Identification of Cell Surface Markers Glypican-4 and CD200 That

Cornea Identiﬁcation of Cell Surface Markers Glypican-4 and CD200 That Differentiate Human Corneal Endothelium From Stromal Fibroblasts

Yuen Kuen Cheong,1 Zi Xian Ngoh,1 Gary Swee Lim Peh,2 Heng-Pei Ang,2 Xin-Yi Seah,2 Zhenzhi Chng,3 Alan Colman,3 Jodhbir S. Mehta,2 and William Sun1,4

1Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 2Singapore Eye Research Institute, Singapore 3Institute of Medical Biology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 4Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

Correspondence: William Sun, Ex- PURPOSE. There is a lack of definitive cell surface markers to differentiate cultured human perimental Therapeutic Centre, 31 corneal endothelial cells (HCECs) from stromal fibroblasts, which could contaminate HCEC Biopolis Way, Singapore 138669; cultures. The aim of our study is to discover cell surface antigens on HCECs that can be used [email protected]. to identify and purify HCECs from stromal fibroblasts. Submitted: January 28, 2013 METHODS. RNA sequencing (RNA-seq) was used to find differentially overexpressed genes in Accepted: May 25, 2013 HCECs and commercial antibodies against these overexpressed antigens were screened by Citation: Cheong YK, Ngoh ZX, Peh immunofluorescence assay. Similarly, 242 commercial antibodies against cell-surface antigens GSL, et al. Identification of cell surface also were screened. Selected antibodies were used to sort HCECs from stromal fibroblasts by markers Glypican-4 and CD200 that fluorescence-activated cell sorting (FACS). differentiate human corneal endothelium from stromal fibroblasts. Invest RESULTS. Two monoclonal antibodies, anti-GPC4 and anti-CD200, were identified to stain Ophthalmol Vis Sci. 2013;54:4538– HCECs specifically. FACS was used successfully to sort HCECs away from stromal fibroblasts. 4547. DOI:10.1167/iovs.13-11754 Recovery efficiency of HCECs after sorting using anti-GPC4 antibody was higher compared to anti-CD200 antibody, but purity of HCECs culture using either antibody was comparable.

CONCLUSIONS. Taken together, the anti-GPC4 and anti-CD200 antibodies can be useful for purification and identification of HCECs in cultures containing stromal fibroblasts. Keywords: antibodies, corneal endothelial cells, stromal fibroblast, biomarker

he human corneal endothelium is a monolayer of cells that The isolation of HCECs from cadaveric donors involves Tfunctions as a barrier between the aqueous humor and peeling of the DM together with the corneal endothelium layer, corneal stroma via the formation of focal tight junctions to and subsequent enzymatic digestion with collagenase to release prevent excessive fluids from entering the stromal layer.1 The the corneal endothelial cells from the DM.11 However, in some Naþ/Kþ and Mg2þ ionic pumps on the corneal endothelial cell cases excessive manipulation may result in tearing into and co- surface regulate corneal hydration2,3 to maintain corneal isolation of small amounts of stromal tissue. Exposure to transparency. As the human corneal endothelial cells (HCECs) culture medium required for HCECs growth would transform do not regenerate in vivo,4,5 the integrity of the cellular these stromal keratocytes into fast-growing stromal fibro- monolayer is sustained through migration and enlargement of blasts,17 which outgrow the less proliferative HCECs.18,19 existing cells in the event of cell loss due to disease or surgical Although an L-valine–free selection medium could be used to trauma.6 However, decompensation of the endothelium occurs arrest growth of stromal fibroblasts, they could not be when corneal endothelial cell density falls below a critical eliminated.18 To abolish fibroblastic contamination, the culture threshold, resulting in its inability to maintain stroma deturges- would have to be passaged several times to dilute away the cence, thereby affecting visual acuity.7 stromal fibroblasts. However, the selection media is not optimal Endothelial dysfunction is the second leading cause of visual for long-term cultivation of HCECs.18 Alternatively, stromal blindness worldwide.8 Endothelial keratoplasty involving dis- fibroblasts could be depleted from contaminated cultures sected Descemet’s membrane (DM) with the endothelium layer through a negative cell–selection strategy by magnetic affinity can be performed to restore vision. However, global supply of cell separation (MACS) using antifibroblast magnetic micro- transplant-grade corneal tissues is limited and this restricts the beads.20 A positive cell–selection approach, using magnetic number of corneal transplantation performed annually.9 To microbeads or a more sensitive fluorescence-activated cell circumvent the shortage of corneal tissues and reliance on sorting (FACS), is plausible, but this is limited by the specificity human donor material, development of suitable tissue-engi- of cell-surface markers for HCECs. For example, the two highly neered constructs using cultured HCECs from cadaveric used markers reported for cultured HCECs are pump-associated donors10–13 or HCECs derived from multipotent progenitor protein Naþ/Kþ-ATPase (NaK) and tight-junction protein ZO-1. cells are being explored by various groups as potential graft In the cornea, the coexpression of these proteins indicates the alternatives.14–16 presence of functional components on the corneal endotheli-

