CD200 facilitates the isolation of corneal epithelial cells derived from human pluripotent stem cells

The in vitro induction of corneal epithelial cells (CECs) from human induced pluripotent stem cells (iPSCs) represents a new strategy for obtaining CE stem/progenitor cells for the surgical reconstruction of a diseased or injured ocular surface. The clinical promise of this strategy is considerable, but if the approaches’ potential is to be realised, robust methods for the purification of iPSC-derived CE lineage cells need to be developed to avoid contamination with other cells that may carry the risk of unwanted side effects, such as tumorigenesis. Experiments conducted here revealed that during CEC isolation, CD200-negative selection using a cell sorter considerably reduced the contamination of the cell population with various non-CECs compared with what could be achieved using TRA-1-60, a conventional negative marker for CECs. Furthermore, CD200-negative sorting did not affect the yield of CECs nor that of their stem/progenitor cells. Single-cell gene expression analysis for CEC sheets obtained using CD200-negative sorting showed that all analysed cells were CE-lineage cells, expressing PAX6, delta-N p63, and E-cadherin. Non-CECs, on the other hand, expressed non-CEC genes such as FGFR1 and RPE65. CD200, thus, represents a robust negative marker for purification of induced CE lineage cells, which is expressed by undifferentiated iPSCs and non-CECs, including iPSC-derived neural and retinal cells.

outermost SEAM zone 4. This diversity with multiple cell lineages in the same culture, however, means that the differentiation efficiency of CE lineage cells is not very high 14 . Nevertheless, sufficient numbers of CE stem/progenitor cells could be obtained and expanded into functional epithelial multi-layers via our use of the cell surface markers SSEA-4 and ITGB4 as positive markers of CE lineage cells in conjunction with TRA-1-60 as a negative marker. Using this approach, we were able to isolate CECs, including their stem/progenitor cells, from the differentiated iPSCs 13,14 . Although this technique enabled isolation of CE lineage cells, they were not sufficiently pure, prompting the current investigation to investigate ways of removing untargeted cells and enhancing the purity of CECs induced from SEAMs using a cell surface marker. To achieve this we screened cell surface markers that can enhance the purity of CECs by cell sorting and identified CD200 as a new and effective negative marker to remove non-target cells from differentiated human iPSC cultures, permitting superior purification of CE lineage cells.

Results
CEC induction and isolation from human iPSCs. CEC differentiation from human iPSCs via SEAM formation was conducted according to our previously reported methods 14 , and by weeks 3 to 4 of differentiation SEAMs comprising typical cellular multi-zones were formed (Figs 1a and S1a). After removing the inner non-epithelial zones of the SEAM (i.e. zones 1 and 2), CE lineage cells differentiated in zone 3 were isolated by cell sorting at weeks 10 to 15 after initiation of differentiation. The majority of cells isolated using the conventional combination of cell surface markers, including TRA-1-60, SSEA-4, and ITGB4, were found to be CE lineage cells. Nevertheless, several non-CEC colonies comprising cells not expressing stratified epithelial cells and CEC markers, such as p63 and K12, were obtained also (Fig. 1b). Interestingly, our analyses revealed that many of these non-epithelial cells, which showed negative staining for pan-cytokeratin and K14, expressed CD200 (Fig. 1c,d) CD200 expression during human iPSC differentiation. Based on the identification of CD200 expressed in non-CEC cultures, we sought to explore its use as a potential marker for CE lineage cell isolation from human iPSCs differentiated using the SEAM method. Initially, the expression of CD200 with time during iPSC differentiation was examined (Fig. 2a). Flow cytometry demonstrated that CD200 is highly expressed by all undifferentiated iPSCs before differentiation and by 80% of iPSCs even after 6 weeks of differentiation. After removing non-epithelial-like cellular zones from the SEAM at week 7, CD200 expression decreased considerably, but remained at around 20% even at week 12 (Fig. 2b). In contrast, TRA-1-60 expression was below 10% immediately after the early differentiation steps, at week 1, ultimately dropping to approximately 1% at week 12 (Fig. 2b). Immunostaining indicated that CD200 was strongly expressed by all undifferentiated iPSCs and non-epithelial cells (i.e. such as those in zones 1 and 2 of the SEAM at week 4), but was only faintly expressed in zone 3 ( Fig. 2c). At 12 weeks of culture, the majority of differentiated CEC-like cells showed no CD200 expression, while some non-CEC-like cells did. The expression of TRA-1-60 was limited to the undifferentiated iPSCs, with negligible expression of this molecule during the differentiation (Fig. 2c).

