Nieto-Nicolau et al. Stem Cell Research & Therapy (2019) 10:374 https://doi.org/10.1186/s13287-019-1501-9 RESEARCH Open Access Xenofree generation of limbal stem cells for ocular surface advanced cell therapy Nuria Nieto-Nicolau1,2†, Eva M. Martínez-Conesa1,2†, Alba M. Velasco-García3, Caterina Aloy-Reverté1,2, Anna Vilarrodona1,2 and Ricardo P. Casaroli-Marano1,2,3* Abstract Background: Limbal stem cells (LSC) sustain the corneal integrity and homeostasis. LSC deficiency (LSCD) leads to loss of corneal transparency and blindness. A clinical approach to treat unilateral LSCD comprises autologous cultured limbal epithelial stem cell transplantation (CLET). CLET uses xenobiotic culture systems with potential zoonotic transmission risks, and regulatory guidelines make necessary to find xenofree alternatives. Methods: We compared two xenofree clinical grade media and two feeder layers. We used CnT07, a defined commercial medium for keratinocytes, and a modified xenofree supplemented hormonal epithelial medium with human serum (XSHEM). Optimal formulation was used to compare two feeder layers: the gold standard 3T3 murine fibroblasts and human processed lipoaspirate cells (PLA). We tested the expressions of ΔNp63α and cytokeratin 3 and 12 by qPCR and immunofluorescence. Morphology, viability, clonogenicity, proliferation, and cell growth assays were carried out. We also evaluated interleukin 6 (IL-6) and stromal-derived factor 1 (SDF-1) by qPCR and ELISA. Results: XSHEM maintained better LSC culture viability and morphology than CnT07. Irradiated PLA feeder cells improved the undifferentiated state of LSC and enhanced their growth and clonogenicity stimulating IL-6 secretion and SDF-1 expression, as well as increased proliferation and cell growth when compared with irradiated 3T3 feeder cells. Conclusions: The combination of XSHEM and PLA feeder cells efficiently sustained LSC xenofree cultures for clinical application. Moreover, PLA feeder layers were able to improve the LSC potential characteristics. Our results would have direct clinical application in CLET for advanced therapy. Keywords: Cultured limbal stem cells transplantation, XSHEM, CnT07, Processed lipoaspirate cells, 3T3, Clinical grade, Limbal stem cells deficiency Background cytokeratin (CK) 12 and CK3 [2]. The loss of LSC pro- The transparency and the integrity of the cornea are duces new vessel formation, corneal conjunctivalization, maintained by a subset of stem cells located at the epi- and scarring, leading to corneal blindness [4]. Limbal thelial basal layer of the limbus, an anatomic circumfer- stem cell deficiency (LSCD) can be caused by chemical, ential area that separates the transparent cornea from traumatic, and infectious insults and also by genetic eti- the conjunctiva [1]. These stem cells are called limbal ologies [4]. Its prevalence is increasing, due to the use of stem cells (LSC), and they are defined by their small size, corrosive cleaners in the household field [5]. Approxi- high nucleus-to-cytoplasm ratio [1, 2], and positivity for mately, LSCD affects approximately 10 million people the putative stemness marker ΔNp63α [2, 3] as well as worldwide [6, 7]. negativity for corneal epithelial differentiation markers Although the technique for the treatment of unilateral LSCD has evolved with time, the current gold standard * Correspondence: rcasaroli@ub.edu treatment for unilateral LSCD is cultured limbal epithelial † Nuria Nieto-Nicolau and Eva M. Martínez-Conesa are co-first authors and stem cell transplantation (CLET) [8]. For CLET, the LSC contributed equally to this work. 1 Barcelona Tissue Bank, Banc de Sang i Teixits (BST), Barcelona, Spain can be cultured by explant or cell suspension systems [9]. 2 Institute of Biomedical Research (IIB-Sant Pau; SGR1113), Barcelona, Spain In the first system, a small biopsy of the healthy limbus is Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Nieto-Nicolau et al. Stem Cell Research & Therapy (2019) 10:374 Page 2 of 12 seeded on amniotic membrane. The cells grow, and the cells (PLA) from fresh human lipoaspirates were collected sheet is transplanted onto the damaged eye. In the cell from healthy donors, during plastic liposuction procedures, suspension approach, LSC obtained from a minimally inva- in planned lipoaspiration surgeries. PLA were obtained by sive limbal biopsy are enzymatically disaggregated and stromal vascular fraction isolation, and cultured and charac- ex vivo expanded on an inactivated feeder layer of 3T3 terized as previously described [24]. PLA cells accomplished murine fibroblasts until sub-confluence. Then, the cells are the criteria for mesenchymal stem cell characterization (data detached and seeded on a