Propagation and Phenotypic Preservation of Rabbit Limbal Epithelial Cells on Amniotic Membrane

Wang, Der-Yuan; Hsueh, Yi‐Jen; Yang, Vivian; Chen, Jan‐Kan

doi:10.1167/iovs.03-0272

Cited by 44 publications

(43 citation statements)

References 43 publications

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“…34 Although the exact mechanism has not been fully elucidated, the 3T3 fibroblast culture system has successfully applied in the limbal epithelial cell culture. [35][36][37][38] Gruenterich et al demonstrated that the addition of 3T3 feeder cells in their cultivation system may be beneficial for preventing limbal epithelial differentiation when denuded AM is used as substrates. 20 The fact that the regenerated epithelial cells produced by the Method 1 were less stratified with poor polarity in the present study may be related to the above-mentioned notion.…”

Section: Discussionmentioning

confidence: 99%

Factors Influencing Outcomes in Cultivated Limbal Epithelial Transplantation for Chronic Cicatricial Ocular Surface Disorders

Shimazaki

Higa

Morito

et al. 2007

American Journal of Ophthalmology

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Cultivated limbal epithelial transplantation (CLET) is a recently developedsurgical method to reconstruct the ocular surface in eyes with limbal dysfunction. The concept of the CLET is to produce a limbal epithelial sheet containing progenitor cells of the corneal epithelium, and then to transplant to the ocular surface following excision of cicatricial tissues. The method was first reported by Pellegrini et al in 1997, followed by several other investigators including our group. [1][2][3][4][5][6][7][8][9][10][11][12] In our previous report in 2002, the short-term outcomes of CLET were comparable with conventional limbal epithelial transplantation. 7 We found that the method did not produce excellent outcomes in patients with severe cicatricial ocular surface disorders such as Stevens-Johnson syndrome (SJS) and chemical/thermal burns of the cornea. 7 This may be due to the fact that many of the patients had risk factors for poor epithelialization such as decreased tear production, lid abnormalities, persistent inflammation. [13][14][15] It is not clear whether the relatively poor outcomes of our previous report can be improved by the modification of the epithelial sheet preparation. There are a variety of factors that may influence the quality of the epithelial sheet, which include; source of cells, preparation of the cells, types of substrates used, cultivation medium, the use of feeder cells, and culture conditions. Our group has made some modification of the cultivation/preparation methods including the use of feeder cells, cell suspension technique and air-lifting of the cell sheet.In order to investigate whether the modification resulted in improvement in surgical outcomes, we retrospectively studied the mid-term outcomes of CLET. The mid-term outcomes of CLET for chronic cicatricial ocular surface disorders were compared with the different cultivation methods. Also, other factors were analyzed to see if they influenced the results. METHODS PatientsTwenty nine eyes of 27 patients that had CLET between June 1999 and November 2003 were retrospectively analyzed in this study. Patients consisted of 16 males and 11 females, with a mean age of 50.2 + 20.7 years (15-82 years). All eyes had total limbal dysfunction. Conjunctivalization was confirmed preoperatively by impression cytology in 10 of 27 eyes. Mean corrected visual acuity was counting fingers, and two-third of the eyes had less than 20/2000. Schirmer`s test was performed in 21 eyes and decreased tear secretion (Schirmer`s value < 5 mm) was noted in 12 eyes. As for preoperative complications, glaucoma, macular degeneration, and lid deformity were noted in 4, 1, and 3 eyes, respectively.

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Section: Discussionmentioning

confidence: 99%

Factors Influencing Outcomes in Cultivated Limbal Epithelial Transplantation for Chronic Cicatricial Ocular Surface Disorders

Shimazaki

Higa

Morito

et al. 2007

American Journal of Ophthalmology

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“…The variability in integrin staining has been very well profiled across human corneal epithelium (Schlotzer-Schrehard and Kruse, 2005). In the cornea, ␤1, ␤4, and ␣6 integrins have been implicated in adhesion to the basement membrane, migration, and maintenance of a less differentiated phenotype (Watt, 2002;Wang et al, 2003;Zhou et al, 2004). Recently, Pajoohesh-Ganji et al (2005) identified a correlation between ␤1 and ␤4 localization and slow epithelial cell cycling in the mouse model.…”

Section: Discussionmentioning

confidence: 99%

Localization of candidate stem and progenitor cell markers within the human cornea, limbus, and bulbar conjunctiva in vivo and in cell culture

Vascotto

Griffith

2006

Anat. Rec.

