showed that the size and morphology of the clone could notpredict whether it would eventually be holoclone or mer-oclone.

Immunocytochemistry for limbal progenitor markerp63abright cells quantified at the first passage showed morep63abright cells in cultures grown with low calcium Xeno-freemedia with 10 ng/mL EGF and less cells in X-MCBD medium (P¼ 0.01, Fig. 3F, Supplementary Fig. S3A), but no significantdifference among the rest of the media and the baselinecondition, which had an average number of 8% 6 2.3% of

p63abright cells cultured with X-KGM. Immunocytochemistry

expression for limbal stem cell markers was high for p63a,

ABCG2, C/EBPd, and K15, and low or absent for K12 andMUC1 at the first passage for all conditions evaluated. We

found it difficult to predict the amount of passages from

evaluating LSC marker expression under the microscope,

except in extreme cases of only 1 to 2 passages in which wecould see more K12 expression and less p63abright cells at P1.

The quantified p63abright expression in cytospins showed a

positive correlation with the number of passages that was

FIGURE 6. Further evaluation of human fibroblast feeders MRC-5 and F-HDF with Xeno-free medium. Human lung fibroblasts MRC-5 and F-HDFfeeders supported LEC cultures of different morphology at first passage; colonies looked more round and separated from the 3T3-J2 or MRC-5feeders with either Xeno or Xeno-free medium, while F-HDF feeders supported colonies that were more difficult to identify among the feeders (35,tissue culture phase microscope, [A] first column). In higher magnification (320), phase photographs and immunocytochemistry for progenitor cellmarker p63a (red), with nuclear counterstain DAPI (blue) overlay ([A], right column) showed presence of limbal progenitor cells growing in more(3T3-J2 or MRC-5) or less (F-HDF) distinct colonies ([A], right column). The RT-PCR ratio of p63a/K12 showed that MRC-5 feeders with XF mediumcould support favorable ratio for progenitor cell expansion comparable to 3T3-J2 feeders with X-KGM medium for multiple weekly passages (B).Points represent mean and SEM of four replicates, normalized to isolated limbal epithelial cells before seeding (P0) in parallel in the four differentconditions under 20% O2. Xeno-free (XF) medium had calcium 0.1 mM and EGF 10 ng/mL.

Culture Conditions for Limbal Stem Cell Function IOVS j January 2014 j Vol. 55 j No. 1 j 381

close, but not statistically significant (q 0.34, P ¼ 0.063). TheRT-PCR ratio of p63a/K12 differentiation marker expressionshowed no significant difference among all the media used (P¼0.79, Fig. 3G, Supplementary Fig. S3B).

Oxygen tension of 20% supported more passages with lowcalcium Xeno-free medium, while 5% O2 supported morepassages with baseline X-KGM media (P ¼ 0.0001, Fig. 4A).Xeno-free medium under 20% O2 also showed more p63abright

cells with immunocytochemistry (P ¼ 0.01, Fig. 4B), and nosignificant difference in RT-PCR ratio p63a/K12 (Fig. 4C) oraborted colonies (Fig. S2G). Univariate analysis on p63abright

cells showed higher expression in 20% than 14% or 5% O2 (P¼0.031), while there was no significant effect of oxygen on RT-PCR ratio of p63a/K12 (P ¼ 0.12).

Xeno-free medium with low calcium 0.1 mM and EGF 10ng/mL was selected as the best performing Xeno-free mediumto be evaluated with human feeders under 20% and 5% oxygentension.

Human Feeders With Xeno-Free Medium

Five different human fibroblast feeders, namely lung fibroblastsMRC-5; dermal fibroblasts, either fetal (F-HDF), neonatal (N-HDF), adult (A-HDF); and adult limbal fibroblasts were tested asalternatives to murine fibroblast 3T3-J2 feeders. Limbalepithelial specimens from 14 human volunteers of age 66 64 years (range, 25–87), 2 male and 12 female, were dissociatedinto single cell suspension, and seeded in parallel ontodifferent feeders with either X-KGM or Xeno-free mediumwith 0.1 mM calcium and 10 ng/mL EGF, and grown under 20%or 5% O2. The condition of 3T3-J2 feeders with X-KGM at 20%O2 was the baseline condition included in every experiment.