biocompatible carrier for trans- not shown) [25]. Then, both 3T3 and PLA cells were inacti- plantation [8, 9]. This approach is more advantageous than vated by irradiation with 6000 rads. After this, cells were explant systems, since it reduces the risk of contamination plated onto culture dishes at 2 × 104 cells/cm2 for feeder- of the culture by other limbal cells (such as stromal fibro- layer use or downstream experiments. blasts) [10] and increases the amount of cells that can be obtained due to higher proliferation rates [11–13]. More- Human LSC and corneal epithelial cells over, the cell suspension cultures are an optimized option Cadaveric adult human limbal tissues from six different since they are more enriched in stem cell progenies than donors were obtained from the Barcelona Tissue Bank explant culture methods [10, 12, 13] leading to improved (BTB-BST, Barcelona, Spain; http://www.bancsang.net/ outcomes [14]. en_index/). LSC were isolated as previously described Since the first use of CLET for human LSCD treat- [26, 27]. LSC from each donor were equally divided and ment in 1997 [15], xeno-products have been used for cultured until sub-confluence with xenofree supple- the ex vivo expansion of LSC both in explant and cell mented hormonal epithelial media (XSHEM) or CnT07 suspension systems [16]. However, European regulatory medium (CellnTec, Bern, Switzerland) on 3T3 or PLA guidelines [17, 18] for the safety and quality of human feeder layers that were seeded 24 h before. XSHEM com- tissues and cells encourage the implementation of standard position consisted of the following: Dulbecco’s modified operating procedures to prevent the use of xenogeneic Eagle’s medium/Ham’s and F-12 (2:1 vol:vol) (DMEM/ compounds and the potential associated contamination. Al- F12; Invitrogen, Carlsbad, CA) supplemented with 2 mM though the use of xeno-products supposes a risk for human L-glutamine (Lonza, Verviers, Belgium), 5 μg/ml human health by virus, prions, and zoonoses transmission [19], lit- insulin (Sigma Aldrich, Munich, Germany), 10 ng/ml hu- tle research has been made to avoid xenobiotics during man epidermal growth factor (hEGF, Sigma-Aldrich), LSC culture in suspension systems [20–22]. Besides, trad- 0.5% dimethyl sulfoxide (DMSO, Sigma-Aldrich), 0.4 μg/ itional LSC media for clinical application contain cholera ml hydrocortisone (Sigma-Aldrich), 2 nM triiodothyronine toxin [15, 23], increasing the risk of disease transmission. (Sigma-Aldrich), 0.18 mM adenine (Sigma-Aldrich), and So, substitution of the LSC culture medium containing 10% Human AB Serum (Corning, Manassas, VA). After serum and xenobiotics from animal origin, along with the the isolation passage, cells were used for downstream replacement of the feeder layer of murine 3T3 fibroblasts applications. Human corneal epithelial cells (CO) were by xenofree alternatives, is of utmost significance. obtained by mechanical scrapping of the central corneal To study clinical grade xenofree alternatives that could epithelium of five different donors, avoiding the perilimbal serve to maintain LSC cultures for cell therapy in region, and used as control for qPCR experiments. humans, we tested two xenofree media and two feeder layer approaches. We compared a defined commercial Colony-forming assay (CFA) and doubling population medium, designed to sustain keratinocyte growth, with a time (DPT) supplemented hormonal epithelial medium complemen- For CFA determination, LSC that were previously cul- ted with human serum (XSHEM). Then, we performed a tured on 3T3 or PLA feeder layers were seeded in 35- comparison between the gold standard murine 3T3 fi- mm-diameter plates and cultured for 14 days [28] with broblasts [16] and human processed lipoaspirate cells 3T3 feeder-layer support. Colonies were fixed and stained (PLA) as a feeder layer for LSC culture growth. Our re- with 0.5% crystal violet in methanol. Analysis was per- sults propose a clinical grade alternative free of xenobi- formed according to previous criteria and presented as a otics to sustain optimal LSC culture growth with direct percentage after applying the previously described formula clinical application for advanced therapy. [29]. The diameter of each colony was measured using ImageJ software [30]. DPT was calculated as described Materials and methods elsewhere [31]. Cell culture of feeder layers Murine 3T3 Swiss Albino fibroblasts were obtained from Immunofluorescence (IF) Kerafast (3T3-J2, EF3003). Cells were cultured with DMEM Cells of each experimental group (5 × 105) were added 4.5 g/l (Thermo Scientific, MA, USA) supplemented with to ThinPrep® PreservCyt solution (Hologic Iberia SL, 10% FCS and 1% antibiotics. Human processed lipoaspirate