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um, but do not define the identity of cultivated HCECs because that, although most of the corneal epithelium spontaneously these proteins are expressed ubiquitously in the heart,21 sloughed off the cornea surface during the processing of the brain,22 and kidney.23 cornea, remnant basal corneal epithelium cells still might be Currently, no HCEC-specific cell surface antigens have been present on the corneal stromal button used for RNA extraction. described. The goal of our study is to identify cell-surface Tissues were homogenized before addition of chloroform. Total antibodies expressed on the corneal endothelium and cultivat- RNA was extracted with RNeasy kit (Qiagen, Inc., Valencia, CA) ed HCECs that can differentiate them from stromal keratocytes with DNAse digestion step. and fibroblasts. Additionally, cell-surface antibodies identified RNA sequencing (RNA-seq) libraries were prepared using an in our study were assessed in its efficacy in purifying cultivated AB Demonstrated Protocol.25 For each sample, 100 pg of total HCECs from a culture contaminated with stromal fibroblasts RNA was transcribed with SuperScript III reverse transcriptase using FACS. (Invitrogen Corp., Carlsbad, CA), using a poly(T) primer with a UP1 anchor sequence. Exonuclease I removed free primers. Subsequently, a poly(A) tail was added to the 30 end of the first MATERIALS AND METHODS strand cDNA by terminal deoxynucleotidyl transferase. This The following protocols conformed to the tenets of the product was used for the synthesis of the second strand cDNA, Declaration of Helsinki, and written consent was acquired using a poly(T) primer with anchor sequence UP2. The from the next of kin of all deceased donors regarding eye product then was amplified separately using primers UP1 donation for research. The study was approved by the and UP2. Amplified cDNA was purified with QIAquick columns institutional review board of the Singapore Eye Research (Qiagen, Inc.), and re-amplified using NH2-UP1 and NH2-UP2 Institute/Singapore National Eye Centre. primers. This subsequently was pooled, purified, and size- selected (500 base pairs [bp] to 3 kilobase pairs [kb]) by gel Isolation and Growth of HCECs purification. Cultivation of HCECs isolated from pairs of donor corneas were RNA Sequencing (RNA-seq) done as described previously,8 with some modification. Briefly, the DM and corneal endothelial cells were peeled off from the The ABI standard library preparation protocol (Chapter 2 of AB stroma under a dissecting stereomicroscope. The DM-endothelial SOLiD 4 system library preparation guide—AB PN 4445673, layer was treated with collagenase for 6 hours and dissociated available in the public domain at http://www.lifetechnologies. further into smaller clumps with TrypLE Express (TE; Life com by Life Technologies) was used for nonbarcoded library Technologies, Carlsbad, CA). Isolated cells were cultured on preparation. The SOLiD fragment library barcoding kit module fibronectin and collagen (FnC) coated culture wares in F99 protocol (AB PN 4443045) was used for barcoded library medium (1:1 Ham’s F12 and M199), supplemented with 5% fetal preparation. The libraries were generated using the SOLiD bovine serum (FBS), 20 lg/mL ascorbic acid, 1 3 ITS, 1 3 fragment library construction (AB PN 