Isolation and characterization of human iPSC-derived CE lineage cells using CD200.
To investigate CD200 as a potential negative marker for CE lineage cells, cell sorting using an anti-CD200 antibody was performed. After 10 to 15 weeks of differentiation, iPSC-derived CE lineage cells were isolated by using anti-ITGB4, anti-SSEA-4, and anti-CD200 or anti-TRA-1-60 antibodies (Fig. 3a). The collection rate for CE lineage cells, defined as ITGB4 + /SSEA-4 + cells, was 36.9 ± 5.2% (n = 6) for CD200-negative cells, and 33.2 ± 5.2% (n = 6) for TRA-1-60-negative cells, with no statistically significant difference between both methods (Fig. 3b). Isolated cells were analysed by qPCR to determine the potential contamination of the sorted population (Figs 3c and S1b) and this revealed a significant decrease in the expression of non-target markers, including FGFR1 (expressed by non-epithelial cells including fibroblasts, neuronal cells, and human iPSCs) [15][16][17] , RPE65 (retinal pigment epithelial cell) 18 , HOXs (non-ocular trunk cells) 19 , COL1A1 and A2 (fibroblasts) 20 , PDGFRA and PDGFRB (mesodermal and vascular cells) 21,22 and CD200 by the isolated cells selected for their lack of CD200 expression. In contrast, no changes in the expression of CEC-associated genes, such as PAX6 and K12, were observed. Non-epithelial-like colonies developed from the sorted CE lineage cell samples indicated that there were significantly fewer non-CECs in CD200-negatively sorted cells compared with the TRA-1-60-negative samples (Fig. 3d). Phase-contrast analyses demonstrated that after CD200-negative sorting, almost no recognizable non-target colonies developed from the CD200-negative iPSC-derived CE lineage cells, unlike the situation using samples obtained by TRA-1-60-negative sorting (Fig. 3e).

CE stem/progenitor cells and CD200 in vitro and in vivo.
To assess the presence of CE stem/progenitor cells in each cell population, colony-forming assay (CFA) experiments were performed. These disclosed no differences in colony-forming efficiency (CFE) between CD200-and TRA-1-60-negative sorting samples (3.6 ± 1.2 vs. 3.8 ± 1.0%, respectively; n = 4) (Fig. 4a). To evaluate the proliferative potential further, a serial cell passaging assay was conducted (Fig. 4b). The results showed that iPSC-derived CE stem/progenitor cells isolated by both CD200 and TRA-1-60-negative sorting exhibited similar levels of population doubling. These data indicated that CD200-negative sorting successfully allowed the harvesting of not only differentiated CECs, but of stem/progenitor cells too. Moreover, iPSC-derived CE stem/progenitor cells isolated by CD200-negative sorting became stratified and were able to successfully form an epithelial cell sheet. This cell sheet expressed CEC-specific makers, including K12, PAX6, K3, and MUC16, but showed no expression of CD200 or conjunctival epithelial markers, K4 and K13 (Fig. 4c). K15, p63 and p40 (delta-N p63 isoform), stratified epithelial stem/progenitor cell markers, were expressed in some basal cells (Fig. 4c) 60% K12 + differentiated CECs (Figs 4d and S1d). Immunostaining of corneal tissue revealed no CD200 expression in the corneal epithelium, including the limbal region at the edge of the cornea where epithelial stem cells are believed to reside (Fig. 4e). Similarly, limbal epithelial cells maintained in vitro showed no CD200 expression (Fig. 4e), and flow cytometric analyses demonstrated minimal CD200 expression in human limbal epithelium in vivo (Fig. 4f). We further examined CD200 expression in murine embryonic and adult eyes (Fig. 4g). Immunostaining data showed that CD200 was expressed in ocular tissues, including corneal epithelium and

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Discussion
