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.

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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. Serum containing 1:1 media was prepared by mixing DMEM eter, Grodig, Austria) at a test wavelength of 450 nm. The and Ham’s F12 at a ratio of 1:1 supplemented with 5% FBS, 10 respective plain basal medium was used as negative control. ng/mL human EGF, 5 lg/mL insulin, 0.5 lg/mL hydrocortisone, The optical density (OD) reading for the negative control was 0.1 nM cholera toxin, 50 IU/mL penicillin, 50 lg/mL subtracted from the sample readings. This gave us a measure of streptomycin, and 50 ng/mL amphotericin B, while 3:1 the degree of proliferation of cells, termed the proliferation medium contained a 3:1 mixture of DMEM and Ham’s F12 index. supplemented with 10% FBS, 10 ng/mL human EGF, 5 lg/mL insulin, 0.5 lg/mL hydrocortisone, 0.1 nM cholera toxin, 50 Number of Population Doublings IU/mL penicillin, 50 lg/mL streptomycin, and 50 ng/mL amphotericin B. The cells were incubated at 378C under 5% The cells were subcultured by enzymatic disaggregation using CO2 and 95% air, with a medium change every 2 days. Cultures 0.25% trypsin/0.02% EDTA for a period of 10 minutes upon were monitored under an inverted phase-contrast microscope reaching 80% confluence. The single-cell suspensions were (Axiovert; Carl Zeiss Meditec, Inc., Oberkochen, Germany). plated at a density of 3 3 104 cells/cm2. The number of The study protocol complied with the Declaration of Helsinki population doublings, x, was calculated as follows: x ¼ log2(N/ and was approved by the Institutional Review Board of the N0), where N is the total number of cells harvested at Singapore National Eye Center and Singapore General Hospital. subculture, and N0 is the number of viable cells seeded.

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FIGURE 1. Representative phase-contrast images of CLEC-muc in different culture media (A–D). (A) Morphology of the CLEC-muc cultivated in PTTe-1, (B) 1:1 media, (C) 3:1 media, and (D) serum-free media (SFM) on day 3. Morphology and growth proﬁle of freshly collected limbal epithelial cells (P0) cultivated with 3T3 (E) and CLEC-muc (F) on day 6. Passage 1 limbal cells were further grown with 3T3 (G) and CLEC-muc (H). Experiments were performed in triplicate. Scale bar: 100 lm.