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Corneal diseases are some of the most prevalent causes of blindness worldwide. While the most common treatment for corneal blindness is the transplantation of cadaver corneas, expanded limbal stem cells are finding recent application. Unknown, however, is the identity of the actual repopulating stem cell fraction utilized in both treatments and the critical factors governing successful engraftment and repopulation. In order to localize potential stem cell populations in vivo, we have immunohistochemically mapped a battery of candidate stem and progenitor cell markers including c-Kit and other growth factor receptors, nuclear markers including ⌬Np63, as well as adhesion factors across the cornea and distal sclera. Cell populations that differentially and specifically stained for some of these markers include the basal and superficial limbal/conjunctival epithelium and scattered cells within the substantia propria of the bulbar conjunctiva. We have also determined that the culture of differentiated cornea epithelial cells as dissociated and explant cultures induces the expression of several markers previously characterized as candidate limbal stem cell markers. This study provides a foundation to explore candidate corneal stem cell populations. As well, we show that expression of traditional stem cell markers may not be reliable indicator of stem cell content during limbal stem cell expansion in vitro and could contribute to the variable success rates of corneal stem cell transplantation. Anat Rec Part A, 288A: 921-931, 2006.

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“…AM-based limbal explant culture has been shown to be a suitable culture system for the growth and maintenance of limbal explant outgrowth in a phenotypic characteristic similar to that of the in vivo limbal tissue (Wang et al, 2003). This culture system is therefore also suitable for studying the effect of growth factors on limbal epithelial cell growth and differentiation.…”

Section: Kgf Promotes Limbal Epithelial Outgrowth Through the Inductimentioning

confidence: 99%

The growth-promoting effect of KGF on limbal epithelial cells is mediated by upregulation of ΔNp63α through the p38 pathway

Cheng

Wang

Kao

et al. 2009

Journal of Cell Science

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Corneal epithelial stem cells are thought to reside in the limbus, the transition zoon between cornea and conjunctiva. Keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) are two paracrine factors that regulate the proliferation, migration and differentiation of the limbal epithelial cells; however, the underlying mechanisms are still poorly understood. In an ex vivo limbal explant culture, we found that KGF is a more potent growth stimulator for the epithelial outgrowth than HGF. Immunofluorescence studies of the epithelial outgrowth from cells treated with HGF or KGF showed similar expression patterns of keratin-3 and keratin-14. Interestingly, p63 was highly expressed in KGF-treated limbal epithelial sheets but not in those treated with HGF. Kinase inhibitor studies showed that induction of ΔNp63α expression by KGF is mediated via the p38 pathway. The effect of KGF on limbal epithelial outgrowth was significantly reduced when endogenous ΔNp63α was suppressed, suggesting that KGF-induced limbal epithelial outgrowth is dependent on the expression of ΔNp63α. Our findings strongly suggest that limbal keratocytes regulate limbal epithelial cell growth and differentiation through a KGF paracrine loop, with ΔNp63α expression as one of the downstream targets.

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Propagation and Phenotypic Preservation of Rabbit Limbal Epithelial Cells on Amniotic Membrane

Abstract: The epithelial cell sheet grown from a limbal explant on amniotic membrane exhibited a phenotype similar to that of the limbus, suggesting that amniotic membrane is a substrate capable of supporting the propagation and preservation of p63-positive limbal epithelial cells.

Cited by 44 publications

References 43 publications

Factors Influencing Outcomes in Cultivated Limbal Epithelial Transplantation for Chronic Cicatricial Ocular Surface Disorders

Factors Influencing Outcomes in Cultivated Limbal Epithelial Transplantation for Chronic Cicatricial Ocular Surface Disorders

Localization of candidate stem and progenitor cell markers within the human cornea, limbus, and bulbar conjunctiva in vivo and in cell culture

The growth-promoting effect of KGF on limbal epithelial cells is mediated by upregulation of ΔNp63α through the p38 pathway

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