Cornea - ARVO

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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneatransient amplifying (TA) progeny, we compared miRNA expressionpatterns in the stem cell-enriched limbal epithelium versus the TAcell-enriched corneal epithelium at several developmental stages.Methods: Laser capture microdissection (LCM) was used toprecisely isolate fresh populations of limbal and corneal epitheliumfrom unfixed cryosections, thereby acquiring miRNA expressionprofiles from relatively “quiescent” cells. LCM was performed onmouse limbal and corneal epithelium from postnatal (PN) mice atdays: 3, 7, 14 and 28. Total RNA was isolated from triplicate samplesand miRNA expression profiles were obtained using qRT-PCR arrays(Exiqon). We conducted loss-of-function experiments with miR-103and -107 in cultured human limbal keratinocytes (HLEKs), inconjunction with Northern, Western, and immunohistochemicalanalyses. We used luciferase reporter assays in HeLa cells to identifypotential targets of miR-103 /107.Results: miR-103 /107 were preferentially expressed in the limbalepithelium at each of the developmental time points. Northern blotanalysis revealed higher expression of miR-103 /107 in HLEKsversus human corneal epithelial keratinocytes. Treatment of HLEKswith antagomirs to either miR-103 or miR-107, resulted in an initialloss of cell-cell contacts, which yielded small, rounded cellscompared to control-treated (antago-124) HLEKs. Consistent with aloss in cell-cell contact, E cadherin (E-cad) was no longerimmunohistochemically detectable at the cell-cell junctions, 3hr postantagotreatment. A marked downregulation was noted in p120catenin and E-cad protein, essential components of adherensjunctions. NEDD9, a non-catalytic scaffolding protein that negativelyregulates localization of E-cad and promotes its degradation, wasshown to be a target of miR-103/107.Conclusions: Maintenance of cell-cell contact and communicationbetween stem cells and TA cells are crucial for stem cell nichehomeostasis. The positive role that miR-103/107 plays in E-cadherinmediatedlimbal epithelial cell-cell contacts via down-regulation ofNEDD9 helps stabilize this aspect of the stem cell niche and maintainstem cell-TA cell integrity.Commercial Relationships: Julia V. Katsnelson, None; JongKook Park, None; Wending Yang, None; Han Peng, None; RobertM. Lavker, NoneSupport: NIH Grants EY06769, EY019463Program Number: 989 Poster Board Number: B0294Presentation Time: 1:00 PM - 2:45 PMComparison of Different Limbal Epithelial Stem Cell IsolationMethods to Improve the Epithelial Sheet Quality forTransplantationSHEYLA GONZALEZ, Martin N. Nakatsu, Hua Mei, Sophie X. Deng.Jules Stein Eye Institute UCLA, Los Angeles, CA.Purpose: To investigate different preparation methods of humanlimbal stem cells (LSCs) for cultivation using dispase II, collagenaseA and the explant culture.Methods: Limbal tissues were incubated with dispase II at 37°C for2 hours (h) or with collagenase A at 37°C or 4°C for 2h or overnightto obtain limbal epithelial cell sheets. Half of the cell sheets werefurther incubated with trypsin to achieve single cell suspension. Theexplant culture was done using a 2x2 mm limbal biopsy. Limbalepithelial cells and limbal tissues were cultured on growth-arrested3T3-J2 mouse cells in SHEM5 medium. The phenotype assessmentincluded stem/progenitor morphology and expression level of LSCputative markers including ABCG2, p63α, N-cadherin andcytokeratin (K) 14, the differentiation marker K12, and the stromalcell markers vimentin (Vim) and α-smooth muscle actin by qRT-PCRand immunohistochemistry.Results: No LSC growth on 3T3-J2 cells was achieved when limbaltissues were incubated with collagenase overnight at 37°C. Amongthe other collagenase treatments tested, the most efficient wasincubation at 37°C for 2h in the incubator. This isolation methodyielded the highest K14 + Vim - progenitor cell population (86.2%) anda moderate K14 - Vim + stromal cell number (9.7%). The LSCs had thebest stem/progenitor-like morphology and the highest level ofputative LSC markers. Collagenase but not dispase isolated the entireepithelial sheet with the underneath stromal cells. When thephenotype of cultured LSCs was compared among all three culturemethods, all LSCs displayed a stem/progenitor-like morphology.However, LSCs cultured from collagenase digestion had the highestmRNA level of putative LSC markers and the highest percentage ofp63α bright cells (21.7% vs. 6% from explant and 12.7% fromdispase, P