Previous work has used the SEAM method to induce the formation of ocular primordia from human iPSCs 13,14 . This results in the generation of a concentric cellular multi-zone in which cells in SEAM zone 3 possess the characteristics of CE lineage cells. But, because a range of cell types exists within a single colony, cell sorting is required to isolate cells with the desired genotype/phenotype, and non-contamination with other cell types is a priority if the intended use is for clinical purposes. In our previous research, to discard undifferentiated iPSCs we used TRA-1-60 as a PSC marker. However, the expression of this molecule decreases considerably before cell sorting, so it is not the most effective agent for the removal of non-target cells and a novel more effective cell surface maker is required. We performed screening for a cell surface marker to remove non-targeted cells and enhance the purity of CECs via the use of previously used microarray databases (GSE73971) 13 . We searched for negative markers that are expressed in undifferentiated iPSCs and in cells other than CECs, but which were negative for CECs. This analysis identified CD200, FGFR1, and PDGFRB ( Supplementary Fig. S2a), and among these three markers only anti-CD200 antibody (OX-104, commercially available) stained human iPSCs (but not CECs) and could detect an extracellular region of the antigen in flow cytometry as shown in Figs 1c,d, 2 and 4f.
CD200 is a glycoprotein widely expressed in somatic cells. It is a marker of breast cancer, leukaemia, and colon cancer cells, as well as being a PSC marker 12,25,26 . Here, we demonstrate that CD200 is uniformly expressed by undifferentiated human iPSCs, and during differentiation, CD200 expression is sustained by more than 80% of iPSCs, even after four weeks of culture. Its expression is thus more stable than that of TRA-1-60, which drops considerably in the early differentiation period. Furthermore, by the end of the differentiation culture, TRA-1-60 expression had become drastically decreased to around 1% of cells. In contrast, after the same differentiation period, CD200 was still maintained in approximately 20% of differentiated iPSCs. These findings show that TRA-1-60 is specific to the undifferentiated state of PSCs, but is no longer expressed in differentiating iPSCs, which probably maintain multi-lineage differentiation potential as well as tumour formation capability. Therefore, after the differentiation culture, TRA-1-60 is no longer useful in removing these differentiating non-CECs because the expression would have already been lost. In contrast to TRA-1-60, CD200 expression was maintained in the differentiating iPSCs even after 12 weeks, implying that CD200 is broadly expressed not only in undifferentiated iPSCs, but also in differentiating or differentiated iPSCs other than CE lineage cells. The immunostaining data clearly indicates that during differentiation culture, CD200 expression was particularly well maintained in non-CEC zones such as zone 1 and 2, indicating that CD200 could be a positive selector of various non-CEC lineages obtained from differentiated iPSCs.
Regarding ITGB4 and SSEA-4 used in combination with CD200, each solo marker cannot specifically select corneal epithelial cells although we previously found that corneal and limbal epithelial cells express both ITGB4 and SSEA-4. In fact, previous reports indicated that each marker is expressed even in some non-epithelial cells in culture 27,28 and our present result further indicates that ITGB4 + /SSEA-4 + cells without CD200-negative sorting contained small numbers of non-epithelial cells. Collectively, these facts suggest that CD200 possibly compensates for the limitations when using ITGB4 and SSEA-4 as selective markers for CE lineage cells.