Barcelona, Spain) for fixation and preservation. Cells Nieto-Nicolau et al. Stem Cell Research & Therapy (2019) 10:374 Page 3 of 12 were then transferred to slides using ThinPrep 3000 pro- Viability calculation cessor (Hologic), which allowed the cells to be seeded in a Viability was tested using live/dead assay (Invitrogen) be- single plane without forming clumps. Slides were pre- fore and after detachment of the cultures following the served in methanol until use, permeabilized, blocked, and manufacturer’s instructions. Moreover, viability calcula- then incubated with primary antibodies. After several tion was performed using trypan blue exclusion assay on a washes in 100 mM PBS solution, proper secondary anti- Neubauer chamber after detachment of the cells with Try- body was added for 60 min at 37 °C in a humidified cham- pLE Select® (Sigma-Aldrich). ber. The antibodies and concentrations used are detailed in Additional file 2: Table S1. Cells were observed in an Statistical analysis epifluorescence microscope (BX61; Olympus R-FTL-T; Experiments were performed in triplicate. A two-tailed Olympus America Inc., Center Valley, PA), coupled with a Student’s t test was run, and p values < 0.05 were consid- program for digital image acquisition (Olympus DP Con- ered statistically significant (PRISM, version 6.0 Graph- troller Program). Images were processed with ImageJ soft- Pad Software, San Diego, CA). Results are presented as ware [30]. the mean ± standard error (MD ± SE) or, in the case of the qPCR analysis, mean ± standard deviation (MD ± SD). mRNA extraction and quantitative polymerase chain reaction (qPCR) analysis Results Total RNA was extracted from co-cultures of LSC with ei- XSHEM produced cells with LSC morphology and higher ther 3T3 or PLA feeder layers or from monocultures of the viability feeder layers at the last day of LSC culture. The extraction We compared the culture features and the morphology was performed using RNA Purelink Mini Kit (Ambion, of LSC when cultured with CnT07 and XSHEM Invitrogen), following the manufacturer’s instructions. The medium. Moreover, we determined their doubling popu- RNA concentration was measured using NanoDrop lite lation time (DPT) and the viability at the end of the spectrophotometer (Thermo Scientific). RNA (1 μg) was culture (Fig. 1). Cells cultured in CnT07 were small and reverse-transcribed using Superscript III (Invitrogen) ac- cuboidal with a characteristic cobblestone morphology cording to the manufacturer’s instructions. Then, cDNA and grew forming a single monolayer (Fig. 1d, f). Usually, (1 μl) was used for qPCR in a final volume of 18 μl with these cultures showed bigger cells with lower nucleus-to- Lightcycler 480 Sybr Green I Master (Roche, Barcelona, cytoplasm ratio, interspersed between the smaller cells Spain) and a 0.2-μM primer concentration. The qPCR was (Fig. 1h). Cells cultured with XSHEM were small and pol- performed using Lightcycler 480 II (Roche) hardware and ygonal (Fig. 1e), and grew from colonies that rapidly software. The expression level of target genes was normal- merged forming thicker stratifications (Fig. 1c, e, g). At the ized to internal 18s (rrn18s, TATAA Biocenter, Sweden) end of the culture, before recombinant protease detach- and represented as relative expression using 2-ΔΔCt formula. ment, live/dead assay showed that cells had similar viability The sequences and annealing temperatures of PCR primers (Additional file 1: Figure S1). After recombinant protease are listed in Additional file 2: Table S2. detachment, cells cultured with CnT07 presented a more impaired viability than those in XSHEM medium, as showed by trypan blue exclusion assay (Fig. 1a) and by live/ ELISA assay dead assay (Additional file 1: Figure S1). However, DPT did Cell culture medium was recovered at every change of not show differences between both culture media (Fig. 1b). medium and was centrifuged at 13,000 rpm during 5 min. Supernatants were stored at − 80 °C until analysis. LSC in XSHEM and in CnT07 were positive for p63 and ELISA assay for interleukin-6 (IL-6) was performed with negative for corneal differentiation markers a specific human ELISA kit for IL-6 (Biosource Europe, We evaluated the expression of ΔNp63α, other characteris- Medgenix, Nivelles, Belgic) according to the manufac- tic stem cells, and corneal markers such as Bmi1, ABCG2, turer’s instructions. CK15, CK19, PAX6, CK12, and CK3, by qPCR and im- munofluorescence (Fig. 2). Both XSHEM and CnT07 lead to LSC highly positive for ΔNp63α as demonstrated by im- Metabolic assay munofluorescence (Fig. 2d), with similar expression by Cell growth was tested using WST-1 assay (Abcam, qPCR (Fig. 2b). Meanwhile, the expression of