Colony Forming Efficiency (CFE) (Roche Life Science). All assays were run in duplicate for three or four individual samples. Relative expression levels were Limbal epithelial cells were seeded on 6-cm culture plates (BD calculated using the DCt method, normalizing to glyceralde- Biosciences, San Jose, CA, USA) in 1:1 medium at a density of hyde-3-phosphate dehydrogenase (GAPDH) transcription. The 500 cells per culture dish. After 10 days, the cultures were PCR products were verified by melting curve analysis and/or fixed with 4% paraformaldehyde and stained with 1% 2% agarose gel electrophoresis. The specific primers were rhodamine B. Colony formation was then assessed under a designed as follows: ABCG2, For, 50-TGGCTTAGACTCAAGCA dissecting microscope (Olympus, Miami, FL, USA). Colonies CAGC-30, Rev, 50-TCGTCCCTGCTTAGACATCC-30; BMI-1, For, having diameters larger than 2.5 mm were considered ‘‘large’’ 50-CTGGTTGCCCATTGACAGC-30, Rev, 50-CAGAAAATGAATGC while smaller ones were considered ‘‘small.’’ The CFE was GAGCCA-30; CK19, For, 50-CTGCGGGACAAGATTCTTGGT-30, determined as follows: Rev, 50-CAGAAAATGAATGCGAGCCA-30; DNp63,For,50- 0 0 Average CFE ð%Þ CTGGAAAACAATGCCCAGAC-3 , Rev, 5 -GGGTGATGGAGAGA GAGCAT-30; KRT14,For,50-TCCGCACCAAGTATGAGACA-30, Colonies Formed at the End of Growth Period 3 100 0 0 0 ¼ Rev, 5 -GGCTCTCAATCTGCATCTCC-3 ; HES1,For,5-GCGGA Total Number of Viable Cells Seeded CATTCTGGAAATGACA-30, Rev, 50-AGCGCAGCCGTCATCTG-30; GAPDH,For,50-GCCAAGGTCATCCATGACAAC-30,Rev,50- GTCCACCACCCTGTTGCTGTA-30; Tp63, Cat. no. PPH01032E Immunocytochemistry (SABiosciences, Frederick, MD, USA). Limbal cells cultivated with CLEC-muc and 3T3 feeders were fixed in cold methanol for 15 minutes at room temperature ESULTS before blocking and permeabilizing with 2% BSA in PBS with R 0.4% Triton X-100. Primary antibodies were incubated over- Morphology of CLEC-muc Cultivated in Various night at 48C in blocking buffer. After washing with PBS, cells Media were further incubated with the appropriate fluorophore- conjugated secondary antibodies (Alexa Fluor; Invitrogen) for 1 We cultivated the cells in three different serum-containing hour at room temperature and counterstained with DAPI media (Figs. 1A, 1B, 1D). In PTTe-1 medium, umbilical CLEC- contained in the mounting medium. Control experiments were muc grew as adherent cells and showed excellent proliferative performed with blocking buffer without primary antibodies. behavior. The cells mostly appeared with a cuboidal shape and Immunofluorescence images were observed on a Zeiss they remained linked to each other as found earlier (Fig. 1A).14 Axioplan 2 microscope (Carl Zeiss Meditec, Inc.). All experi- In 1:1 medium (DMEM:glycerol), cells exhibited similar ments were carried out in triplicate. proliferative capacity while they took on an elongated shape giving rise to a fibroblastic pattern of expansion (Fig. 1B). In Quantitative Reverse Transcription–Polymerase 3:1 medium, cells displayed a mixed morphology; some were Chain Reaction (qRT-PCR) cuboidal and some were elongated (Fig. 1C). In all serum- containing media, cultures became confluent shortly. We also RNAs were obtained from cells using the RNeasy Mini kit observed the growth pattern of CLEC-muc in a serum-free (Qiagen, Valencia, CA, USA), and cDNAs were generated using medium; however, the cells did not grow well in serum-free SuperScript III First Strand Kits (Invitrogen). Quantitative PCR medium. Most of the cells exhibited a thin and elongated was conducted using LightCycler 480 SYBR Green I Master kit morphology. These cells underwent limited mitosis (Fig. 1D).

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FIGURE 3. Limbal epithelial cells formed colonies in two types of feeder cells. Representative images showing colony formation with CLEC-muc (A) and 3T3 (B) as feeder cells. Graph represents the levels of large- and small-sized colonies formed in two types of feeder cells. Data are represented as mean 6 SD of three independent experiments performed in triplicate.