4443471) and barcoding antibiotic/antimycotic,24 and 10 ng/mL basic fibroblast growth (AB PN 4444836) kit. factor. At 90% confluency, HCECs were exposed to Endothelial- Briefly, 1 lg of amplified cDNA was sheared to approxi- SFM supplemented with 5% FBS and 1 3 antibiotic/antimycotic mately 165 bp, end repaired, and P1, P2 adapters ligated. The (AA) for 5 days before they were subcultured. Seeding density product was nick translated and amplified (5 cycles), and size was approximately 13105 cells/cm2. Cultured HCECs were used selected between approximately 240 to 270 bp. The libraries at the first, third, or fifth passage. Incubation and cell culture were processed using the AB SOLiD 4 system templated bead were done in a humidified 5% CO2 incubator at 378C. Cell culture preparation guide (PN 4448378) and sequenced on SOLiD medium was refreshed every other day. slide. The resulting RNA-seq reads were processed with ABI Isolation and Growth of Human Corneal Stromal Bioscope pipeline. The reads were aligned to the hg19 human Fibroblasts reference genome. Gene expression was measured by count- ing the number of reads mapping uniquely to both strands of An 8.5 mm stromal button was obtained by trephination after each gene footprint, and normalized to the total uniquely the DM-endothelial layer was peeled off. Remnant corneal mapped reads to the entire genome. Values were adjusted to epithelial layer was scraped off with a scalpel. Stromal buttons show reads per million. were washed in PBS-AA solution and digested in collagenase overnight. Stromal keratocytes released from the stromal Quantitative PCR (qPCR) button were washed with PBS, and cultured in F99 medium. Corneal stromal keratocytes were transformed into corneal To measure the relative abundance of each gene transcribed in stromal fibroblasts in serum-supplemented medium. Culture HCECs and stromal fibroblasts, RNA was extracted with medium was refreshed every two days and subcultured at a 1:5 mRNeasy kit (Qiagen, Inc.). One lg of total RNA from HCECs ratio. and stromal fibroblasts was used to synthesize cDNA (Fermen- tas; Thermo Scientific). Each real-time PCR reaction was Sectioning of Cornea Tissue performed with iQsyber green supermix (Bio-Rad Laboratories, Hercules, CA) in the CFX96 thermocycler (Bio-Rad Laborato- Human donor cornea was rinsed in PBS twice and immersed in ries). Technical triplicates for each gene were performed. The OCT, and frozen at 808C until sectioning. Sections 8-lm thick D-CT value is normalized to Actin or GAPDH. The DD-CT value were cut using a MicromHM550 cryostat (Thermo Scientific, of each gene is normalized to values obtained from stromal Kalamazoo, MI), collected on glass slides, air dried, and stored fibroblasts. The average DD-CT value of each gene is calculated at 808C. from 3 independent experiments. Primers to each gene were RNA Extraction, Transcription, and Amplification synthesized by 1st Base Pte Ltd. (Singapore). for RNA Sequencing BD Lyoplate Screening Two different donor corneas were used. Isolated HCEC-DM and The BD Lyoplate human cell surface marker screening panel the remaining cornea containing the corneal stroma cells were (BD Biosciences, Franklin Lakes, NJ) contains 242 purified rinsed in PBS, and placed in Trizol reagent. It should be noted monoclonal antibodies against human cell-surface antigens.

