15824934, 2021, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16501 by Readcube (Labtiva Inc.), Wiley Online Library on [05/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Received: 24 November 2019 | Revised: 17 March 2021 | Accepted: 18 March 2021 DOI: 10.1111/jcmm.16501 ORIGINAL ARTICLE Priming human adipose-­derived mesenchymal stem cells for corneal surface regeneration Núria Nieto-­Nicolau1,2,3 | Eva M. Martínez-­Conesa2,3 | Sherezade Fuentes-­Julián4 | Francisco Arnalich-­Montiel4 | Ignacio García-­Tuñón4 | María P. De Miguel4 | Ricardo P. Casaroli-­Marano1,2,3,5 1 CellTec-­UB, Department of Cell Biology, University of Barcelona, Barcelona, Spain Abstract 2 Barcelona Tissue Bank (BTB), Banc de Sang Limbal stem cells (LSC) maintain the transparency of the corneal epithelium. Chemical I Teixits (BST), Barcelona, Spain burns lead the loss of LSC inducing an up-­regulation of pro-­inflammatory and pro-­ 3 Institute of Biomedical Research IIB-­Sant Pau (SGR1113), Barcelona, Spain angiogenic factors, triggering corneal neovascularization and blindness. Adipose 4 Cell Engineering Laboratory, La Paz tissue-­derived mesenchymal stem cells (AT-­MSC) have shown promise in animal Hospital Research Institute (IdiPAZ), Madrid, models to treat LSC deficiency (LSCD), but there are not studies showing their effi- Spain 5 cacy when primed with different media before transplantation. We cultured AT-­MSC Department of Surgery & Hospital Clinic de Barcelona, School of Medicine, University of with standard medium and media used to culture LSC for clinical application. We Barcelona, Barcelona, Spain demonstrated that different media changed the AT-­MSC paracrine secretion show- Correspondence ing different paracrine effector functions in an in vivo model of chemical burn and Ricardo P. Casaroli-­Marano, Hospital Recinto in response to a novel in vitro model of corneal inflammation by alkali induction. Maternitat (2nd floor, ophthalmology), Calle Sabino de Arana 1, E-­0 8028 –­Barcelona Treatment of LSCD with AT-­MSC changed the angiogenic and inflammatory cytokine (Spain). profile of mice corneas. AT-­MSC cultured with the medium that improved their cy- Email: rcasaroli@ub.edu tokine secretion, enhanced the anti-­angiogenic and anti-­inflammatory profile of the Funding information treated corneas. Those corneas also presented better outcome in terms of corneal Fondos de Investigaciones Sanitarias del Instituto Carlos III, Grant/Award Number: transparency, neovascularization and histologic reconstruction. Priming human AT-­ FIS14-­PI00196 and FIS18-­PI00355; MSC with LSC specific medium can potentiate their ability to improve corneal wound Ministerio de Salud y Política Social, Grant/ Award Number: AP2010-­0659; Fundació healing, decrease neovascularization and inflammation modulating paracrine effec- Marató TV3, Grant/Award Number: tor functions in an in vivo optimized rat model of LSCD. 20120630-­3 0-­31 KEYWORDS cell culture priming, cytokines, directed differentiation media, limbal stem cells, limbal stem cells deficiency, paracrine secretion 1 | I NTRO D U C TI O N cell deficiency (LSCD) results in new-­vessel formation and loss of transparency, leading to blindness [2]. Several pathologic conditions Limbal stem cells (LSC) reside in the basal layer of sclerocorneal can cause LSCD [3]. Among them, chemical ocular burns produce limbic region, and they maintain ocular surface integrity allowing an abnormal wound healing process caused by an immediate det- the visual function [1]. LSC divide giving rise to more differentiated rimental inflammatory cascade [4–­ 6]. Several pro-­ inflammatory progenies that form the transparent corneal epithelium. Limbal stem and pro-­angiogenic cytokines are up-­regulated, leading to corneal This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd. 5124 |  wileyonlinelibrary.com/journal/jcmm J Cell Mol Med. 2021;25:5124–5137. | 15824934, 2021, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16501 by Readcube (Labtiva Inc.), Wiley Online Library on [05/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License NIETO-­NICOLAU et al. 5125 vascularization and opacity [4,7,8]. Chemical burns represent the 2.2 | Cell isolation and culture most common cause of LSCD. This kind of chemical aggression to the eye surface represents 7.7%–­18.0% of all ocular traumas [9], and Human adipose tissue aspirates were obtained from three healthy their frequency has augmented by the use of corrosive cleaners in donors and collected by elective liposuction. AT-­MSC cells were iso- the house field. All in all, LSCD affects approximately 10 million peo- lated and cultured as described [24]. Cells (106 cells from each donor) ple worldwide [10]. were pooled. LSC from 7 human cornea donors were isolated as pre- Cell therapy with LSC for the treatment of unilateral LSCD is viously described [25,26] and cultured with supplemented hormonal a well-­consolidated treatment [11]. In this treatment, LSC of the epithelial media (SHEM): DMEM/Ham's and F-­12 (2:1 vol : vol) mixture healthy eye are expanded ex vivo and transplanted on a biocompat- (DMEM/F12; Invitrogen, Carlsbad, CA) supplemented with 2 mmol/L ible scaffold onto the injured eye of the patient [12]. Nevertheless, L-­glutamine (Lonza, Verviers, Belgium), 5 μg/mL insulin (Sigma-­Aldrich, the treatment of bilateral LSCD supposes a more challenging situa- Munich, Germany), 10 ng/mL human epidermal growth factor (hEGF, tion. It relies on allogeneic LSC transplantation, requiring systemic Sigma-­Aldrich), 0.5% dimethyl sulfoxide (DMSO, Sigma-­Aldrich), immunosuppression [13] that can lead to adverse side effects [14]. 