for 6 days and we observed the cell morphology. Limbal cells grew as colonies with distinct boundaries; some were large and some small, with a regular cuboidal shape for both types of feeder cells (Figs. 1E, 1F). These cells were subcultured after 6 FIGURE 2. Growth profiles of CLEC-muc from different donors. Graph days (P1) and again seeded on freshly prepared feeder layers, in (A) shows the levels of BrdU incorporation determined for CLEC- which further exhibited a substantial number of colonies that muc of P5 collected from five donors. Graph in (B) shows the cell maintained small, compact, and uniform cell morphology (Figs. numbers in each passage, and graph in (C) shows the number of cell 1G, 1H). In both feeder layers, we found a similar growth pattern doublings. Data are represented as mean 6 SD of three independent experiments performed in triplicate. n, donor; P, passage. of P0 and P1 limbal cells. No remarkable change in expanded cell shape and size was observed, suggesting that CLEC-muc possess appreciable potential as feeder layer to support ex vivo culture of Cell Proliferation and Cell Doubling Assay human limbal cells, which can be compared with that of the 3T3 feeder layer. We performed a cell proliferation assay to assess the proliferative capacity of the CLEC-muc collected from different donors. Results from BrdU incorporation assay with CLEC-muc Colony-Forming Efficiency from five donors indicated that the proliferative competence of To compare the capacity of CLEC-muc with 3T3 cells as feeder this cell type across the donors was very similar (Fig. 2A). We layer to aid colony formation, CFEs were studied using P1 further carried out cell doubling assay from P3 to P11 using limbal cells. With both types of feeder layers, a good number of CLEC-muc from four donors and observed no remarkable colonies with diameter larger than 2.5 mm were formed, while difference in cell doublings among the donors (12.217 6 many were smaller (Figs. 3A–C). We continued the cultures for 0.320, Figs. 2B, 2C). These results suggest that there is no 7 to 8 days and observed some giant colonies (Figs. 3A, 3B, significant batch-to-batch variation of CLEC-muc between arrows). CLEC-muc raised from a frozen stock or later-passaged different donors. cells (P15) showed a similar outcome (data not shown). These results suggest that CLEC-muc or 3T3 retain comparable Cultivation of Primary Human Limbal Epithelial potential to promote clonal growth of limbal cells. Cells With CLEC-muc and NIH/3T3 Expression of Putative Stem Cell Markers in CLEC-muc and 3T3 cells were cultured and treated with Expanded Limbal Cells mitomycin C to make feeder layers as described in Materials and Methods. Primary limbal cells (P0) were seeded on two types In order to understand the cultured cells’ retention of their of freshly prepared feeder layers. The cultures were continued traits, we performed immunocytochemistry (Fig. 4) and qRT-

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FIGURE 4. Expression profile of adult stem cell markers in limbal cells cocultured with CLEC-muc (first through third columns) and 3T3 (fourth through sixth columns) as feeder cells. Limbal cells cultured with CLEC-muc express HES1 (A–C), ABCG2 (G–I), DNP63 (M–O), and BMI1 (S–U). A similar expression pattern was observed in cells cultured with 3T3 for HES1 (D–F), ABCG2 (J–L), DNP63 (P–R), and BMI1 (V–X). Merged images are shown in columns 3 and 6. DAPI depicts cell nuclei (second, third, fifth, and sixth columns). Experiments were performed in triplicate. Scale bar: 100 lm.

PCR (Fig. 5) to determine several putative limbal stem cell Although we observed similar expression level of CK3 in the markers. Previous studies have shown that many limbal basal cultivated limbal cells with both CLEC-muc and 3T3 feeder cells that are thought to be limbal stem cells express HES1, layers (Figs. 6S–X), CK4 expression was notably less in cells ABCG2, DNP63, and BMI1.15–18 These marker genes are cultured with CLEC-muc feeder layer as compared with 3T3 essentially downregulated in differentiated corneal cells. (Figs. 6Y–Z2, 6Z3–Z5, respectively), suggesting that CLEC-muc Immunofluorescence staining revealed that cells cultivated can be a better substitute of xenologic 3T3 cells for feeder with CLEC-muc strongly expressed HES1, ABCG2, DNP63, and layer. BMI1 (Fig. 4, first through third columns). Similar patterns of expression of these molecules were also observed in cells cultured with 3T3 feeder layer (Fig. 4, fourth through sixth columns). Polymerase chain reaction detection further con- firmed the expression of HES1, ABCG2, DNP63, TP63, and BMI1 genes in cultured cells grown with both types of feeder layers (Fig. 5).

Expression of Cytokeratins in Cultivated Limbal Cells The expression of several cytokeratins at the protein level was examined in cultivated limbal epithelial cells to determine their stemness and differentiation status. We ﬁrst explored basal cytokeratins CK14, CK15, and CK19 as these cytokeratins have been previously reported to be expressed in basal epidermal and limbal/progenitor cells.14,16,19,20 Immunostaining against CK14, CK15, and CK19 antibodies revealed a positive expression of these proteins in limbal cells cocultured with CLEC-muc (Figs. 6A–C, 6G–I, 6M–O). Quantitative RT-PCR also showed consistent ﬁndings at the RNA level (Fig. 5). This result indicates that cells maintained stemness during expansion with the CLEC-muc feeder layer. A similar observation was also noted with the 3T3 feeder layer (Figs. 6D–F, 6J–L, 6P–R). We FIGURE 5. Comparison of gene expression of selected genes. DCt values of real-time RT-PCR from at least three independent experiments then studied the presence of differentiation markers CK3 and reveal similar levels of gene expression of ABCG2, BMI1, TP63, DNP63, CK4 to determine the extent of differentiation occurring in the HES1, KRT14, and CK19. Higher DCt value corresponds to lower gene cultured cells with both type of feeder layers, since this is an expression and vice versa. GAPDH was used as endogenous internal obvious phenomenon observed in a coculture system. control.