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TABLE 1. List of Primary Antibodies

Name Company Species and Type Dilution Used

CD104 Integrin b4 Cat. No. 555719 BD Pharmingen, Franklin Lakes, NJ Rat mAb 1:100 CD200 OX-2 membrane glycoprotein Cat. No. 552023 BD Pharmingen Mouse mAb 1:100 CNTN6 Contactin-6 Cat. No. T3253 Epitomics, Burlingame, CA Rabbit pAb 1:200 GPC4 Glypican-4 Cat. No. NBP1-45286 Novus Biologicals, Littleton, CO Mouse mAb 1:250 NaþKþATPase sodium-potassium pump Cat. No. SC71638 Santa Cruz Biotechnology, Inc. Mouse mAb 1:40 PVRL3 Poliovirus receptor-related 3 Cat. No. SC28637 Santa Cruz Biotechnology, Inc. Rabbit pAb 1:100 SLC4A4 Solute carrier family 4, sodium bicarbonate cotransporter, member 4 Cat. No. SC1622 Santa Cruz Biotechnology, Inc. Goat pAb 1:100 SLC9A7 Solute carrier family 9 member 7 Cat. No. AB104868 Abcam, Cambridge, MA Rabbit pAb 1:100 Z0-1 Zonaoccludens protein BD Pharmingen Mouse mAb 1:50

The antibodies were used to stain cells in 96-well plates (Nunc fresh media for 30 minutes and the CFMDA would become a Multiwell Plates; Sigma-Aldrich Corp., St. Louis, MO). Antibod- cell-impermeant, fluorescent green product. HCECs and ies were reconstituted in 110 lL of sterile PBS and 20 lLof stromal fibroblasts were dislodged from culture with cell each antibody was used for each reaction. Both HCECs and dissociation buffer (Gibco, Carlsbad, CA). An approximately stromal fibroblasts were fixed in 4% paraformaldehyde for 10 equal amount of HCECs and stromal fibroblasts were mixed minutes, washed and incubated with the primary antibodies together and blocked with 10% goat serum (Millipore) for 10 for 1 hour. The cells were washed in PBS and incubated with minutes at 48C. The mixture was incubated with either anti- 1.25 lg/mL of the appropriate secondary antibodies for 1 hour. GPC4 or anti-CD200 antibody at the appropriate dilution The cells were analyzed with InCell Analyzer 2000 (GE (Table 1) for 20 minutes at 48C. Cells were washed and Healthcare Biosciences, Pittsburgh, PA). incubated with anti-mouse antibody conjugated with Alexa Fluor 488 (Invitrogen Corp.) for 20 minutes at 1:1000 dilution. Antibodies and Immunofluorescence Thereafter, the cells were washed and resuspended in 200 lL of PBS. Cells either were analyzed by flow cytometry (Accuri Antibodies were obtained from commercial sources (Table 1). C6; BD Biosciences) or sorted (MoFlo XDP; Beckman Coulter, HCECs and stromal fibroblasts were seeded on multi-test slides Inc., Indianapolis, IN). Sorted cells were seeded onto a 48-well (MP Biomedicals, Solon, OH). Cells or tissue sections were plate and observed under a microscope 24 hours later. Three blocked with 5% goat serum (Millipore, Billerica, MA) or 3% independent sorting experiments were performed to obtain bovine serum albumin (Santa Cruz Biotechnology, Inc., Dallas, TX) and incubated with primary antibodies for 1 hour, and the average percentage of recovery and purity. Unpaired washed with PBS with 0.05% Tween (PBST). Subsequently, the Student’s t-test was applied to calculate statistical significance cells or tissue sections were incubated with secondary in recovery and purity between using anti-GPC4 and anti- antibodies for 1 hour and washed with PBST. Cells were CD200 antibody for sorting. counterstained with Hoeschst (Invitrogen Corp.). A mouse, rabbit, goat, or rat IgG isotype antibody was used as negative RESULTS control (Santa Cruz Biotechnology, Inc.). Slides were viewed under a fluorescence microscope (Nikon, Inc., Melville, NY). RNA-seq and Quantitative PCR of Relatively Overexpressed Genes in HCECs Flow Cytometry and FACS RNA-seq was performed on two donor corneas to determine Stromal fibroblasts were labeled with CellTracker green, 5- the relative expression of genes in human corneal endothelium chloromethylfluorescein diacetate (CFMDA; Invitrogen, Corp.), and stromal keratocytes. Genes that were expressed at least 20- before dissociation from culture according to manufacturer’s fold higher in the endothelium were crossed referenced with protocol. Briefly, stromal fibroblasts were incubated with 5 lM Genbank database to select those that code for membrane of CFMDA in serum-free media at 378C for 30 minutes. associated proteins. A selected list of candidate genes is shown Subsequently, the CFMDA-containing media was replaced with in Table 2. In addition, quantitative PCR analysis of extracted RNA from cultured HCECs and stromal fibroblasts was TABLE 2. Relative Fold Difference of RNA Expression in Cornea Tissue performed to investigate if relative overexpression of some Section genes also would be reflected in cultured cells. The top 5 Fold Difference by RNA Seq Gene (HCEC Over Stromal Keratocytes) TABLE 3. Relative Fold Difference of RNA in Cultured HCEC Over Stromal Fibroblast PVRL3 1700 TRO 1508 Fold Difference by Quantitative PCR SLC9A7 889 (HCEC Over Stromal Fibroblast) 796 PARD6A Gene Normalized to Actin Normalized to GAPDH SCAMP5 776 CNTN6 636 GPC4 262.0 6 55.3 70.3 6 12.2 GPC4 490 CNTN6 163.0 6 21.5 43.80 6 6.3 PTPRO 315 SLC9A7 8.30 6 1.52 2.2 6 0.92 PTCH1 125 PVRL3 2.83 6 0.79 0.76 6 0.22 SLC4A4 88 SLC4A4 2.33 6 0.56 0.62 6 0.10