Bmi1, ABCG2, Cambridge, UK) attending the manufacturer’s recom- and CK15 was higher in XSHEM (Fig. 2c). The mRNA ex- mendations at every medium change. Plates were read at pression of CK3, CK12, and PAX6 was negligible when 450 nm with a reference wavelength of 680 nm in an ab- compared to corneal epithelial cells (CO); the positive con- sorbance plate reader (Biotek). trol obtained directly scrapped form central corneas (Fig. 2a, Nieto-Nicolau et al. Stem Cell Research & Therapy (2019) 10:374 Page 4 of 12 Fig. 1 Comparison of the DPT, viability, and morphology of LSC grown with XSHEM or CnT07. a Evaluation of the viability by trypan blue exclusion assay. Cell viability was higher with XSHEM medium after cell detachment. b DPT did not show differences between both media. c Morphology of a starting culture of LSC cultured in XSHEM, corresponding to one clone in expansion. d Morphology of a starting culture of LSC cultured in CnT07. No clones could be observed. e Morphology of LSC cultured in XSHEM. Cells were small and polygonal. f Morphology of LSC cultured in CnT07. Cells had a cobblestone morphology, but differentiated cells could be observed (arrows). g XSHEM cultures showed stratifications as clones grew. h CnT07 cultures only grew forming monolayers and did not form any stratification. Results are presented as mean ± SE from three independent experiments. Statistical analysis was performed using two-tailed Student’s t tests (***p < 0.001). DPT, doubling population time; LSC, limbal stem cells; XSHEM, xenofree supplemented hormonal medium. Bar = 100 μm for c to g. Bar = 50 μm for h c). LSC cultured in CnT07 showed higher mRNA expres- PLA increased the clonogenicity and growth of LSC sion for CK3 and PAX6 than those cultured in XSHEM Taking into account previous results, XSHEM medium (Fig. 2a). However, both cultures were negative for CK3 and was selected to assay the potential of PLA as feeder CK12 as assayed by immunofluorescence (Fig. 2d). layers for LSC. To this end, we compared LSC cultured Nieto-Nicolau et al. Stem Cell Research & Therapy (2019) 10:374 Page 5 of 12 Fig. 2 Expression of stemness and differentiation markers in LSC grown in XSHEM and CnT07. a Analysis of the mRNA expression levels for corneal differentiation markers CK3 and CK12. Their expression was minimal when compared with CO, although LSC cultured in CnT07 expressed more CK3 than those cultured in XSHEM. b Analysis of the mRNA expression levels for the stemness marker ΔNp63α did not show differences between LSC cultured in XSHEM or CnT07. c Analysis of the mRNA expression levels for Bmi1, ABCG2, CK19, CK15, and PAX6. d Immunofluorescence for p63, CK3, and CK12. Cultures showed a broad positivity for p63, while were widely negative for CK3 and CK12. Results are presented as mean ± SE from three independent experiments. Statistical analysis was performed using two-tailed Student’s t tests (*p < 0.05; ***p < 0.001; n.s, not significant). CK, cytokeratin; CO, human corneal epithelial cells; XSHEM, xenofree supplemented hormonal medium. Bar = 10 μm for p63. Bar = 25 μm for CK3 and CK12 either with PLA or with 3T3, the gold standard feeder confluence than the LSC grown on 3T3 (LSC-3T3). layer for CLET, for clonogenicity, DPT, and cell growth Therefore, the DPTs were lower in these LSC (Fig. 3c), using WST-1 assay (Fig. 3). The LSC cultured on PLA indicating faster cell growth. In fact, cell growth of LSC- feeder layer (LSC-PLA) took less time to reach sub- PLA increased with culture time up to reaching sub- Nieto-Nicolau et al. Stem Cell Research & Therapy (2019) 10:374 Page 6 of 12 Fig. 3 Cell growth, viability, and clonogenicity of the LSC cultured on murine 3T3 or human PLA feeder layers. a Cell growth evaluated by WST-1 was higher in LSC grown on PLA feeder layers during culture. b Evaluation of the LSC viability by trypan blue exclusion assay did not show differences after cell detachment. c DPT was higher in LSC cultured on PLA feeder cells. d Clonogenicity was increased in LSC cultured on PLA feeder cells (n = 6). e The colony diameters were larger in LSC cultured on PLA feeder cells. f CFA results are presented as mean ± SE from at least three independent experiments. Statistical analysis was performed using two-tailed Student’s t tests (**p < 0.01; ***p < 0.001). CFA, colony forming assay; D, day; DPT, doubling population time; LSC, limbal stem cells; LSC-3T3, co-cultures of LSC with 3T3 feeder layers; LSC-PLA, co-cultures of LSC with PLA feeder layers; PLA, processed lipoaspirate cells confluence at day 6 (D6). Instead, co-cultures of LSC- points in the supernatants of co-cultures and in the PLA 3T3 showed decreased cell growth at day 4 of culture and 3T3 cultures (Fig. 