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Aaron Yeung, None; AndrewHopkinson, None; Harminder S. Dua, NoneProgram Number: 991 Poster Board Number: B0296Presentation Time: 1:00 PM - 2:45 PMCharacterization of Biomaterial free Cell Sheets Cultured fromHuman Oral Mucosal Epithelial CellsMee Kum Kim 1, 2 , Joo Hee Lee 3 , Ja Young Lee 1, 2 , Ah Young Koh 2 ,Yun Hee Kim 3 , Won Ryang Wee 1, 2 , Saewha Jeon 3 . 1 Ophthalmology,Seoul National University Hospital, Seoul, Republic of Korea;2 Laboraory of Corneal Regenerative Medicine and OcularImmunology, Seoul Artificial Eye Center, Seoul, Republic of Korea;3 Cutigen Research Institute, Tego Science Inc., Seoul, Republic ofKorea.Purpose: To investigate the characteristics of biomaterial free cellsheets cultured from oral mucosal epithelial cells both in vitro and ina limbal deficient animal model.Methods: Both human oral mucosal epithelial cells and limbalepithelial cells were cultured to form cell sheets with or withoutfibrin glue for 2 weeks. The resulting sheets were detached using abuffer containing 1% dispase and transplanted to limbal deficientrabbits which had been chemically injured with lamellar limbectomy.The adhesion stability of these biomaterial-free cell sheets wereevaluated three days after transplantation. In parallel, ColonyForming Efficiency(CFE) as well as the immunohistochemistry usingantibodies specific to proliferation and differentiation of epithelialcells were performed to characterize the cell sheets.Results: CFE in Human oral mucosal epithelial cells and limbalepithelial cells were measured to be 57.5% and 14%, respectively.K1, K3, and K4 were expressed in mucosal epithelial sheets while K3and K12 were in limbal epithelial sheets. The cell sheets werecomposed of three to six layers of stratified, well differentiatedmucosal epithelial cells with 87.5% viable cells present. The in vivoadhesion stability of biomaterial-free cell sheets of oral mucosalepithelial cells was comparable to that of fibrin-based counterpart.Conclusions: Our results suggest that biomaterial free cell sheets oforal mucosal epithelial cells can be a good candidate in the treatmentof limbal deficient diseases.Commercial Relationships: Mee Kum Kim, None; Joo Hee Lee,None; Ja Young Lee, None; Ah Young Koh, None; Yun Hee Kim,None; Won Ryang Wee, None; Saewha Jeon, Tego Science Inc. (E)Program Number: 992 Poster Board Number: B0297Presentation Time: 1:00 PM - 2:45 PMHuman adipose-derived stem cells promote wound healing ofcorneal epithelial cells in vitroLadan Espandar 1 , Tomas Blanco 1 , Rose Mathew 1 , Natalie A.Afshari 2 , Bruce Bunnell 3 , Daniel R. Saban 1 . 1 Ophthalmology, DukeUniversity, Durham, NC; 2 University of California San Diego, SanDiego, CA; 3 Tulane University, New Orleans, LA.Purpose: Human adipose-derived stem cells (hASCs) have beenshown to promote wound healing in the skin, and may thus havepotential application for ocular surface regenerative therapy andwound healing. The purpose of this study is to investigate the effectof hASCs on corneal epithelial cells (CECs) using an in vitro woundhealing model.Methods: An established in vitro epithelial wound-healing modelwas utilized in which a 0.5 mm scratch was made on a monolayer ofconfluent human corneal epithelial cells (hCECs). hASCs seeded on atranswell membrane was added to determine their effect on epithelialwound healing. Controls included scratched CECs without theaddition of hASCs, or mitomycin-C-treated scratched hCECs. Woundclosure was evaluated by time-lapse inverted microscopy for aminimum of 24 hours. The average wound width and migration speedat seven random areas were assessed and quantified digitally usingMetaMorph® software.Results: Wound closure in the condition containing CECs alone tookas long as 18 hours, whereas the addition of hASCs decreased thistime significantly to 13 hours. Likewise, without hASCs the averagewound width (166.6 µm) was significantly higher than with hASCs(138.7 µm, p
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