0.4 μg/mL hydrocortisone (Sigma-­Aldrich), 2 nmol/L triiodothyronine This entails the need for alternative treatments. (Sigma-­Aldrich) and 0.18 mmol/L adenine (Sigma-­Aldrich), with 10% In this framework, somatic stem cells are a promising tool. Human FCS and 1% antibiotics. Human corneal epithelial cells were obtained adipose tissue-­derived mesenchymal stem cells (AT-­MSC) can be by mechanical scrapping of the central corneal epithelium. easily obtained from liposuction aspirates and have been applied in tissue-­engineering applications for wound healing repair [15] and other cell based therapies [16]. AT-­MSC exert a paracrine action that 2.3 | AT-­MSC culture characterization benefit the regenerative processes through trophic factors secretion [15,17] suppressing, the inflammation and immune reaction signal- Cultures were fixed and blocked with 10% foetal calf serum (FCS) in ling [18]. In fact, their immunomodulatory properties are explored 100 mmol/L PBS for 10 minutes and then incubated with conjugated for the treatment of immune disorders [19] and the therapeutic ef- antibodies for 30 minutes at room temperature. Mouse monoclo- fects of their secretome are being increasingly studied on several nal immunoglobulin (IgG) isotype was used as a negative control. diseases [17]. Moreover, in vitro and in vivo approaches have shown Samples were analysed by fluorescence-­activated cell sorting (FACS; that AT-­MSC cells have potential plasticity to differentiate into sev- FACSCalibur™ Flow Cytometer, BD Biosciences, San José, CA), and eral lineages and to integrate in the regenerated tissue [20,21]. data were evaluated by flow cytometry software (Summit, version Recently, MSC have demonstrated to be effective and safe in 3.1; Cytomation, Fort Collins, CO). Antibodies used are detailed in human clinical application in comparison with allogeneic LSC trans- Table S1 (supplemental material). plantation in cases of LSCD [22]. In addition, AT-­MSC demonstrated their ability to improve total LSCD in rabbit models [23]. With this encouraging outcome in mind, there is a need to not only study the 2.4 | AT-­MSC adipogenic and osteogenic mechanisms through AT-­MSC could exert their pleiotropic action, differentiation but also to optimize culture conditions to increase their therapeutic potential. Our leading aim was to evaluate the therapeutic effective- Cells (104 cells/cm2) were plated and cultured in an AT-­MSC medium ness of AT-­MSC primed with different LSC media in the regener- (DMEM, 10% FCS, 2 mmol/L L-­glutamine, 10 mmol/L HEPES and anti- ation, inflammatory and angiogenic cytokines profile of the ocular biotics) for 24 hours. The medium was changed to adipogenic or oste- surface of an optimized rat model for total LSCD. ogenic medium, as described [24,27,28] and maintained for 4 weeks, changing the medium every 48 hours. All media and supplements were supplied by Invitrogen (Invitrogen, Carslbad, CA, USA), and other rea- 2 | M ATE R I A L A N D M E TH O DS gents were supplied by Sigma-­Aldrich (Munich, Germany). Adipogenic differentiation was confirmed by Oil red staining, and osteogenic dif- 2.1 | Ethical considerations ferentiation was confirmed by alkaline phosphatase histochemistry. Samples were obtained according to the principles outlined in the Declaration of Helsinki. Informed consent was obtained from pa- 2.5 | Human amniotic membrane (AM) preparation tients, and active transmissible infections were excluded by sero- logic analyses. The local Ethics Committee approved this study for Human placenta samples were obtained at the time of elective cae- Clinical Research (UASP, Hospital Clinic de Barcelona, Barcelona, sarean and prepared at the facilities of the Barcelona Tissue Bank Spain). Animals were treated in accordance with the ARVO state- (BTB-­BST, Barcelona, Spain). Placenta was cleaned of blood clots ment for the Use of Animals in Ophthalmic and Vision Research. The with saline solution containing penicillin (50 μg/mL; ICN, Costa Committee for Animal Research at Hospital La Paz (Madrid, Spain) Mesa, CA, USA), streptomycin (50 μg/mL; ICN) and amphotericin B also approved the experimental procedure. (2.5 μg/mL; Bristol Myers Squibb Co, Princeton, NJ, USA). The AM | 15824934, 2021, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16501 by Readcube (Labtiva Inc.), Wiley Online Library on [05/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 5126 NIETO-­NICOLAU et al. was separated from the chorion by blunt dissection. Approximately 2.8 | Histopathology 4 × 4 cm of the AM was placed on Millipore filters in a plastic sterile container with DMEM (Invitrogen) and glycerol (ICN) at a 1:1 ratio Enucleated eyes were obtained after 30 days of cell implant pro- and preserved at −80°C until use. cedures. Half of each cornea was immediately frozen in liquid N2 and used for molecular analysis. The other half was fixed in a 4% paraformaldehyde buffered solution and prepared for conventional 2.6 | Treatment of AT-­MSC cells with specific media histopathologic studies. Material was embedded in paraffin blocks, sectioned (6–­8 μm) and stained with haematoxylin and eosin for light AT-­MSC cells were cultured (10 4 cells/cm2) under the following microscopy examination. conditions: AT-­MSC medium; SHEM medium prepared as described above; and CnT30 medium (CellnTech Advanced Cell System, ZenBio, NC, USA). All culture media were supplemented with 2% 2.9 | In vitro model of corneal inflammation by FCS. After 7 days, cells were harvested and 106 cells/ml were seeded alkali treatment on 4 × 4 cm sections of AM. Implants were observed by phase con- trast microscopy to confirm cellular adhesion. Comparison of the Human corneal epithelial cells (HCE) were seeded at a density of 3 × 105 expression of progenitor and corneal markers was performed at dif- cells/cm2 and cultured in DMEM, 10% FCS, 2 mmol/L L-­glutamine, ferent time-­points by qPCR as described below. 