The MRC-5, F-HDF, N-HDF, and baseline 3T3-J2 feeders hadmuch lower percentage of aborted colonies than A-HDFfeeders (P ¼ 0.0001, Fig. 5A). The MRC-5, F-HDF, and A-HDFfeeders with Xeno and Xeno-free media, and N-HDF with Xenomedia showed more p63abright cells (P ¼ 0.022, Fig. 5B) andhigher RT-PCR ratio p63a/K12 (P¼ 0.03, Fig. 5C). The A-HDFcould not support more than 3 passages of LECs in culture,while MRC-5, F-HDF, and 3T3-J2 supported at least eightpassages, and N-HDF supported approximately five passages.Limbal fibroblasts could support only minimal growth of LECs,with very few and small differentiated colonies that could beonly passaged once or twice. Limbal fibroblasts, therefore,were not analyzed further (results not shown). No significantdifference was found between 20% and 5% O2 (P¼ 0.35). TheMRC-5 and F-HDF feeders at 20% O2 were selected for furtheranalysis.

Colonies grown on MRC-5 feeders with Xeno-free mediumat 20% O2 were compact, round, and separated from thefeeders, similar to colonies grown on 3T3-J2 feeders, whilecolonies grown on F-HDF feeders were more difficult toidentify among the feeders (Fig. 6A). Cell size analysis duringmultiple passages showed no statistically significant differencebetween MRC-5 or F-HDF feeders with XF media and thebaseline condition (Supplementary Fig. S4, results from MRC-5and baseline condition shown). The CFE showed a gradualdecrease during multiple passages that was not statisticallydifferent between MRC-5, F-HDF feeders, and baseline condi-tion (Supplementary Fig. S5, results from MRC-5 and baselinecondition shown). Colonies on all feeders showed goodexpression of p63a at P1 (Fig. 6A), which lasted throughoutmultiple passages. The RT-PCR ratio of p63a/K12 over multiplepassages showed similar results of sustained high ratio forMRC-5 feeders with Xeno-free medium and for baseline 3T3-J2feeders with X-KGM, while F-HDF feeders with Xeno-freemedium had comparable levels, but with less stable course,and 3T3-J2 with Xeno-free medium dropped the ratio earlier

than with X-KGM medium (Fig. 6B). Colonies grown on MRC-5,F-HDF, and 3T3-J2 feeders with Xeno-free medium demonstrat-ed high expression of p63a, ABCG2, C/EBPd, and K15, withminimal or no expression of cornea (K12) and conjunctiva(MUC1) differentiation markers from the first passage (Supple-mentary Fig. S6, results from MRC-5 feeders shown). Expres-sion of LSC genes with immunocytochemistry persisted topassage 7 and it was more robust in colonies grown on MRC-5feeders than F-HDF, and similar to expression in coloniesgrown on 3T3-J2 feeders with Xeno medium (Figs. 7A–D,results from MRC-5 feeders shown). The RT-PCR for p63a,ABCG2, C/EBPd, Bmi1, and K12 during multiple passagesshowed robust upregulation of mostly p63a and ABCG2 withdownregulation of K12, with a pattern similar in coloniesgrown with MRC-5 or F-HDF, or 3T3-J2 feeders, with F-HDFfeeders showing a less stable expression pattern (Fig. 7E,results from MRC-5 feeders shown). A summary of all theresults obtained is presented in Supplementary Table S5.

Overall, MRC-5 feeders with Xeno-free low calcium mediumat 20% O2 were as robust as the baseline 3T3-J2 feeders withXeno medium in supporting limbal epithelial stem cell culture.Cells grown in this condition exhibited appropriate colony andcell morphology, sustained expression of appropriate markerswith immunocytochemistry and RT-PCR, low percentage ofaborted colonies, and the ability to support multiple passages,indicating preservation of LSCs during cultivation.

