneal limbus stem cells autograft in groupA.In sixty three cases of 64 eyes were stage pterygium resection followed by corneal limbus stem cells autograft with amniotic membrane autograft in group B.Results Only 8 eyes recurrent in A group recurrent rate 11.8%,only 3 eyes recurrent in B group recurrent rate 4.7%,There was statistically difference(P＜0.05).Conclusion Corneal limbus stem cells autograft with amniotic membrane autograft showed successful in treating active stage pterygium decreased recurrent rate increased visual ability. Key words: Stage pterygium; Corneal limbus cells autograft; Amniotic membrane autograft