10 mmol/L HEPES and 1% antibiotics medium. After 24 hours, medium was changed by 10 mmol/L, 25 mmol/L or 50 mmol/L NaOH that were dissolved in the same medium. Medium without NaOH was used as 2.7 | In vivo rat model for LSCD control. Treatment was applied for 10 minutes, 3 washes with PBS were carried out and cells were cultured with new medium for 24 hours. Thirty Wistar albino rats (n = 6 per group; weighing 250–­350 g) were Then, medium was collected for paracrine secretion analysis. Treatment anaesthetized with intramuscular ketamine hydrochloride (35 mg/ was repeated again for 10 minutes. Supernatants were collected at kg; Phoenix Scientific Inc, USA) and xylazine hydrochloride (5 mg/kg; 24 hours from the second round for paracrine secretion analysis and Phoenix Scientific). Study eyes (left eyes) were topically anaesthe- further experiments with AT-­MSC. Cell toxicity assay was carried out. tized with 1% proparacaine hydrochloride (Bausch & Lomb, Madrid, HCE cells were seeded and alkali-­treated as explained above on 96 well Spain) and decontaminated with 5% povidone iodine drops for three plates. WST-­1 assay (Abcam, Cambridge, UK) was performed following minutes before procedures. Each cornea was enclosed with a plastic manufacturer’s instructions 72 hours after seeding. Absorbance was cylinder (3-­mm diameter), and several drops of n-­heptanol (Sigma) read at 450 nm with a reference wavelength of 680 nm. were placed on the centre of the cornea for 2 minutes to injure the corneal epithelium. Mechanical debridement was performed, fol- lowed by 360° surgical destruction of a lamellar superficial limbal 2.10 | Multiplex analysis ring, defined as a 1.5-­mm segment on either side of the anatomic junction between the cornea and the conjunctiva [29]. Topical anti- Supernatants were collected, centrifuged at 13 000 g for 5 min- biotics (levofloxacin 0.5%) and steroids (betamethasone 0.1%) were utes and stored at −80°C until analysis. Concentrations of MMP-­ applied twice daily. Rats were examined using a handheld light daily 2, VEGF-­A (isoform A), MCP-­1, IL-­6 and TGF-­β were analysed using and a slit lamp on days 3 and 7. After one week, n-­heptanol applica- Luminex multiplex immunobead assays from Millipore according to tion with mechanical debridement was repeated before ocular sur- the manufacturer's protocols. The reactions were detected with a face treatment as described above. Luminex 100 IS 2.3 system (Luminex, Austin, TX). Animals were classified into five study groups (n = 6), as follows: controls for corneal injury; AM ocular surface implants; AM ocular surface implants with AT-­MSC cells cultured with standard medium 2.11 | mRNA isolation and reverse transcription (AT-­MSC); AM ocular surface implants with AT-­MSC cultured in CnT30 medium (CnT30); AM ocular surface implants with AT-­MSC cultured Total RNA from rat corneas was extracted using phenol/chloroform in SHEM medium (SHEM). AM fragments measuring 7 × 7 mm were purification with TriPure Isolation Reagent® (Roche Diagnostics GmbH, sutured with six episcleral nylon 8.0 sutures, with implants of AM-­cells Mannheim, Germany), following the manufacturer's instructions. RNA placed face down. We also performed a protective partial tarsorrha- was purified using an RNeasy MinElute Cleanup Kit (Qiagen, Madrid, phy to avoid scratching. Topical antibiotics and steroids were applied Spain), following the manufacturer's instructions. The concentration was twice daily during a 1-­week period. Animals were then killed at day 30 measured using Tecan infinite m200 pro (Tecan, Männedorf, Switzerland) after the transplantation and eyes prepared for both molecular and and adjusted to 100 µg/µL. Then, 8 μL was reverse transcribed using histopathologic studies. Contralateral eyes were used as controls for SuperScript® III Reverse Transcriptase (Thermo Fisher Scientific, healthy eyes. Clinical evaluation was performed with a scale of 0–­12 Invitrogen) in a final volume of 20 μL, following the manufacturer's recom- based in the observation of haze, oedema and neovascularization [30]. mendations. Total RNA from AT-­MSC, LSC and CO were extracted using | 15824934, 2021, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16501 by Readcube (Labtiva Inc.), Wiley Online Library on [05/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License NIETO-­NICOLAU et al. 5127 F I G U R E 1 Schematic representation of the experimental design. A, In vivo assay. Upper timeline represents the LSCD generation. Corneas were damaged with n-­heptanol followed by mechanical debridement and surgical destruction of the limbal ring. Rats were examined on days (d) 3 and 7. The procedure was repeated at d7. Transplantation was performed at d14 from the first damage. At d44, corneas were analysed by qPCR for inflammatory markers and by histology. Bottom timeline represents the culture of AT-­MSC under different conditions (standard medium, SHEM medium and CnT30 medium). AT-­MSC seeding on amniotic membrane (AM) was performed at d7. Corneal and limbal markers were assayed by qPCR. B, In vitro assay. Upper timeline shows the culture of AT-­MSC with the different media. Inflammatory markers were analysed by multiplex assay at d1, 3, 5 and 7, and by qPCR at d7. At d7 of culture, media were changed by conditioned medium (CM) of alkali-­treated HCE. AT-­MSC were cultured for 72 hours (h) with this medium. Multiplex assay was performed after 48 and 72 h from the medium change. Bottom timeline exemplifies the in vitro inflammation model by alkali treatment. NaOH treatments were applied after 24h from HCE seeding. A second round of NaOH treatment was applied after 48h from the seeding. Media were recovered at 48 and 72 h and analysed by multiplex assay for inflammatory markers. CM of alkali-­treated HCE was recovered at 72 h to culture AT-­MSC cells that were grown previously in the 3 different culture media PureLink RNA Mini Kit (Ambion, Invitrogen) following the manufacturer's internal 18s (RRN18S, TATAA Biocenter, Sweden) and represented as recommendations. One μg was reverse transcribed as explained above. the relative expression. The sequences and annealing temperatures of PCR primers are listed as supplemental data (Table S2, supplemental material). 