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FIGURE 1. Immunofluorescence microscopy of HCECs and stomal fibroblasts. Cell cultures of HCECs and stromal fibroblasts seeded onto a 12-well multi-slide was stained with antibodies against markers identified in Table 3 (A) or from screening the BD Lyoplate (B). The primary antibodies were detected with the appropriate secondary antibodies conjugated to Alexa Fluor 488 (Invitrogen Corp.). Nuclei of cells were stained with Hoechst.

genes that were overexpressed in HCECs relative to stromal them (CNTN6, Fig. 2). It also was observed that the DM was fibroblasts are shown in Table 3. stained by some of the antibodies (Fig. 2, white arrowhead). In all, 7 potential cell surface markers were identified (5 Screening of Antibodies on Cell Culture from RNA-seq and 2 from BD Lyoplate) that might be specific for HCECs only. Since CD200 was identified through BD Quantitative PCR data indicated that these genes - CNTN6, Lyoplate screen, we asked if CD200 is expressed more GPC4, PVRL3, SLC4A4, and SLC9A7, were significantly more abundantly in cultured HCECs than cultured stromal fibro- abundant in HCECs compared to stromal fibroblasts in culture blasts. Quantitative RT-PCR analysis for CD200 indicated that it (Table 3). Commercial antibodies to these proteins were used was, indeed, expressed more abundantly in HCECS than in to stain cultures of HCECs and stromal fibroblasts to confirm stromal fibroblasts (Supplementary Table S1). their expression on HCECs. It was observed that only anti- Taken together, only anti-GPC4 and CD200 antibodies GPC4 antibody stained HCECs, but not stromal fibroblasts (Fig. specifically stained both cultured HCECs and the endothelium 1A). in tissue sections, and hence, they were used for subsequent The BD Lyoplate human surface cell marker panel provided experiments. a total of 242 monoclonal antibodies for screening on HCECs and stromal fibroblasts. From the screen, only CD104 and Flow Cytometry With Anti-GPC4 and Anti-CD200 CD200 appeared to stain HCECs specifically (Fig. 1B). There on Cultured Cells may be differences in gene expression between HCECs in culture and cells in the corneal endothelium within the cornea Flow cytometry with HCECs specific antibodies potentially tissue (Tables 2, 3); therefore, these antibodies were used to could be used to purify HCECs from contaminating fibroblasts. stain cornea tissue sections, too. It was observed that anti- To test if these antibodies are amenable for FACS, HCECs and GPC4, CD200, and SLC4A4 antibodies stained the corneal stromal fibroblasts were labeled with anti-GPC4 and anti- endothelial layer specifically. The rest of the antibodies either CD200 antibodies in separate experiments, and analyzed by were found to be positive on the corneal endothelium and flow cytometry. Consistent with the staining results, both stromal layers (SLC9A7, PVRL3 and CD104), or on neither of antibodies labeled HCECs only, but not stromal fibroblasts,