4a). Interestingly, at the first 24 h (D4), and then, cell growth increased until the end of (D1), PLA feeders secreted their highest amount of IL-6. the culture at day 10 (D10) (Fig. 3a). Accordingly, LSC- At day 2 (D2), the IL-6 levels decayed in these PLA cul- PLA presented more ki67-positive cells at the end of the tures. At this time point, the initiated co-cultures of LSC- culture (Additional file 1: Figure S2), showed higher clo- PLA started to increase their expression of IL-6. These nogenic capacity, and exhibited clones with bigger diam- levels of IL-6 secretion continued to increase remarkably eter (Fig. 3d, f). Moreover, viability tests by trypan blue until the end of the culture, at day 7 (D7). Conversely, assay and live/dead assay did not show differences after PLA cultures retained lower levels of IL-6 until the end. the protease detachment of the cultures (Fig. 3b and LSC-3T3 secreted lower levels of IL-6 in comparison with Additional file 1: Figure S1). No obvious cell death could LSC-PLA. IL-6 secretion by LSC-3T3 reached their max- be observed at the end of the cultures as well. imum secretion at day 7 (D7), 2 days before reaching sub- confluence. As expected, due to the specificity for human LSC-PLA expressed more IL-6 and SDF-1 than LSC-3T3 IL-6, no IL-6 could be detected in the 3T3 cultures. So, a To elucidate if the possible mechanism through PLA qPCR was run with specific primers for mouse IL-6 could favor the improvement of clonogenicity and cell mRNA. The results showed that 3T3 cells expressed sig- growth of LSC, we analyzed the expression of IL-6 and nificantly lower mRNA expression than PLA (Fig. 3b). In SDF-1 (Fig. 4). The secretion of IL-6 was evaluated by spe- addition, qPCR results for IL-6 confirmed ELISA data re- cific human IL-6 ELISA assay at different culture time garding LSC-PLA and LSC-3T3 co-cultures. Nieto-Nicolau et al. Stem Cell Research & Therapy (2019) 10:374 Page 7 of 12 PLA showed a markedly increased expression of SDF-1 than the co-cultures of LSC and 3T3. Both PLA and 3T3 feeder generated cells with LSC characteristics Both LSC co-cultured on PLA and on 3T3 feeder layers were analyzed for their morphology and the expression of ΔNp63α, Bmi1, ABCG2, CK15, CK19, PAX6, CK3, and CK12 by qPCR and immunofluorescence. There were no differences regarding the morphology of LSC in both co-culture approaches; they were smaller, expanded from clones, and grew showing stratifications, until reaching sub-confluence (Fig. 5). Moreover, there were also no differences in the percentage of p63-positive cells in co-cultures, showing a broad positivity for ΔNp63α, as assayed by immunofluorescence (Fig. 6b, c). Results from qPCR corroborated similar mRNA expression for ΔNp63α between conditions (Fig. 6a). However, in- creased mRNA expression for Bmi1 and CK15 was de- tected in LSC-PLA without significant differences regarding ABCG2, CK19, and PAX6 expression (Fig. 7a). Negativity for CK3 and CK12 was observed by immuno- fluorescence and qPCR in LSC-PLA or LSC-3T3, and lower mRNA expression of PAX6 was detected in com- parison with CO (Fig. 7a, b). Discussion The search for clinical grade xenofree alternatives to culture LSC for advanced therapy is a need. Here, we demonstrated that LSC maintained an undifferentiated state and an adequate morphology, and improved its doubling population time and stemness when cultured in XSHEM on PLA feeder layer, without the interven- tion of any xenobiotic. The maintenance of the size and the morphology of LSC have deep implications in their characterization [1, 2, 32]. LSC cultured with XSHEM media were Fig. 4 Expression of IL-6 and SDF-1. a ELISA assay detected increased expression of human IL-6 during the co-cultures of LSC smaller, with higher nucleus-to-cytoplasm ratio, and with PLA. PLA monocultures also secreted higher levels of IL-6 the cultures showed stratifications. Although stratifi- during the first day of seeding. b Analysis of mRNA expression levels cation in LSC cultures is prevented in cell therapy be- of IL-6 in 3T3 and PLA monocultures, and in LSC co-cultures with cause they can induce cell differentiation by cell both feeder layers. c Analysis of mRNA expression levels of SDF-1 in confluence, this is a well-known trait of LSC cultures 3T3 and PLA monocultures, and in LSC co-cultures with both feeder layers. Statistical analysis was performed using two-tailed Student’s t and an indication of the quality of the culture [33]. tests (**p < 0.01 compared to LSC-3T3; ***p < 0.001 compared to The cultures of CnT07 grew in monolayers, had an LSC-3T3; &p < 0.001 compared to PLA; %p < 0.01 compared to 3T3). impaired viability after detachment, and had lower ex- D, day; IL-6, interleukin-6; LSC, limbal