2.12 | Quantitative real-­time polymerase chain reaction 2.13 | Polymerase Chain Reaction One μL of cDNA was used for qPCR in a final volume of 18 µL with analysis and sequencing SYBR® Green PCR Master Mix (Thermo Fisher Scientific, Invitrogen). The qPCR was performed with StepOne™ Real-­Time PCR Systems To analyse the presence of human CK12 in the rat corneas, we (Applied Biosystems, Life Technologies, Glasgow, UK) hardware and used a specific primer for human CK12. Reactions were car- software. The expression level of target genes was normalized to ried out as commented above using Taq Platinum PCR SuperMix | 15824934, 2021, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16501 by Readcube (Labtiva Inc.), Wiley Online Library on [05/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 5128 NIETO-­NICOLAU et al. A a b c d e f B a b c d e f F I G U R E 2 In vivo rat model for LSCD and epithelial regeneration. A, Representative images of the corneal surface after 30 days of the transplantation. (a) normal non-­damaged cornea without pharmacological mydriasis; (b) control for corneal injury; (c) AM ocular surface implants; (d) AM ocular surface implant with AT-­MSC cultured in standard medium; (e) AM ocular surface implant with AT-­MSC cells cultured with CnT30 medium; (f) AM ocular surface implant with AT-­MSC cells cultured on SHEM medium. AM ocular surface implants with AT-­MSC cells (d–­f ) showed superficial neovascularization and improved transparency of the cornea, with the best results for SHEM treatment (f). B, Haematoxylin-­eosin histology of the corneas. (a) Normal control cornea; (b) Injured cornea without treatment; (c) AM ocular surface implant; (d) AM ocular surface implant with AT-­MSC cultured in standard medium; (e) AM ocular surface implant with AT-­MSC cells cultured with CnT30 medium; (f) AM ocular surface implant with AT-­MSC cells cultured with SHEM medium. Cell inflammatory infiltrates were observed in the anterior stroma of the cornea (b–­f ) and associated with fine newly formed capillaries (b–­e). Satisfactory structural epithelial regeneration was observed only with treatments carried out with AM ocular surface implants with AT-­MSC induced on SHEM medium (f). Asterisks mark goblet cells (d and f). Bar = 25 μm for a, b, d and e; bar = 50 μm for c and f. Abbreviation: AM, intact amniotic membrane (Thermo Fisher Scientific, Invitrogen) following manufacturer's in- PCR products were purified using QIAquick PCR Purification Kit structions. Products were electrophoresed in 2% agarose gel for (Qiagen, Madrid, Spain), following the manufacturer's instructions. 1 hour. Ethidium bromide was used to visualize the PCR bands. The product was sequenced using the ABI Prism 3730 Genetic | 15824934, 2021, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16501 by Readcube (Labtiva Inc.), Wiley Online Library on [05/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License NIETO-­NICOLAU et al. 5129 Analyzer (ABI Applied Biosystems, Life Technologies). Finally, se- and decreased the neovascularization in comparison with non-­treated quences were aligned with mRNA using the ClustalW software and amniotic membrane-­ treated groups (Figure 2A-­ D). Corneas (http://www.ebi.ac.uk/Tools/​clust​alw2). treated with CnT30-­cultured AT-­MSC presented similar results. Corneas treated with SHEM-­cultured AT-­MSC showed better regular- ity of the corneal surface, more transparency and less vascularization 2.14 | Statistical analysis (Figure 2A-­F) than the corneas of the rest of the groups (Figure 2A-­E). Histopathological evaluation supported these observations. Corneas Data are reported as mean ± standard error of the mean (MD ± SE). treated with SHEM-­cultured AT-­MSC demonstrated successful regen- Statistics were analysed using GraphPad Prism software (version 4; eration of the epithelium structure. Although the epithelium showed GraphPad Software Inc, CA, USA). Statistical significance was deter- more layers than the control corneas, the epithelium preserved the mined by one-­way analysis of variance with Bonferroni's post hoc stratification and showed better regularity than corneas treated with analysis. p-­values ≤ 0.05 were considered statistically significant. control AT-­MSC or CnT30-­cultured AT-­MSC (Figure 2B-­F). A comparison of the degree of conjunctivalization between groups could not be done because specific markers for conjunctiva 3 | R E S U LT S were not used. However, goblet cells (Figure 2B, asterisks), that in- dicate the degree of conjunctivalization, could be observed in the 3.1 | Corneal epithelium showed improved corneal epithelia of all of the groups except the control corneas regeneration with SHEM-­cultured AT-­MSC (Figure 2B-­D and 2B-­E). However, these cells were rare in corneas treated with SHEM-­cultured AT-­MSC (Figure 2B-­F ). We evaluated the efficiency of AT-­MSC cells cultured with differ- ent media to regenerate the damaged ocular surface on an in vivo model of total LSCD in rats following the schematic representation 3.2 | SHEM-­treated AT-­MSC regenerated the depicted in Figure 1. As the use of NaOH has been reported to in- corneas and modulated murine cytokines duce stromal damage along with severe corneal ulcers, hyphema and hypopyon [31], which could mislead the interpretation of the out- Non-­ treated damaged corneas showed increased expression of come, we used n-­heptanol accompanied