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FIGURE 2. Staining of cornea tissue sections. Formaldehyde-ﬁxed cornea tissue sections were incubated with various primary antibodies. The primary antibodies were detected with the appropriate secondary conjugated to Alexa Fluor 488. A white arrow indicates the corneal endothelial layer. A white arrowhead indicates the DM.

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FIGURE 3. Flow cytometry analysis of antibody-labeled HCECs and stromal fibroblasts. (A) HCECs or stromal fibroblasts were labeled with anti- GPC4 antibody, anti-CD200 antibody, or a mouse IgG isotype control antibody. Anti-mouse antibody conjugated to Alexa Fluor 488 (AF488) was used to detect the primary antibodies. The histogram for HCECs or stromal fibroblasts labeled with the isotype control antibody (arrow, black)is overlaid with the histogram for HCECs or stromal fibroblasts labeled with either anti-GPC4 or anti-CD200 antibodies (pink). (B) CFMDA-labeled stromal fibroblasts were mixed with HCECs in a 1:1 ratio before incubation with either anti-GPC4 of anti-CD200 antibodies. The mixture subsequently was incubated with anti-mouse antibody conjugated to Alexa Fluor 647 (AF647). (I) and (IV) show detection of fluorescence

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produced by CFMDA, and (II) and (V) show the detection of AF647. M1 and M2 indicate the percentage of HCECs and stromal ﬁbroblasts in the mixture, respectively. (III) and (VI) show a dot plot of all the events on a CFMDA versus AF647 graph. Each graph is divided into 4 quadrants, and the percentage of events captured in each quadrant is indicated.