stem cells; LSC-3T3, co-cultures pression of progenitor markers Bmi1 and ABCG2. of LSC with 3T3 feeder layers; LSC-PLA, co-cultures of LSC with PLA This pointed out that XSHEM maintains better LSC feeder layers; PLA, processed lipoaspirate cells; SDF-1, stromal derived factor culture characteristics than CnT07, although there were no differences in the expression of the putative stemness marker p63 by qPCR and immunofluores- cence. In addition, both medium conditions prevented When assayed by qPCR, monocultures of PLA showed the expression of corneal differentiation epithelial higher expression of SDF-1 than monocultures of 3T3 markers CK3 and CK12. Our results also support pre- cells (Fig. 3c). Accordingly, the co-cultures of LSC and vious data showing that LSC express lower levels of Nieto-Nicolau et al. Stem Cell Research & Therapy (2019) 10:374 Page 8 of 12 Fig. 5 Morphology of LSC cultured on 3T3 or PLA feeder layers. a, b LSC cultured either on PLA or on 3T3 formed clones when the culture started. c, d In both cultures, cells were small and polygonal. e–f Clones of both cultures grew, merged, and formed stratifications. LSC, limbal stem cells; LSC-3T3, co-cultures of LSC with 3T3 feeder layers; LSC-PLA, co-cultures of LSC with PLA feeder layers; PLA, processed lipoaspirate cells. Bar = 50 μm for a and b. Bar = 100 for c to f PAX6 when compared to more differentiated progen- conditions produce grafts enriched in differentiated cells, ies [34]. Moreover, our results are consistent with positive for CK3 and CK12, indicating the loss of stem previous research showing that serum supplemented cell progenies [21]. This highlights the importance of the media maintain better survival and enrichment of LSC feeder layer to culture LSC. Although 3T3 feeder cells than commercial defined keratinocyte media [35]. are necessary to maintain better cumulative LSC cell The use of non-defined media has drawbacks, such as numbers even with xenofree medium [20], the risk of batch to batch serum variations [36]; however, “in murine fibroblast use is the potential inflammatory re- house” formulations allow independence of commercial sponses against the graft generated by the presence of companies overcoming the need of further GMP valida- xeno-antigens in LSC transplant [39]. This would impair tions if the product became discontinued. Another draw- the results of the transplantation since any inflammatory back of a non-defined medium is the potential risk of reaction could lead to detrimental outcomes after graft- disease transmission [36]. However, this risk can also be ing [40–43]. Avoiding this risk, one study changed the managed effectively by the application of the European 3T3 feeder cells by human embryonic fibroblast cell line regulatory controls [17, 18]. Thus, microbiologic purity [22], demonstrating the same feasibility to support LSC of the media can be tested and further validated to avoid cell growth. Since the use of human embryonic cells en- disease transmission and assure an aseptic manufactur- tails ethical implications and is related to teratogenic ing process [37]. risks [44], somatic stem cell lines, such as PLA [45, 46], LSC growth under an undifferentiated state is proved may be more advantageous for clinical applications. Hu- with defined media only when supplemented with either man PLA are easy to obtain and isolate [45] and could human serum or synthetic supplements [20, 38]. Here, be used for allogenic or autologous feeder layer pur- we showed that our defined medium without serum sup- poses. Allogenic PLA feeder cells could be well charac- plementation hinders the viability of LSC, making the terized following international recommendations [25], culture less cost-effective. One explanation for the viabil- screened for paracrine secretion, optimized to sustain ity loss could be that the recombinant protease used for LSC growth, and GMP-banked [46–48]. Moreover, PLA cell detachment was not completely inhibited by defined cells do not express HLA DRII [49] being invisible to media, which usually contain low concentration of pro- the immune system and avoiding rejection in allogenic teins and other elements such as calcium. Then, the cell clinic application [50]. viability loss in CLET could impair the outcomes of the Successful clinical outcomes with CLET approach are re- transplantation by decreasing the survival of undifferen- lated to the presence of undifferentiated progenies [14]. tiated progenies [14]. For all these reasons, XSHEM Since our culture system with XSHEM medium and PLA medium represented a better alternative to culture LSC feeder layer generates undifferentiated LSC with higher clo- for human application and was chosen to