Human Umbilical Cord Lining Cells As Novel Feeder Layer for Ex Vivo Cultivation of Limbal Epithelial Cells

Nanotechnology and Regenerative Medicine Human Umbilical Cord Lining Cells as Novel Feeder Layer for Ex Vivo Cultivation of Limbal Epithelial Cells Leonard Pek-Kiang Ang,1,2 Preeti Jain,3 Toan Thang Phan,4 and Hasan Mahmud Reza3 1Lang Eye Centre, Singapore 2Singapore National Eye Centre, Singapore 3Department of Pharmaceutical Sciences, North South University (NSU), Dhaka, Bangladesh 4Department of Surgery and Faculty of Dentistry, National University of Singapore, Singapore Correspondence: Hasan Mahmud PURPOSE. To determine the effectiveness of human umbilical cord-derived mucin-expressing Reza, Department of Pharmaceutical cord lining epithelial cells (CLEC-muc) as feeder cells in a coculture system for the cultivation Sciences, North South University, of human limbal stem cells. Bashundhara, Dhaka 1229, Bangla- desh; METHODS. Human CLEC-muc were cultured in PTTe-1 medium and treated with mitomycin C [email protected]. to arrest their growth to make the feeder layer. Single-cell suspension of limbal cells was LP-KA and HMR contributed equally prepared from corneal rim collected from the Singapore Eye Bank. Limbal cells were cultured to the work presented here and in a coculture system with CLEC-muc as well as 3T3 cells as feeder layer. We compared the should therefore be regarded as colony-forming efficiency and cell morphology of the limbal cells cultured in the two different equivalent authors. feeder layers. We also compared the expression level of several putative limbal stem cell Submitted: October 29, 2014 markers, such as HES1, ABCG2, DNP63, and BMI1, in the cultured limbal cells by Accepted: May 5, 2015 immunostaining and quantitative (q)RT-PCR. Expression of cytokeratins CK14, CK15, CK19, CK3, and CK4 was further compared. Citation: Ang LP-K, Jain P, Phan TT, Reza HM. Human umbilical cord lining RESULTS. Human limbal epithelial cells cultured in both types of feeder layers showed cells as novel feeder layer for ex vivo comparable cell morphology and colony-forming efficiency. These cells exhibited a similar cultivation of limbal epithelial cells. expression pattern of HES1, ABCG2, DNP63, BMI1, CK14, CK15, CK19, and CK3 as detected Invest Ophthalmol Vis Sci. by immunostaining and PCR. 2015;56:4697–4704. DOI:10.1167/ iovs.14-15965 CONCLUSIONS. Human CLEC-muc may be a suitable alternative to conventional mouse 3T3 feeder cells, which may reduce the risk of zoonotic infection. Keywords: cord lining epithelial cell, feeder cells, limbal cells, ocular surface tem/progenitor cells located at the basal layer of limbal replacement for 3T3 cells.9–13 We hypothesized that mucin- S epithelium play crucial role in maintenance of corneal expressing cord lining epithelial cells (CLEC-muc) could be a transparency and repairing of damaged corneal surface.1,2 Several better candidate since this cell is of human origin, is ocular surface diseases are associated with significant loss of stem nonimmunogenic, has high proliferative capacity, and can be cells, which results in a decrease in turnover of the required cryopreserved and passaged according to the need.14 CLEC- number of corneal cells.3 As a consequence, the cornea lacks muc was isolated as a novel cell type from human umbilical functional integrity and the person proceeds toward blindness. cord and is different from mesenchymal cells. This cell is Ex vivo expansion of autologous limbal epithelial stem cells or unique with regard to p63 expression.14 oral mucosal cells can produce the necessary cell population, In this study, we cultivated limbal stem cells in a coculture which has been used as a surgical graft to treat patients with total system using CLEC-muc as well as mouse 3T3 cells as feeders or partial limbal stem cell deficiency.2,4,5 This strategy, however, and compared relevant characteristics of the expanded limbal involves the use of growth-arrested murine 3T3 fibroblast feeder cells in order to assess whether CLEC-muc can be used as layers in a coculture system to support the epithelial stem/ feeder cells, avoiding zoonotic hazards. progenitor growth.6,7 As 3T3 feeder cells originate from a nonhuman source, their use in ex vivo expansion of human cells to be transplanted carries the risk of transmission of zoonotic MATERIALS AND METHODS diseases from animal feeders to human cells. It has been reported that human embryonic stem cells cultured on mouse feeder Chemical Reagents and Cell Culture Media layers generate an immunogenic nonhuman sialic acid.8 There- Dulbecco’s modified Eagle’s medium (DMEM), Ham’s F12, fore there is a growing need to substitute 3T3 cells with a human keratinocyte serum-free medium (KGM), bovine pituitary extract, equivalent to avoid xenotoxicity. human epidermal growth factor (EGF), penicillin, streptomycin, Several studies demonstrate that amniotic epithelial cells, amphotericin B, dispase, and trypsin-EDTA were purchased from human fibroblast cells, human mesenchymal stem cells, limbal Invitrogen-Gibco (Grand Island, NY, USA); Medium-171 was mesenchymal cells, and adipocytes can be challenged to purchased from Cascade Biologics (Portland, OR, USA). Insulin, replace 3T3 feeder cells in a coculture system; however, none hydrocortisone, cholera toxin, insulin-like growth factor-1 (IGF- of these candidates have been so far suggested to be an ideal 1), and rhodamine B were purchased from Sigma-Aldrich Corp. Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc. iovs.arvojournals.org j ISSN: 1552-5783 4697 Downloaded from iovs.arvojournals.org on 09/29/2021 CLEC-muc for Culture of Limbal Epithelial Cells IOVS j July 2015 j Vol. 56 j No. 8 j 4698 TABLE. Primary Antibodies and Sources Antibody Category Dilution Source ABCG2 Mouse monoclonal 1:100 Abcam, Cambridge, UK DNp63 Rabbit polyclonal 1:100 Biolegend, San Diego, CA, USA BMI1 Rabbit polyclonal 1:100 ProSci, Inc., Poway, CA, USA HES1 Rabbit polyclonal 1:200 Millipore, Temecula, CA, USA Cytokeratin 3 Mouse monoclonal 1:100 Acris, San Diego, CA, USA Cytokeratin 4 Mouse monoclonal 1:100 Acris Cytokeratin 14 Mouse monoclonal 1:100 Millipore Cytokeratin 15 Mouse monoclonal 1:100 Leica Biosystems, Newcastle, UK Cytokeratin 19 Mouse monoclonal 1:100 DakoCytomation, Glostrup, Denmark (St. Louis, MO, USA); fetal bovine serum (FBS) was purchased Feeder Cell Preparation from Hyclone (Logan, UT, USA); transforming growth factor-b1 (TGF-b1) and platelet-derived growth factor BB (PDGF-BB) were The 3T3 cells and CLEC-muc were maintained in DMEM with obtained from R&D Systems (Minneapolis, MN, USA) and 10% FBS and 1:1 medium, respectively. At 70% to 80% PeproTech (Rocky Hill, NJ, USA), respectively. Mitomycin C confluence, both types of cells were treated with 4 lg/mL was purchased from Roche Life Science (Indianapolis, IN, USA). mitomycin C for 2 hours at 378C under 5% CO2 and 95% air to Primary antibodies used in this study and their sources are listed arrest cell growth. After incubation, the cells were washed in the Table. All secondary antibodies were obtained from with PBS three times for 5 minutes each, then trypsinized using Invitrogen (Carlsbad, CA, USA), and mounting medium was 0.25% trypsin and 0.02% EDTA for 5 minutes, and replated at a purchased from DakoCytomation (Carpinteria, CA, USA); opti- density of 2.4 3 104 cells/cm2. mal cutting temperature (OCT) freezing compound (Tissue-Tek) and 40,6-diamidino-2-phenylindole (DAPI) contained in the Cultivation of Limbal Epithelial Cells mounting media (Vectashield) were purchased from Sakura Finetek (Torrance, CA, USA) and Vector Laboratories (Burlin- Human limbal rims were obtained from the Singapore Eye Bank game, CA, USA), respectively. after the central corneal button was used for corneal transplantation. After washing with PBS, the limbal rims were CLEC-muc Culture exposed to 1.2 U/mL dispase and incubated at 378C for 2 hours. The epithelial sheet was removed by gentle scraping CLEC-muc were isolated from human cord lining tissues and separated into single cells by 0.25% trypsin and 0.02% obtained from healthy women undergoing delivery after EDTA for 8 minutes. Limbal cells were plated at 3 to 4 3 104 obtaining proper informed consent as described previously.13 cells/cm2 in cell culture dishes containing mitomycin C-treated Briefly, surgical dissection of the umbilical cord was first 3T3 and CLEC-muc feeder cells. performed to separate the umbilical cord lining membrane from the enclosed Wharton’s jelly and other internal struc- tures. The isolated cord lining membrane was then divided into Bromodeoxyuridine (BrdU) ELISA Cell small squares (0.5 cm2) for cell isolation. For CLEC-muc Proliferation Assay separation/cultivation, explant tissue samples were placed on We determined proliferative capacity of CLEC-muc by BrdU- cell culture plastic surfaces and submerged in 5 mL Medium- ELISA cell proliferation assay (RPN250; Amersham Biosciences, 171 (Cascade Biologics). Outgrowing cells were harvested by Freiburg, Germany). Cells were seeded in 96-well plates at a trypsinization (0.0125% trypsin/0.05% EDTA) in Medium-171 density of 2000 cells/well with 100 lL culture medium. and cryopreserved. The cryopreserved CLEC-muc were Cultured cells were incubated with 10 lM BrdU labeling thawed and cultured in PTTe-1 medium containing Medium- solution for 20 hours at 37 C, followed by washing with 200 L 171 supplemented with 2.5% FBS, 50 lg/mL IGF-1, 50 lg/mL 8 l PDGF-BB, 5 lg/mL EGF, 2 lg/mL TGF-b1, and 5 lg/mL insulin. PBS containing 10% serum. Then the cells were fixed and We used cells from different passages (P5–P15). Serum-free incubated with 100 lL monoclonal antibody against BrdU for 2 media included KGM supplemented with 5 ng/mL human EGF, hours, followed by 100 lL peroxidase substrate per well. The 5 lg/mL insulin, 0.5 lg/mL hydrocortisone, 50 IU/mL BrdU absorbance in each well was measured directly using a penicillin, 50 lg/mL streptomycin, and 50 ng/mL amphotericin spectrophotometric microplate reader (Tecan spectrophotom- B.

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