causing a shift in the histogram when compared to the isotype retained the expression of these proteins after sorting (Fig. 5). controls (Fig. 3A). In contrast, the anti-NaK antibody caused a These markers also are retained on HCECs that were cultured smaller shift in the histogram with HCECs relative to the for five passages without undergoing any sorting procedure isotype controls, and it also caused a slight shift with stromal (Supplementary Fig. S3). fibroblasts (Supplementary Fig. S1). This suggests that anti-NaK antibody may not be suitable for sorting. The anti–ZO-1 antibody did not cause any shift in the histogram in either cell DISCUSSION type relative to the isotype control (Supplementary Fig. S1). Since all data until now were derived from HCECs in their Expansion of HCECs from cultured donor corneal endothelium third passage, FACS analysis was performed on HCECs in their could provide an alternative source of transplantable material first passage to ensure that GPC4 and CD200 were present. for endothelial keratoplasty. However, isolation of HCECs for in Consistent with previous observations, both antibodies caused vitro culture potentially can be contaminated by stromal a shift in the histogram when compared to the isotype control. keratocytes/fibroblasts during the isolation process. If HCECs- (Supplementary Fig. S2). specific cell-surface markers are identified in the context of the Next, we asked if anti-GPC4 and anti-CD200 antibodies cornea, they can be used to identify and purify HCECs from were able to identify differentially HCECs within a mixed cultures contaminated with stromal fibroblasts. Hence, cell- population of cells. Stromal fibroblasts were prelabeled with surface antibodies that are specific for HCECs would be CFMDA before mixing with the HCECs at a 1:1 ratio. This was valuable tools in this regard. done to differentiate them from HCECs during the analysis. The Through gene expression profiling and screening of a panel mixed population of cells was incubated with either anti-GPC4 of commercial antibodies, we identified two cell-surface or anti-CD200 antibodies. Although both antibodies were able antibodies that are specific for the corneal endothelium and to stain HCECs, labeling with anti-GPC4 antibodies resulted in cultivated HCECs. Although a recent transcriptomic study also a better histogram separation (Figs. 3BII, 3BV). FACS analysis has identified 4 markers for adult HCECs, they are intracellular showed that within the mixed population, 1% of the anti-GPC4 proteins, and it was not demonstrated if these markers could positive cells and 1.7% of the anti-CD200 positive cells also be used to differentiate HCECs from stromal fibroblast cells in 26 were CFMDA positive, indicating a low level background culture. Moreover, being intracellular proteins, they cannot staining of these antibodies on stromal fibroblasts (Figs. 3BIII, be used in cell separation procedures to purify HCECs from 3BVI). cultures contaminated with stromal fibroblasts. Results from RNA-seq indicated that some genes were overexpressed in the corneal endothelium relative to the FACS With a Mixed Population of Cells stroma. The relative expression of these genes in cultured The mixed cell population of CFMDA prelabeled stromal HCECs and stromal fibroblasts subsequently were quantified by fibroblasts and unlabeled HCECs, mixed at a ratio of 1:1, was quantitative PCR, since expression levels could have changed incubated with either anti-GPC4 or anti-CD200 antibodies. during propagation in vitro. Discrepancies between the RNA- GPC4- and CD200-positive cells were sorted (Figs. 4AI, 4AIV) seq and qPCR data are expected, since these are two different and seeded into wells of a 48-well plate (Figs. 4AIII, 4AVI). methods used on two different types of samples. Differences in Consistent with earlier results (Fig. 3B), cells labeled with anti- gene expression did not always translate to differences in GPC4 antibodies were better separated from stromal fibro- protein expression as shown by immunohistochemistry. blasts (Fig. 4AI) than cells labeled with anti-CD200 (Fig. 4AIII). Nonetheless, immunohistochemistry on cultured HCECs and On average from three independent experiments, approxi- corneal tissue sections identified anti-GPC4 and anti-CD200 mately 20% of HCECs were not recovered when sorted with antibodies that specifically stain HCECs and the corneal anti-CD200 antibody (Figs. 4AIV, 4BI). In contrast, 96% of the endothelium. cells were recovered when sorted with anti-GPC4 antibody To our knowledge, GPC4 and CD200 have not been (Figs. 4AII, 4BI). The difference in recovery between using anti- described as markers for HCECs. Glypicans are heparan sulfate GPC4 or anti-CD200 antibody is significant (P 0.05). Flow proteoglycan bound to the outer surface of plasma membrane cytometry analysis of cells sorted by either antibodies indicated by a glycosyl-phosphatidylinosiol anchor. Glypicans can be that a small percentage of the cells were CFMDA-positive (Figs. released into the extracellular environment by an extracellular 4AII, V). Low numbers of CFMDA-positive cells were, indeed, lipase known as Notum through the cleavage of the GPI observed in culture after FACS (Figs. 4AIII, 4VI), indicating that anchor.27 This could explain why a signal was observed on the some stromal fibroblasts were co-sorted with the HCECs. There DM when corneal sections were stained for GPC4 (Fig. 2). It is no significant difference (P ‡ 0.05) in terms of the purity also has been reported that glypicans are associated with lipid (>98%) of the cells when sorted by the two antibodies (Fig. rafts and the basolateral membranes of polarized cells.28 4BII). Members of this family are known to regulate Wnts signaling in Drosophila,29 zebra fish embryos,30 and mammalian cells,31 Cells Retain Surface Markers After Sorting as well as signaling of hedgehogs, fibroblast growth factors, and bone morphogenetic proteins.32 CD200, previously To determine if the sorted HCECs express markers indicative of known as OX2, is a membrane glycoprotein belonging to the the human corneal endothelium, sorted cells were plated and immunoglobulin superfamily. It interacts with a receptor, probed for their expression of Naþ/Kþ-ATPase pump, tight CD200R, on myeloid lineage cells and downregulates myeloid junctions Z0-1, GPC4, and CD200 proteins. After sorting and lineage proliferation.33,34 Upregulation of this protein is plating, these cells would be in their fourth passage. Positive associated with certain B-cell lymphoproliferative disorders, staining observed for all these antibodies indicated that HCECs such as chronic lymphocytic leukemia and small lymphocytic