DISCUSSION

To our knowledge, our study is the first to demonstrate thatlimbal stem cells can be expanded in vitro under Xeno-freeconditions, by sustained multiple passages until exhaustion,low percentage of aborted colonies, and appropriate pheno-type with high expression of the limbal progenitor markerp63a. The efficacy of this Xeno-free system is as robust as thebaseline culture condition with xenobiotic murine feeders,bovine serum, and cholera toxin. This baseline conditionreported by Pellegrini et al.3 and Rama et al.,14 based on theGreen’s lab culture method, has been validated extensivelywith clonal analysis as well as clinically, using percentage ofp63bright cells and aborted colonies. Our results matched theseprior reports of this baseline condition and demonstrated thatonly appropriate Xeno-free culture conditions can supportfunctioning LSCs in vitro for multiple passages with appropri-ate phenotype.

In our study human lung fibroblasts MRC-5; dermalfibroblasts of fetal, neonatal, or adult origin; as well as limbalstromal fibroblasts were tested in parallel as feeders. Ourresults agreed with prior publications of MRC-5 feeders used asfeeders for epithelial cells,21,43,44 but for the first time to ourknowledge we demonstrated that these feeders can supportmultiple passages under Xeno-free conditions, while keepingprogenitor cell markers expressed in similar levels withbaseline Xeno condition. The MRC-5 is a diploid cell linederived from human fetal lung tissue that can attain 48population doublings before declining. It is not carcinogenic inanimal models and it has been used extensively for theproduction of human vaccines.45,46 The MRC-5 feeders do notdepend as much on bovine serum, but in our case theyperformed well with 5% human serum, which was shownrecently to be the best alternative to bovine serum for culturingconjunctiva cells.43 Limbal epithelial cultures on MRC-5feeders had higher CFE without serum compared to 10%bovine serum.21 Human dermal fibroblasts, in our hands,showed significant variability in their ability to supportcultures. These results agreed with prior reports using Xenomedia, where only 2 of 4 newborn and 1 fetal dermal fibroblast

Culture Conditions for Limbal Stem Cell Function IOVS j January 2014 j Vol. 55 j No. 1 j 382

FIGURE 7. Limbal epithelial cells express appropriate markers of LSCs in multiple passages under Xeno-free culture conditions. Colonies at passage7 expressed limbal progenitor cell marker p63a or p63 (red, [A–D]) as well as ABCG2 (green, [A]), C/EBPd (green, [B]), cytokeratin K15 (K15,green, [C]), and cornea differentiation marker cytokeratin 12 (K12, green, [D]), and were counterstained with DAPI (blue, [A–D]) for overlays ([A–D]). The RT-PCR expression (mean and SEM from 4 replicates) of limbal progenitor cell markers p63a, ABCG2, Bmi1, C/EBPd, and differentiationmarker K12, was normalized to isolated limbal epithelial cells before seeding (P0), and was preserved for multiple weekly passages (E).

Culture Conditions for Limbal Stem Cell Function IOVS j January 2014 j Vol. 55 j No. 1 j 383

lines could support growth of LECs,13 and human dermalfibroblasts of unknown donor age supported growth of LECson amniotic membrane.17 Human limbal stromal fibroblastshave been shown to support limbal epithelial culture,18 andthey would have the advantage of origin next to the LSC niche,and readily availability from cadaveric donor tissue, but in ourhands they could not support robust colonies or multiplepassages. Our results are in agreement with results from theonly other group to our knowledge that tried limbal fibroblastsas feeders, where they could support only one to two passages(Harkin DG, personal communication, 2013).18 Despite theadvantage of limbal fibroblasts’ origin next to the native niche,these cells are not specialized niche cells, and the specificculture conditions that have been formulated using differentkinds of fibroblasts likely make it difficult to reproduce thelimbal niche and support LSC cultures in multiple passages.Overall, MRC-5 feeders showed the most consistent support ofLSCs in Xeno-free culture in levels similar to baseline Xenoconditions, when evaluated by multiple passages, percentageof aborted colonies, and expression of limbal progenitormarkers.