The findings of a gene expression analysis also suggest that CD200 can be used to effectively remove non-CECs such as neuronal cells, fibroblasts, retinal cells, and keratinocytes from the SEAM colony. The most pronounced CD200-instigated negative selection was discovered to be for FGFR1-expressing cells. FGFR1 is expressed by PSCs and by a range of non-epithelial cells, including neuronal cells and fibroblasts, suggesting that it may be a useful indicator of possible contamination of non-stratified epithelial cells. FGFR1 is a cell-surface protein, but when we assessed its potential use for the negative marker for selection of CECs using commercially available anti-FGFR1 antibodies, it transpired that the cell sorting we performed was not successful. Perhaps this is because extracellular regions of FGFR1 protein were digested by enzymatic treatment at cell harvesting. In contrast to that of the non-CEC genes, no remarkable changes in the expression of CEC-associated makers, including PAX6, delta-N p63, and K12, were observed. Flow cytometry and CFA results, moreover, disclosed no difference in the yields of CECs and their stem/progenitor cells obtained by CD200-or TRA-1-60-negative sorting.
As shown here, CD200 is primarily expressed by non-epithelial cells, although during differentiation culture some transient expression was detected in ITGB4 + epithelial cells (Supplementary Fig. S1e). This expression, however, is down-regulated by week 12, and in adult corneal tissue and in limbal epithelial cells grown in vitro almost no CD200 expression was detected (Fig. 4e,f). Moreover, we confirmed that CD200 expression was found only in developmental CECs, not in adult CECs (Fig. 4g). These data suggest that CECs transiently express CD200 during their development, but not when fully differentiated. Previous reports indicated that CD200 represents a hair follicle stem cell marker 29,30 , suggesting that the CD200 + iPSC-derived epithelial fraction might contain other types of epithelial cells, including hair follicle stem cells.
Single-cell gene expression analysis results showed that all analysed cells (n = 151) isolated by CD200-negative selection showed PAX6, delta-N p63, and E-cadherin expression; non-epithelial cell markers, such as FGFR1, RPE65, and MLANA, on the other hand, were essentially not expressed. Other non-target cell-related genes were slightly expressed by a very low number of cells in the isolated population. Use of anti-CD200 antibodies, therefore, allows us to eliminate non-target cells and enrich CECs in CD200-negatively isolated cell populations, creating a high purity iPSC-derived CE lineage cell population. In the current experiments a small number of the analysed CE lineage cells (8/151) expressed the CE stem/progenitor cell specific marker, N-cadherin, with majority of these cells (6/8) not expressing corneal differentiation markers, K3 and K12, nor non-CEC makers, such as FGFR1, CRYAA, and PDGFRA. This suggests that a number of undifferentiated CE stem/progenitor cells can be found among CD200-negatively isolated CE lineage cells. Single-cell gene expression analysis and immunostaining for iPSC-derived CEC sheets demonstrated different expression patterns between K3 and K12, even though they should form a pair. Our previous data indicated that long-term culture (i.e. more than 10 weeks) promoted CEC maturation-as evidenced by an enhancement of stratification and keratin expression-over and above that seen within shorter culture periods (3 weeks) 14 . Additionally, a previous report has suggested that K3 and K12 expression in embryonic cornea is not coincident 31 , whilst another report has pointed to a differential regulation of K3 and K12 expression by PAX6 isoforms 32 . These reports of a discordant expression of K3 and K12 during developmental, may indicate the possibility of an incomplete maturation of our CECs in vitro due to an insufficient culture period. The corneal epithelium is maintained by stem cells located in the basal region of the limbal epithelium at the edge of the cornea 33 . Regenerative medicine involving the transplantation of iPSC-derived CEC sheets including stem/progenitor cells presents a promising strategy for the treatment of stem cell deficiencies caused by CE dysfunction. However, to be clinically applicable, contamination with non-target cells must be avoided because of the potential of unwanted side effects. The results obtained here show that CD200 is preferentially expressed by undifferentiated iPSCs and non-CECs, identifying it as a robust negative selector of hiPSC-derived CE lineage cells. CD200 is also a cancer-associated marker [31][32][33] , thus, CD200-negative sorting may also limit the risk of tumorigenesis. CD200-negative sorting is therefore viewed as a favoured approach for first-in-human clinical trials of corneal epithelial regenerative medicine.