by epithelial debridement pro-­inflammatory and angiogenic markers when compared with and excision of the superficial limbal ring for LSCD. Our approach is normal control corneas (Figure 3A). Corneas treated with am- generally used in the generation of LSCD because allows specifically niotic membrane without cells only decreased the expression of a complete LSC niche removal and corneal epithelium denudation MMP-­2 and IL-­6 . Further, corneas treated with control AT-­MSC without further stromal damage [23,29]. showed reduced expressions of TNF-­α , MMP-­2 , IL-­6 and MCP-­ Non-­treated damaged corneas showed severe neovascularization 1. Corneas treated with AT-­MSC cultured with CnT30 or SHEM and opacification (Figure 2A-­B) in comparison with unwounded cor- down-­ regulated the expression of TNF-­ α , MMP-­2 , IL-­6 , MCP-­1 neas (Figure 2A-­A). Histologically, these corneas showed epithelial and and VEGFA. Interestingly, only the corneas treated with SHEM-­ stromal disorganization as well as inflammatory cell infiltration. There cultured AT-­MSC increased IL-­10 expression. were spaces among epithelial cells, indicating oedema (Figure 2B-­A). The ocular surface of the group treated with the amniotic membrane implant did not show differences with the ocular surface of non-­treated 3.3 | AT-­MSC were detected on the corneas after damaged corneas group (Figure 2A-­B. Amniotic membrane-­treated 30-­days after implantation corneas histologic sections demonstrated stromal disorganization, cell invasion and loss of epithelium stratification (Figure 2B-­B). AT-­MSC Human CK12 amplicons of the expected size (0.2 kb) were identified cultured with standard medium improved the corneal transparency in AT-­MSC cultured with different media and in the corneas treated F I G U R E 3 mRNA expressions on rat corneas. A, Cytokine and growth factor expression on treated corneas. The mRNA expression levels for some molecules related to angiogenic (MMP-­2 and VEGF-­A), pro-­inflammatory (MCP-­1, IL-­6, TGF-­β and TNF-­α) and anti-­inflammatory (IL-­ 10) events were analysed in treated corneas. Cellular extracts were obtained from corneas after 30 days. MD ± SE from three independent experiments. *P < .05; **P < .01, ***P < .001 in comparison with DC. B, Evidence of human CK12 in treated corneas. Upper row shows CK12 mRNA expression in cultured AT-­MSC. H2O was used as the negative control for amplification. Middle row shows actin expression as control for DNA load. Bottom row (lanes A–­F ) shows that human CK12 was not present in the injured corneas without treatment (lane D), those treated with AM (lane E) or the control normal rat corneas (lane F). Human CK12 was present in the corneas treated with AT-­MSC cells (lane A), with AT-­MSC cultured on CnT30 medium (lane B); and with AT-­MSC cultured with SHEM medium (lane C). Actin expression of each condition is shown below (uncropped image). C, PCR product was purified, sequenced and compared with the human CK12 mRNA sequence exhibiting higher homology (98%). Abbreviations: AM, amniotic membrane ocular surface implants; AT-­MSC, amniotic membrane ocular surface implants with AT-­MSC cultured in standard conditions; CnT30, amniotic membrane ocular surface implants with AT-­MSC cultured in CnT30 medium; Ctl, normal control corneas; DC, damaged corneas without treatment; SHEM, amniotic membrane ocular surface implants with AT-­MSC cultured in SHEM medium 15824934, 2021, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16501 by Readcube (Labtiva Inc.), Wiley Online Library on [05/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | NIETO-­NICOLAU et al. 5130 | 15824934, 2021, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16501 by Readcube (Labtiva Inc.), Wiley Online Library on [05/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License NIETO-­NICOLAU et al. 5131 with AT-­MSC (Figure 3B). Sequencing of the PCR products obtained with 25mM NaOH incremented the secretion of TGF-­β, MMP-­2, from the rat corneas confirmed the specificity (approximately 98%) MCP-­1 and IL-­6. The 10mM NaOH treatment only stimulated the of the amplicons for human CK12 mRNA (Figure 3C), indicating the secretion of MMP-­2 and MCP-­1 at the second round. Neither treat- presence of AT-­MSC on the rat corneas 30 days after implantation. ments generated cytotoxic effects after the second round of treat- ment (Figure 4B), which could be explained by the short exposure to NaOH. However, treatment with higher concentrations of NaOH, 3.4 | Treatment with 25mM NaOH generated such as 50mM, generated obvious cell death at the first round (Figure inflammation without cytotoxicity in an in vitro S1, supplemental material). However, after the double ‘burn’ treat- corneal alkali injury model ment, a morphologic change could be observed on HCE (Figure 4C). Cells appeared more flattened and bigger. Because 25mM NaOH We elaborated a cell culture model of inflammation by alkali treat- treatment raised more the secretion of inflammatory cytokines, su- ment in human corneal cells (HCE) to elucidate the in vitro paracrine pernatants of 25mM NaOH treated HCE were collected for further response of AT-­MSC to their supernatants in further experiments. experiments with AT-­MSC. We analysed the secretion TGF-­β, MMP-­2, MCP-­1, VEGF and IL-­6 in this model by Luminex assay. The culture model involved 2 rounds of alkali treatment to emulate the in vivo model that also comprised 3.5 | Specific media change the paracrine 2 rounds of chemical treatment. Figure 1 shows a schematic repre- secretion of AT-­MSC and prime different paracrine sentation of the model. responses to alkali-­treated HCE conditioned medium After the first round of alkali treatment (48 hours), only HCE treated with 25mM NaOH increased the secretion of IL-­6, TGF-­β and We evaluated