Oxygen tensions of 20%, 14%, and 5% in this study did notshow statistically significantly consistent universal effect onsupporting limbal cultures. Instead, we observed that differentoxygen tensions had varying effects on specific combinationsof feeders and media. For example, 5% O2 supported morepassages on 3T3-J2 feeders with Xeno media, while 20% O2

was better on the same feeders with Xeno-free media. We alsodid not find any statistically significant difference between 5%and 20% O2 among cultures using human feeders with Xeno-free media. We suggested that this differential effect of oxygenis responsible for the conflicting results in the literature, wheresome publications advocate beneficial effect with 2% to 5%O2,

32,37 and others with 14% O2.34 Other studies suggest that

hypoxia promotes terminal differentiation of limbal epithelialcells,33 while hypoxia is a well recognized cause of LSCD.47 Ofnote, our results from an extended number of samples culturedin parallel demonstrated that the effect of oxygen on onecombination of feeders and media does not predict that thesame effect will be true in another combination of feeders-media, likely because they create a different niche-likeenvironment.

A new formulation of Xeno-free medium, based on thestandard keratinocyte growth medium, showed the bestperformance. We found that low calcium and Xeno-freeadditives that have been found to be beneficial in other culturesystems, like albumin, transferrin, selenium, nonessentialamino acids, and pyruvate, allowed us to decrease the amountof human serum to 5%, but 3T3-J2 feeders could not supportgrowth of limbal epithelial cells under serum-free conditions.Our results agreed with the previous report on the effect oflower calcium and EGF on preserving stem cell pheno-type.12,48 This new medium formulation is a Xeno-freealternative to the baseline Xeno medium, since it performedwell with murine 3T3-J2 feeders, but it performed even betterwith human MRC-5 feeders.

Isolation methods with gentle trypsinization showedimproved colony-forming efficiency, likely due to lowertoxicity of the trypsin as well as the potential presence ofmore cell clusters at the beginning of the culture. Donor age, inthis study, showed a statistically significant negative effect onnumber of passages in cultures grown from healthy volunteersand on CFE in cultures grown from cadaveric donor tissue. Forcadaveric tissue, prolonged preservation decreased yield andCFE. These results were in agreement with prior reports ofnegative effect of age on CFE,49 while explant cultures showedno effect of age,50 but a negative effect of increasedpreservation time on CFE51 or a delay on initial expansion of

the culture.50 Of note though, a specimen from an older than90-year-old healthy volunteer, in our study, still grew coloniesthat sustained six passages. So, younger age probably isbeneficial in cases where this option may exist, but older agedoes not preclude good stem cell function.

Limitations of this study arise from our ability to replicatepreviously published methods exactly the way they wereperformed initially. Other limitations include the inherentvariability of individual human tissue, both for limbal samplesas well as for human feeders, which we tried to mitigate byusing multiple samples and growing cultures in parallel, alwaysincluding the same baseline condition.

In conclusion, our results demonstrated that LSC functioncan be preserved in vitro under Xeno-free conditions. Thatallows for the best support of lifelong corneal epithelialregeneration, while minimizing the risk of transmitting anydisease through xenobiotic factors. To our knowledge, this isthe first extensive comparison of Xeno-free methods that hasused rigorous evaluation of stem cell function, namely serialpassages, as well as clinically validated quality controlmeasurements of quantification of p63abright cells, andpercentage of aborted colonies. Our results showed thatcertain Xeno-free conditions can support limbal epithelialstem cell culture under good manufacturing practices facilitat-ing translation into clinical applications.

Acknowledgments

The authors thank the Lions Eye Bank of Delaware Valley, StephenE. Orlin and Michael E. Sulewski for the supply of donor tissue, andJamie L. Ifkovits and Lori D. Kellam for critical reading of themanuscript.

Supported by funding from NIH Grant K12 EY015398, Pennsylva-nia Commonwealth Universal Research Enhancement (CURE)Program 0426/554248/8319, and a Research to Prevent Blindnessunrestricted grant.

Disclosure: K. Stasi, None; D. Goings, None; J. Huang, None; L.Herman, None; F. Pinto, None; R.C. Addis, None; D. Klein,None; G. Massaro-Giordano, None; J.D. Gearhart, None

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