Methods
All experiments were performed in accordance with the relevant institutional and national guidelines and regulations.
Human iPSCs. The human iPSC line 201B7 was purchased from RIKEN Bio Resource Center (Tsukuba, Japan). After passaging the cells on a mitomycin C (MMC)-treated mouse embryonic fibroblast (MEF) feeder layer at least three times, they were cultivated in a feeder-free system using Laminin-511 E8 fragments (LN511E8; Nippi, Tokyo, Japan) and serum-free medium (StemFit; Ajinomoto, Tokyo, Japan) for 7 days 34,35 . All experiments using recombinant DNA were approved by the Recombinant DNA Committee of Osaka University and were performed according to our institutional guidelines.

CFA and cell sheet generation.
For the CFA, CE lineage cells isolated by cell sorting were seeded onto MMC-treated NIH-3T3 feeder cells at the cell density of 2000-5000 cells/well in 12-well plates. After 10 to 14 days, the cells were fixed with 10% formaldehyde neutral buffer solution (Nacalai Tesque, Kyoto, Japan) and stained with rhodamine B (Wako, Osaka, Japan). Colony formation was assessed using a dissecting microscope and the CFE was calculated. Cell sheet generation was performed as previously reported 13 . Briefly, the cells isolated by cell sorting were seeded onto LN511E8-coated dishes at a density of 1 × 10 5 cells/well in 12-well plates or in cell culture inserts and cultivated in CEM and corneal epithelium maturation medium (CMM; i.e. KCM medium containing 10-20 ng/mL KGF and 10 μM Y-27632) for 14-21 days.
Serial cell passaging assay. CE lineage cells isolated by cell sorting were cultivated on MMC-treated NIH-3T3 feeder layers in CMM up to 70-80% confluence. The CECs were harvested using TrypLE Express (Life Technologies) following the removal of feeder cells by manual pipetting. Cells were counted and seeded onto newly prepared feeder layers at a cell density of 20000 cells per well in 12-well plates. These cells were cultivated in CMM until sub-confluence was once more reached. Immunofluorescence staining. Cells or tissues were fixed in 4% paraformaldehyde (PFA, Wako), washed with Tris-buffered saline (TBS, TaKaRa Bio, Shiga, Japan) three times for 10 min and incubated with TBS containing 5% donkey serum and 0.3% Triton X-100 for 1 h to block non-specific reactions. Afterwards, they were incubated with the antibodies listed in Supplementary Table S1 at 4 °C overnight or at room temperature for 3 h. The cells were again washed twice with TBS for 10 min, and incubated with AF488-and 568-conjugated secondary antibodies (1:200; Life Technologies) for 1 h at room temperature. For p40 staining, the slides were incubated with Biotin-SP-conjugated secondary antibody (1:200, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at room temperature. After two washes with TBS for 10 min, the slides were incubated with AF488-conjugated Streptavidin (1:200, Jackson ImmunoResearch Laboratories) for 1 h at room temperature. Counterstaining was performed with Hoechst 33342 (Life Technologies) prior to fluorescence microscopy (Axio Observer.D1, Carl Zeiss, Jena, Germany). For CD200 immunostaining, the PE-conjugated antibody was directly added to the medium and the cells were incubated for 3 h at 37 °C. Research-grade human corneo-scleral tissue was obtained from Northwest Lions Eye Bank (Seattle, WA, USA). To examine mouse eyes, pregnant females (ICR, E12.5) were acquired from SLC Japan (Shizuoka, Japan). All animal experimentation was performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and was approved by the animal ethics committee of Osaka University.