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FIGURE 6. Expression profile of cytokeratins in limbal cells cocultured with CLEC-muc (first through third columns) and 3T3 (fourth through sixth columns) as feeder cells. Limbal cells cultured with CLEC-muc express CK14 (A–C), CK15 (G–I), CK19 (M–O), and CK3 (S–U). A similar expression pattern was observed in cells cultured with 3T3 for CK14 (D–F), CK15 (J–L), CK19 (P–R), and CK3 (V–X). Merged images are shown in columns 3 and 6. Boxed areas shown in the insets depict clear expression of CK15 in the cytoplasm. However, CK4 expression was relatively weak in cells cultured with CLEC-muc (Y–Z2) as compared to that in cells cultured with 3T3 (Z3–Z5). DAPI depicts cell nuclei (second, third, fifth, and sixth columns). Experiments were performed in triplicate. Scale bar: 100 lm.

DISCUSSION limbal cells. We observed that the number and shape of the colonies developed in the two systems were comparable, The use of feeder cells appears to be vital in maintaining larger which signifies the importance of CLEC-muc as feeder cells to amounts of stem/progenitor cells for transplantation. Murine- support limbal stem/progenitor cell growth ex vivo (Fig. 3). We derived 3T3 cells are widely used as feeder layer in a coculture observed a similar growth pattern and proliferative capacity of system.21 It is perceived that feeder cells probably provide an CLEC-muc from different donors and passage numbers (Fig. 2), artificial niche in the culture system that inhibits differentiation demonstrating that CLEC-muc maintain comparable culture while favoring stem cell proliferation. Since 3T3 originates characteristics. Our current study findings are consistent with from an animal source, some ethical and safety issues arise, in our previous studies14,26 in which CLEC-muc were shown to particular when the cultured epithelial cells are to be used proliferate rapidly while maintaining their cell integrity during clinically.22–25 In order to eliminate any risk, researchers have successive passaging. Although CLEC-muc are not immortal- made various attempts to find an effective alternative to 3T3 ized cells, they can be propagated for at least 40 passages in feeder cells. Human placenta-derived fibroblast-like cells (HPC) vitro without a significant loss of proliferative capacity or originating from chorionic villi of women and primary human multipotency.26 amnion epithelial cells (hAEC) have been implicated in culture Although bona fide limbal stem cell markers are yet to be of mouse embryonic stem cells.9 Another study has revealed defined, to assess the quality of the cultivated limbal cells in successful undifferentiated growth of primate ES cells on terms of gene expression pattern, we examined the expression human amniotic epithelial (HAE) feeder cells.10 Autologous of several putative stem cell markers and other genes. It is fibroblasts as feeder cells in a rabbit alkali burn model expected that limbal stem cells cultivated ex vivo should retain suppressed peripheral neovascularization but promoted regu- their stem cell properties to contribute long-term as stem cells lar epithelial cell proliferation.12 A recent study has demon- grafted in a living system. In our study, we checked several strated the use of human-derived marrow adherent stem cells stem cell markers, such as HES1, ABCG2, DNP63, and BMI1. (MASCS) as feeder cells in making cell sheets for transplanta- HES1 and DNP63 are well-recognized putative stem cell tion in the ocular surface.11 markers for both keratinocytes and corneal epithelial cells. We aimed to explore the possibility of using the human- ABCG2 and BMI1 are expressed in hematopoietic stem cells derived CLEC-muc as feeder cells to support the expansion of and in many other tissue-specific stem cells,16,27–29 as well as limbal epithelial cells ex vivo. We compared the CFE between limbal epithelial side population (SP) cells.30 Fluorescence CLEC-muc and 3T3 as feeder cells by seeding equal amounts of immunostaining and qRT-PCR data revealed positive expres-