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FIGURE 4. FACS of HCECs from stromal fibroblasts. (A) A representative sorting experiment with anti-GPC4 and anti-CD200 antibodies. Anti-GPC4 or anti-CD200 labeled cells were sorted away from CFMDA labeled stromal fibroblasts (I) and (IV). Sorted cells (from [I] and [IV]) were gated and plotted on a histogram to quantify the percentage of CFMDA positive cells (indicated by arrows)in(II) and (V). Sorted cells subsequently were seeded on 12-well multi-slide and viewed under fluorescence microscope (III) and (VI). (B) The average recovery yield of HCECs and proportion of CFMDA-positive stromal fibroblasts from 3 independent sorting experiments using either anti-GPC4 or anti-CD200 antibody were tabulated and plotted as a bar graph (*) indicates significant difference, P 0.05). Mean values with SDs are indicated in or above the bars.

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FIGURE 5. Immunoﬂuorescence staining of cells after FACS. After the HCECs were sorted, they were seeded onto a 12-well multi-test slide. Anti- GPC4, CD200, Z0-1, and Naþ/Kþ-ATPase (NaK) antibodies were used to probe the cells. Anti-mouse conjugated to Alexa Fluor 647 was used to detect the primary antibodies.

lymphoma.35 A truncated isoform of CD200 resulting from than the other commonly used markers, such as NaK and Z0-1. alternative mRNA splicing has been found to be soluble,36 and Flow cytometry analysis showed that anti-NaK and Z0-1 this could explain the staining observed on the DM (Fig. 2). antibodies would not be optimal reagents for sorting a Functions of these two proteins in the HCECs have not been heterogeneous culture (Supplementary Fig. S2). investigated. In conclusion, two cell-surface markers, GPC4 and CD200, These two cell-surface antibodies are not just valuable tools previously not described in the corneal endothelium to our for discriminating corneal endothelium/HCECs from stromal knowledge, were identified in our study. These markers could fibroblasts, but they also could be used for purifying HCECs be used to differentiate HCECs from stromal keratocytes and contaminated with stromal fibroblasts. Previously, a negative corneal stromal fibroblasts. We demonstrated the use of these depletion method using antifibroblast magnetic beads was antibodies to separate and purify HCECs from corneal stromal used to purify cultured HCECs from stromal fibroblasts, since fibroblasts by FACS. These antibodies also potentially might be no cell-surface antibody specific for HCECs were described.20 useful as part of a panel of reagents for identifying and In our study, we adopted a positive selection strategy to isolate characterizing a population of putative HCECs derived from HCECs from a mixed population of HCECs and stromal other cell sources, such as multipotent corneal progenitors14 fibroblasts by FACS. Results in our study showed that cells or pluripotent stem cells. separated by FACS were viable and retained pump-associated þ þ marker Na /K -ATPase, tight junction protein ZO-1, as well as Acknowledgments GPC4 and CD200. Although purity of the eventual culture sorted by either antibody was comparable, sorting with anti- Supported by the Biomedical Research Council, Agency for CD200 was found to be less efficient. Hence, anti-GPC4 Science, Technology and Research, Singapore. antibody would be better suited for sorting by FACS. A strategy Disclosure: Y.K. Cheong, None; Z.X. Ngoh, None; G.S.L. Peh, to sort cells using both markers simultaneously might enhance None; H.-P. Ang, None; X.-Y. Seah, None; Z. Chng, None; A. the purity of HCECs. Finally, these two markers are more useful Colman, None; J.S. Mehta, None; W. Sun, None

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