the anti-­inflammatory and anti-­angiogenic paracrine MMP-­2 (Figure 4A). At the second round of ‘burn’, HCE cells treated potential of AT-­MSC cells cultured with different media and their F I G U R E 4 In vitro model of inflammation by alkali treatment. A, Multiplex Luminex assay for IL-­6, VEGF MCP-­1, MMP-­2 and TGF-­β at the first round of treatment (48 h) and at the second round of treatment (72 h) on HCE cells. B, Viability of HCE using WST-­1 assay after the second round of ‘burn’. C, Morphology of HCE. Cells appeared rounder during the 10 min treatment of NaOH. After the first round of NaOH treatment (48 h), there were not morphological changes, but they appeared after the second round (72 h). MD ± SE from three independent experiments. *P < .05; **P < .01, ***P < .001 in comparison with control. Bar = 25 μm | 15824934, 2021, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16501 by Readcube (Labtiva Inc.), Wiley Online Library on [05/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 5132 NIETO-­NICOLAU et al. response after changing these media by the supernatants of alkali-­ after 72 hours in contact with the conditioned media of alkali-­ treated corneal cells in vitro. For that, we carried out an analysis of treated HCE, SHEM-­cultured AT-­MSC presented lower levels of the secretion of the abovementioned cytokines by Luminex assay VEGF than CnT30 condition (Figure 5A). The expression of MMP-­2 and qPCR. was only detectable at 72 hours and 5 days in standard conditions VEGF increased with CnT30-­ and SHEM-­cultured AT-­MSC in and at 24 hours of CnT30 culture. MMP-­2 was inappreciable in comparison with AT-­MSC cultured in control medium. However, SHEM-­cultured AT-­MSC during all the experiment (Figure 5A). The F I G U R E 5 Cytokines expression on AT-­MSC cells. A, Multiplex Luminex assay for VEGF, MMP-­2, MCP-­1, IL-­6 and TGF-­β during culture in standard conditions (AT-­MSC), with SHEM medium (SHEM) and with CnT30 medium (CnT30). After 7 days, media was changed by alkali-­treated HCE conditioned supernatants. B, mRNA expressions for angiogenic (VEGF-­A), pro-­inflammatory (IL-­6, TGF-­β and TNF-­α) and anti-­inflammatory (IL-­10) events were analysed in vitro. The expression level of target genes was normalized to an internal 18 s control and represented as a relative expression. MD ± SE from three independent experiments. *P < .05; **P < .01; ***P < .001 in comparison with AT-­ MSC Abbreviations: d, day | 15824934, 2021, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16501 by Readcube (Labtiva Inc.), Wiley Online Library on [05/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License NIETO-­NICOLAU et al. 5133 F I G U R E 6 Expression of mRNA markers by qPCR on induced AT-­MSC cells. A, Expression of corneal differentiation and progenitor limbal markers by LSC, corneal scrapped cells (CO) and AT-­MSC cultured with standard medium (AT-­MSC), SHEM medium (SHEM) or CnT30 medium (CnT30) during culture up to 14 d. The expression of target genes was normalized to an internal 18 s control. B, AT-­MSC cell phenotypes. No changes were observed in cellular morphology at 24 h (upper row) in AT-­MSC control medium (AT-­MSC), CnT30 medium or SHEM medium. Polygonal and rounded morphologies (bottom row) were evident at day 5 in CnT30, whereas SHEM showed elongated phenotypes. MD ± SE from three independent experiments. *P < .05; **P < .01, ***P < .001 in comparison with AT-­MSC. Bar = 50 μm. Abbreviations: d, day | 15824934, 2021, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16501 by Readcube (Labtiva Inc.), Wiley Online Library on [05/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 5134 NIETO-­NICOLAU et al. secretion of MCP-­1 did not present differences except at 72 hours 3.7 | AT-­MSC cells accomplished mesenchymal stem of HCE conditioned media culture, which was found lower in control cells criteria characterization AT-­MSC (Figure 5A). IL-­6 decreased in SHEM-­ and CnT30-­cultured AT-­MSC, with lower levels secreted in SHEM, also after HCE con- FACS analysis showed that AT-­MSC cells were positive for CD105, ditioned media (Figure 4A) changed the medium. TGF-­β secretion CD90, CD73 and negative for CD34, CD14, CD45 and HLA-­DR was variable in CnT30-­ and SHEM-­cultured AT-­MSC, but this se- markers (Figure S2, supplemental material) which confirm their mes- cretion was always different from the levels of TGF-­β secreted by enchymal characteristics according to the International Society for control AT-­MSC. When the supernatants of alkali-­treated HCE were Cell Therapy (ISCT) [32]. To ensure AT-­MSC cell plasticity and their in contact with the cells, CnT30-­cultured AT-­MSC showed a high ability for multilineage differentiation, cells were induced to differ- pike of TGF-­β secretion at 24 hours whereas SHEM-­cultured AT-­ entiate into adipogenic and osteogenic lineages. Intracellular lipid ac- MSC presented the lowest levels in comparison with control AT-­ cumulation was confirmed using Oil Red O staining and osteogenic MSC and CnT30-­cultured AT-­MSC at 72 hours after the change of differentiation was detected by Alizarin Red staining (Figure S2). media (Figure 5A). Analysis of mRNA levels at 7 days of culture with specific media (SHEM or CnT30) or control medium supported the Luminex assay 4 | D I S CU S S I O N data, further indicating that TNF-­α was lower in SHEM cell culture conditions and that the mRNA expression level of IL-­10 did not differ Currently, different stem cells sources are investigated for the treat- between treatments (Figure 5B). ment of bilateral LSCD. A recent proof of concept clinical trial with MSC has showed promising results for the treatment of this chal- lenging pathologic condition [22]. As far as we know, this is the first 3.6 | AT-­MSC cultured with LSC specific