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sion of all these genes in limbal cells cultivated with both 3T3 9. Park Y, Lee SJ, Choi IY, et al. The efficacy of human placenta as and CLEC-muc as feeder cells in a similar fashion. This suggests a source of the universal feeder in human and mouse that CLEC-muc possesses sufficient potential to help prolifer- pluripotent stem cell culture. Cell Reprogram. 2010;12:315– ation and maintain stemness of cultured limbal cells. It is to be 328. noted that the current study compared only CLEC-muc and 10. Miyamoto K, Hayashi K, Suzuki T, et al. Human placenta feeder 3T3 cells; however, other cells proposed by different studies layers support undifferentiated growth of primate embryonic could be considered. stem cells. Stem Cells. 2004;22:433–440. Further investigation showed that cytokeratins such as 11. Omoto M, Miyashita H, Shimmura S, et al. The use of human CK14, CK15, and CK19 were also expressed in cells cultivated mesenchymal stem cell–derived feeder cells for the cultivation with CLEC-muc; again this comparable to observations in 3T3 of transplantable epithelial sheets. Invest Ophthalmol Vis Sci. cells. CK14, CK15, and CK19 have been demonstrated to be 2009;50:2109–2115. expressed in different stem/progenitor cells and also in limbal 12. Sharma SM, Fuchsluger T, Ahmad S, et al. Comparative analysis basal cells; thus they are considered proposed limbal stem cell of human-derived feeder layers with 3T3 fibroblasts for the ex markers.31 Interestingly, the conjunctival epithelial marker vivo expansion of human limbal and oral epithelium. Stem Cell CK4 was less expressed in limbal cells cultivated with CLEC- Rev. 2012;8:696–705. muc as compared to limbal epithelial cells grown with 3T3 13. Jang IK, Ahn JI, Shin JS, et al. Transplantation of reconstructed cells (Figs. 6Y–Z2, 6Z–Z5, respectively); however, corneal corneal layer composed of corneal epithelium and fibroblasts epithelial marker CK3 expression was similar in both cases. on a lyophilized amniotic membrane to severely alkali-burned Based on this finding, we propose that CLEC-muc may provide cornea. Artif Organs. 2006;30:424–431. a more favorable niche in the culture system for generating the 14. Reza HM, Ng BY, Phan TT, Tan DT, Beuerman RW, Ang LP. corneal cell type rather than the conjunctival type. It is to be Characterization of a novel umbilical cord lining cell with notedthatCK3andCK4areconsidereddifferentiation CD227 positivity and unique pattern of p63 expression and markers; hence, expression of these genes indicates that some function. Stem Cell Rev. 2011;7:624–638. level of differentiation occurs during ex vivo culture of limbal 15. Weissman IL. Translating stem and progenitor cell biology to cells with both types of feeder cells. the clinic: barriers and opportunities. Science. 2000;287: In summary, we propose that CLEC-muc can substitute for 1442–1446. 3T3 fibroblasts as feeder layer for the cultivation of limbal stem 16. Reza HM, Ng BY, Gimeno FL, Phan TT, Ang LP. Umbilical cord cells, which would drive down the potential risk of xenogenic lining stem cells as a novel and promising source for ocular contamination to a large extent. However, in order to use a surface regeneration. Stem Cell Rev. 2011;7:935–947. completely animal-free culture system, culture media should be 17. de Paiva CS, Chen Z, Corrales RM, Pflugfelder SC, Li DQ. improved by avoiding the use of animal serum. ABCG2 transporter identifies a population of clonogenic human limbal epithelial cells. Stem Cells. 2005;23:63–73. Acknowledgments 18. Di Iorio E, Barbaro V, Ruzza A, Ponzin D, Pellegrini G, De Luca M. 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