test different nogenic potential, expressing more Bmi1, highly positive for feeder layers. p63 and negative for the expression of CK3 and CK12, it is LSC cultures obtained in suspension systems directly likely to provide better outcomes in clinical transplantation. on amniotic membrane without feeder layers in xenofree Moreover, we demonstrated that PLA feeders induced faster Nieto-Nicolau et al. Stem Cell Research & Therapy (2019) 10:374 Page 9 of 12 Fig. 6 Expression of stemness and differentiation markers in LSC cultured with PLA and 3T3 feeder layers. a Analysis of the mRNA expression levels for stemness marker ΔNp63α. b The number (%) of p63-positive LSC cultured on PLA or 3T3 feeder layers did not show differences (n = 100). c Immunofluorescence quantification of p63-positive LSC cultured on PLA or 3T3 feeder layers did not show differences (n = 100). Immunofluorescence showing broad positivity for p63. Results are presented as mean ± SE from three independent experiments. Statistical analysis was performed using two-tailed Student’s t tests (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant). CO, corneal epithelial cells; LSC, limbal stem cells; LSC- 3T3, co-cultures of LSC with 3T3 feeder layers; LSC-PLA, co-cultures of LSC with PLA feeder layers; PLA, processed lipoaspirate cells. Bar = 50 μm proliferation and cell growth on LSC, so the cultures were Finally, there is a close interaction between LSC and their more cost-effective. Moreover, the viability of LSC cultured microenvironment, such as their neighboring cells and on both feeders after detachment was similar. This fact extracellular matrix, which regulate their proliferation and makes sense, since both cultures used the same medium differentiation in the limbal niche [51]. When LSC are co- and the protease could be inhibited with the same efficacy. isolated with limbal stromal niche cells, the LSC proliferate Nieto-Nicolau et al. Stem Cell Research & Therapy (2019) 10:374 Page 10 of 12 Fig. 7 Expression of stemness and differentiation markers in LSC cultured with PLA and 3T3 feeder layers. a Analysis of the mRNA expression levels for CK3 and CK12 (corneal epithelial differentiation markers), and Bmi1, ABCG2, CK19, CK15 (putative LSC markers), and PAX6. There was minimal differences between LSC cultured on PLA or 3T3 feeder layers. b Immunofluorescence negative for CK12 and CK3. Results are presented as mean ± SE from three independent experiments. Statistical analysis was performed using two-tailed Student’s t tests (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant). CK, cytokeratin; CO, corneal epithelial cells; LSC, limbal stem cells; LSC-3T3, co-cultures of LSC with 3T3 feeder layers; LSC-PLA, co-cultures of LSC with PLA feeder layers; PLA, processed lipoaspirate cells. Bar = 25 μm faster and form more clones through the expression of SDF- PLA secreted high amounts of IL-6 in the first 24 h of seed- 1 [52], highlighting the importance of the communication ing, and its secretion could possibly influence the fate of between LSC and the stromal cells for their proliferation LSC culture. In addition, it is well known that murine IL-6 and maintenance. Moreover, IL-6 is secreted by limbal and does not have effect on human cells due to conformational stromal cells which act as a mediator between both cell pop- differences within the IL-6 molecules [54]. This fact also ulations, increasing the clonogenic potential and maintain- supports the reasoning of using feeder layers from human ing an undifferentiated state of LSC in in vitro culture origin to avoid species-specific effects. systems [53]. Here, we also demonstrated that co-cultures with PLA induced more secretion of IL-6 and SDF-1, than Conclusions co-cultures with 3T3, explaining the higher clonogenic cap- In summary, we demonstrated that the substitution of ability, and the faster proliferation and cell growth. Human xenobiotics by human-derived alternatives is a feasible Nieto-Nicolau et al. Stem Cell Research & Therapy (2019) 10:374 Page 11 of 12 clinical grade xenofree option for LSC cultures in ad- obtained, processed, and analyzed according to current guidance in relation vanced therapy for ocular surface regeneration. The use of to the collection and preservation of human tissues for clinical use (EEC regulations 2004/23/CE and 2006/17/CE). These samples were treated in XSHEM medium combined with PLA feeder layers not accordance with the protocol and legal requirements for the use of only maintained LSC characteristics but also improved the biological samples and biomedical research in Spain (Law 14/2007 and RD LSC potential. This approach will have a direct clinical ap- 1716/2011) and following the agreement on the