media report showing that priming human AT-­MSC with LSC specific cul- did not transdifferentiate to corneal cells ture medium can potentiate their therapeutic potential to promote corneal wound repair, restore transparency, decrease inflammation As AT-­MSC were cultured on specific LSC media, we aimed to study and modulate paracrine effector functions in an in vivo rat model of whether these conditions can induce the transdifferentiation of AT-­ total LSCD. MSC. To do so, we compared the expression of several progenitor We optimized an experimental model of total LSCD in rats. The and corneal epithelial markers with LSC and scrapped corneal epi- resulting clinical features, evaluated using previous defined stan- thelial cells by qPCR in different time culture points. dards [33], are compatible with the clinical and histopathologic find- In comparison with LSC and CO, AT-­M SC in all conditions ings in LSCD in humans [34,35], other rat LSCD models [31,36,37] showed a marked down-­r egulation of corneal and progenitor LSC and in other species, such as rabbits [23,29]. The limbic deficiency markers, except ITGβ1 and ABCG 2 . CK12 did change with cul- was achieved in all damaged corneas, faster than in other models ture time, as was up-­r egulated at 5 days with SHEM. However, [23,29,36]. Damaged corneas showed loss of transparency, corneal this expression dropped at 14 days of culture. CnT30 did not neovascularization and stromal cell infiltration, indicatives of an up-­r egulate the expression of CK12 (Figure 6A). CK3 also de- inflammatory state that correlated by an increased expression of clined with culture time in all situations, as well as ΔNp63α did pro-­angiogenic and inflammatory cytokines such as VEGF, MMP-­ (Figure 6A). E-­c adherin incremented in standard medium culture 2, MCP-­1, IL-­6, TNF-­α and TGF-­β, consistent with previous data in at 7 days and 14 days, but showed a reduction with LSC specific murine alkali burned corneas [4,38]. Although these cytokines are media (Figure 6A). implied in corneal wound healing [4,39,40], the specific contribution ITGβ1 was elevated in SHEM at 5 days in comparison with con- of each cytokine to corneal re-­epithelization and homeostasis resto- trol AT-­MSC at day 0. However, the expression decreased with ration needs to be well-­defined. CnT30 medium at 5 days (Figure 6A). LSC expressed higher levels Amniotic membrane is used in clinical application to overcome of ABCG2, whereas corneal epithelial cells showed an evident re- the effects of inflammatory events in LSCD [5]. However, our data duction in this expression. In comparison with AT-­MSC at day 0, the showed that amniotic membrane alone did not suffice to avoid cor- highest enhancement in the expression of this marker was found in neal opacification, neovascularization, epithelial and stromal dis- SHEM at 5 days and at 7 days. However, ABCG2 was down-­regulated organization, agreeing with other investigations in animal models in SHEM at 14 days demonstrating a trend to decrease during cul- [23,31]. In fact, amniotic membrane only down-­regulated the ex- ture (Figure 6A). pression of MMP-­2 and IL-­6. AT-­MSC showed a typical spindle shaped morphology at 24 hours Our results agree with previous results showing that MSC play in all treatments. During culture, cells growing with CnT30 medium an advantageous role during wound healing mitigating the adverse showed a smaller polygonal morphology, whereas SHEM-­cultured effects of inflammation and angiogenesis [23,31,41,42], which cells showed an elongated phenotype. Neither treatment changed was supported by the improved corneal transparency, histologic the morphology to epithelial phenotypes (Figure 6B). reconstruction and decreased expression of cytokines related to | 15824934, 2021, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16501 by Readcube (Labtiva Inc.), Wiley Online Library on [05/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License NIETO-­NICOLAU et al. 5135 angiogenesis and inflammation. Moreover, the absence of visible levels of cytokines. The different media, also, primed the cells to signs of rejection in all AT-­MSC treated corneas indicated that human have different paracrine effector responses in front of the same su- AT-­MSC were well tolerated and safe at the end of the follow up. pernatants of alkali-­treated HCE. SHEM-­cultured AT-­MSC showed a AT-­MSC treated corneas, regardless of the culture media, showed better anti-­inflammatory profile in response to the supernatants, de- lower expression of MMP-­2, MCP-­1, IL-­6, TNF-­α and TGF-­β. These creasing their secretion of VEGF, MMP-­2, IL-­6 and TGF-­β after 72h results are consistent with other research demonstrating that MSC with the conditioned media of alkali-­treated HCE, further supporting suppress lymphocyte proliferation by inhibition of TNF-­α and IL-­6, the superiority of SHEM medium. decrease the production of MCP-­1 by macrophages and lessen the Supporting our previous and others investigations [58,59], we expression of TGF-­β and MMP-­2 preventing tissue fibrosis [43–­46]. found that MSC express CK12 and CK3, among other limbal markers, Our results further highlight the importance of the culture media in standard culture conditions. The expression of these and other to improve the potential of AT-­MSC in LSCD treatment. Here, we limbal markers were lost during culture time in all the conditions, in- used SHEM and Cnt30 media to culture AT-­MSC because both media dicating that corneal phenotypes were not achieved. Attending this are used for LSCD cell therapy. Corneas treated with AT-­MSC that fact, the expression of corneal-­specific markers did not mean that were cultured with CnT30 or SHEM demonstrated also a decrease in AT-­MSC transdifferentiated into corneal phenotypes [60]. Rather, VEGF expression. VEGF is a cytokine strongly associated with cor- the expression of CK12 could highlight the heterogeneity of inter- neal neovascularization [47]. Although AT-­MSC cells secrete VEGF mediate filaments expression on MSC, because other epithelium-­ [48], our data paradoxically demonstrated that its expression by AT-­ specific cytokeratins are found in MSC [61]. MSC could be associated with a decrease in corneal neovasculariza- Other research found the presence of MSC after transplantation tion. Further, corneas treated with SHEM-­cultured AT-­MSC showed onto the corneas [23,31,62], as we did. Moreover, we found human an increase in IL-­10. This anti-­inflammatory and anti-­angiogenic cy- CK12 expression on the rat corneas. Other reports confirmed the tokine plays a central role in corneal and epithelial wound healing expression of corneal markers by MSC after transplantation [63,64]. increasing re-­epithelialization [49,50]. This could explain the better However, our results should be interpreted carefully. First, we corneal regeneration, transparency and histologic reconstruction in should consider the route of administration, as well as the time be- comparison with the other treatments. Some studies indicated that cause transplantation to the detection of AT-­MSC could have impli- MSC mediate their anti-­inflammatory effects increasing the secre- cations in MSC permanence on the corneal surface [63,65]. Second, tion of IL-­10 as well as blocking the secretion of TNF-­α by dendritic it should be contemplated the sensitivity of the method to detect cells [51], which also support our results. either the presence of MSC or the expression of corneal markers So, the composition of SHEM medium enhanced the wound by MSC. Here, we evaluated the presence of AT-­MSC after a rela- healing properties of AT-­MSC cells and produced better outcome in tive short period from the transplantation and used a high sensitive vivo. Interestingly, among other trophic factors, EGF was a compo- method such as PCR for the detection of human CK12 already ex- nent of SHEM media which enhances the wound healing therapeu- pressed by AT-­MSC. tic potential of MSC through paracrine secretion [52]. In fact, MSC AT-­MSC were detected in all corneas regardless of the media used preconditioning with growth factors or cytokines constitutes a reli- to culture AT-­MSC. However, the best outcome were found with able approach to enhance their therapeutic properties [53], through SHEM-­treated AT-­MSC that showed an improved anti-­inflammatory activation of trophic signalling pathways [54]. Despite the complex and anti-­angiogenic paracrine profile. This indicated that the thera- mechanisms governing the immunomodulation in AT-­MSC [18,19], peutic effects of AT-­MSC were mediated mainly through paracrine we demonstrated that SHEM-­treated AT-­MSC lowered the levels of effector functions than by a transdifferentiation mechanism on the IL-­6, TGF-­β, TNF-­α and MMP-­2, whereas CnT30 up-­regulated the corneal surface. However, more studies would elucidate whether levels of TGF-­β and VEGF and did not change the levels of TNF-­α . SHEM could prime AT-­MSC to exert therapeutic effects through Overall, SHEM-­treated AT-­MSC showed a better anti-­inflammatory other mechanisms than paracrine regulation, such as mesenchymal profile than AT-­MSC cultured either with CnT30 or standard me- mitochondrial transfer [66] that have demonstrated protective roles dium. This could influence the therapeutic effect of AT-­MSC as the in corneal wound healing [67]. increase in IL-­6 expression hinder the therapeutic activity of MSC We demonstrate that culture media can potentiate the therapeu- [55]. Further studies would determine whether upstream regulators tic potential of AT-­MSC to promote corneal wound repair, restore of immunoregulation, such as nuclear factor-­kappa B (NF-­κB) and its transparency and decrease inflammation on chemical burn corneas regulator Rap 1 [56,57], could be implicated in the paracrine effector modulating paracrine effector functions. The use of LSC specific functions mediated by AT-­MSC treated with different mediums. medium to culture AT-­MSC is a feasible option for cell therapy be- We aimed to demonstrate that AT-­MSC cultured with different cause this medium is already used in LSC ex vivo culture before media react differently when exposed to the same inflammatory transplantation. stimuli from an alkali-­treated corneal cell line. In vitro alkali-­treated HCE presented increased expression of IL-­6, MMP-­2, MCP-­1 and AC K N OW L E D G E M E N T S TGF-­β, indicating an inflammatory state that did not generated cy- This work was funded in part by a grant from Fondos de totoxicity. As said, different media led AT-­MSC to secrete different Investigaciones Sanitarias del Instituto Carlos III (FIS14-­PI00196 and | 15824934, 2021, 11, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16501 by Readcube (Labtiva Inc.), Wiley Online Library on [05/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 5136 NIETO-­NICOLAU et al. FIS18-­PI00355), Fundació Marató TV3 (20120630-­3 0-­31), grant 11. Pellegrini G, Traverso CE, Franzi AT, et al. Long-­term restoration of damaged corneal surfaces with autologous cultivated corneal epi- PI15-­01447 from the Ministerio de Salud y Politica Social (Spain) and thelium. Lancet. 1997;349:990-­993. AP2010-­0 659 fellowship to SF-­J from the Ministerio de Educación, 12. Sacchetti M, Rama P, Bruscolini A, et al. Limbal stem cell trans- Cultura y Deportes (Spain). Research project was co-­financed by plantation: clinical results, limits, and perspectives. Stem Cells Int. the European Regional Development Fund (FEDER) of European 2018;2018:1-­12. Union. 13. Holland EJ. 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