use of human donated tissue for ocular transplantation, research, and future technologies (The plication in cell therapy for LSCD treatment. Barcelona Principles; www.gaeba.org/publications). In addition, the acquisition, processing, and preservation of the tissues of this study are in accordance with the Spanish Laws for the Development and Applications of Supplementary information Organ Transplants (RD 9/2014). All the information provided before donation, Supplementary information accompanies this paper at https://doi.org/10. together with informed consent, stressed that the samples obtained were to 1186/s13287-019-1501-9. be used for clinical application and/or applied research. The use, protection, communication, and transfer of personal data complied with local Additional file 1: Figure S1. Live/dead assay for LSC cultured in all regulations (Law 03/2018). conditions before and after protease detachment. No obvious cell death could be observed at the end of the cultures. After detachment, cultures with Cnt07 medium showed impaired viability. Both conditions cultured Consent for publication with XSHEM, LSC-3T3 and LSC-PLA, did not showed differences in Not applicable. This manuscript does not contain data from any individual viability. Results are presented as mean ± SE from 3 independent person. experiments. LSC, limbal stem cells; LSC-3T3, co-cultures of LSC with 3T3 feeder layers; LSC-PLA, co-cultures of LSC with PLA feeder layers; PLA, processed lipoaspirate cells. Bar = 100 μm. Figure S2. Competing interests Immunofluorescence for ki67 in LSC-3T3 and LSC-PLA. LSC-PLA showed The authors declare that they have no competing interests. higher percentage of ki67-positive cells than LSC-3T3 (n = 300). Results are presented as mean ± SE from 3 independent experiments. Statistical Author details 1 analysis was performed using two-tailed Student’s t-tests (*** p < 0.001). Barcelona Tissue Bank, Banc de Sang i Teixits (BST), Barcelona, Spain. 2 LSC, limbal stem cells; LSC-3T3, co-cultures of LSC with 3T3 feeder layers; Institute of Biomedical Research (IIB-Sant Pau; SGR1113), Barcelona, Spain. 3 LSC-PLA, co-cultures of LSC with PLA feeder layers; PLA, processed Department of Surgery, School of Medicine & Hospital Clinic de Barcelona, lipoaspirate cells. Bar = 25 μm. University of Barcelona, Barcelona, Spain. Additional file 2: Table S1. Primary and secondary antibodies. Table Received: 6 August 2019 Revised: 9 November 2019 S2. Primers and sequences. Accepted: 20 November 2019 Abbreviations CK: Cytokeratin; CO: Human corneal epithelial cells; DPT: Doubling References population time; IL-6: Interleukin-6; LSC: Limbal stem cells; LSC-3T3: Co- 1. Cotsarelis G, Cheng SZ, Dong G, Sun TT, Lavker RM. Existence of slow- cultures of LSC with 3T3 feeder layers; LSC-PLA: Co-cultures of LSC with PLA cycling limbal epithelial basal cells that can be preferentially stimulated to feeder layers; PLA: Processed lipoaspirate cells; SDF-1: Stromal-derived factor; proliferate: implications on epithelial stem cells. Cell. 1989;57(2):201–9. XSHEM: Xenofree supplemented hormonal medium 2. Schlötzer-Schrehardt U, Kruse FE. Identification and characterization of limbal stem cells. Exp Eye Res. 2005;81:247–64. 3. Pellegrini G, Dellambra E, Golisano O, Martinelli E, Fantozzi I, Bondanza S, Acknowledgements et al. p63 identifies keratinocyte stem cells. Proc Natl Acad Sci U S A. 2001; The authors thank Mrs. Engracia Pineda, Mrs. Judit Vela, and Mrs. Adriana 98(6):3156–61 Available from: http://www.pubmedcentral.nih.gov/ Alias from the Cytology Laboratory (Anatomopathology Service, Hospital articlerender.fcgi?artid=30623&tool=pmcentrez&rendertype=abstract. Clinic de Barcelona, Barcelona, Spain) for their expert assistance. They also 4. Ahmad S. Concise review: limbal stem cell deficiency, dysfunction, and thank Ms. Nausica Otero, Mrs. Elba Agustí (Barcelona Tissue Bank, BST, distress. Stem Cells Transl Med. 2012;1(2):110–5 Available from: http://www. Barcelona, Spain), and all technicians working in the Barcelona Tissue Bank. pubmedcentral.nih.gov/articlerender.fcgi?artid=3659689&tool= pmcentrez&rendertype=abstract. Authors’ contributions 5. Bakunowicz-Lazarczyk A, Urban B. Assessment of therapeutic options for EMC and NNN designed and performed the experiments, analyzed the data, reducing alkali burn-induced corneal neovascularization and inflammation. and wrote the manuscript in consultation with RCM. AVG and CAR Adv Med Sci. 2015;61(1):101–12. performed the experiments and analyzed the data. AV contributed to the 6. Menzel-Severing J. Emerging techniques to treat limbal epithelial stem cell critical revision of the manuscript. 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