Xenofree generation of limbal stem cells for ocular surface advanced cell therapy
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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.
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.
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.
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.
KeywordsCultured limbal stem cells transplantation XSHEM CnT07 Processed lipoaspirate cells 3T3 Clinical grade Limbal stem cells deficiency
Human corneal epithelial cells
Doubling population time
Limbal stem cells
Co-cultures of LSC with 3T3 feeder layers
Co-cultures of LSC with PLA feeder layers
Processed lipoaspirate cells
Xenofree supplemented hormonal medium
The transparency and the integrity of the cornea are maintained by a subset of stem cells located at the epithelial basal layer of the limbus, an anatomic circumferential area that separates the transparent cornea from the conjunctiva . These stem cells are called limbal stem cells (LSC), and they are defined by their small size, high nucleus-to-cytoplasm ratio [1, 2], and positivity for the putative stemness marker ΔNp63α [2, 3] as well as negativity for corneal epithelial differentiation markers cytokeratin (CK) 12 and CK3 . The loss of LSC produces new vessel formation, corneal conjunctivalization, and scarring, leading to corneal blindness . Limbal stem cell deficiency (LSCD) can be caused by chemical, traumatic, and infectious insults and also by genetic etiologies . Its prevalence is increasing, due to the use of corrosive cleaners in the household field . Approximately, LSCD affects approximately 10 million people worldwide [6, 7].
Although the technique for the treatment of unilateral LSCD has evolved with time, the current gold standard treatment for unilateral LSCD is cultured limbal epithelial stem cell transplantation (CLET) . For CLET, the LSC can be cultured by explant or cell suspension systems . In the first system, a small biopsy of the healthy limbus is seeded on amniotic membrane. The cells grow, and the sheet is transplanted onto the damaged eye. In the cell suspension approach, LSC obtained from a minimally invasive limbal biopsy are enzymatically disaggregated and ex vivo expanded on an inactivated feeder layer of 3T3 murine fibroblasts until sub-confluence. Then, the cells are detached and seeded on a biocompatible carrier for transplantation [8, 9]. This approach is more advantageous than explant systems, since it reduces the risk of contamination of the culture by other limbal cells (such as stromal fibroblasts)  and increases the amount of cells that can be obtained due to higher proliferation rates [11, 12, 13]. Moreover, the cell suspension cultures are an optimized option since they are more enriched in stem cell progenies than explant culture methods [10, 12, 13] leading to improved outcomes .
Since the first use of CLET for human LSCD treatment in 1997 , xeno-products have been used for the ex vivo expansion of LSC both in explant and cell suspension systems . However, European regulatory guidelines [17, 18] for the safety and quality of human tissues and cells encourage the implementation of standard operating procedures to prevent the use of xenogeneic compounds and the potential associated contamination. Although the use of xeno-products supposes a risk for human health by virus, prions, and zoonoses transmission , little research has been made to avoid xenobiotics during LSC culture in suspension systems [20, 21, 22]. Besides, traditional LSC media for clinical application contain cholera toxin [15, 23], increasing the risk of disease transmission. So, substitution of the LSC culture medium containing serum and xenobiotics from animal origin, along with the replacement of the feeder layer of murine 3T3 fibroblasts by xenofree alternatives, is of utmost significance.
To study clinical grade xenofree alternatives that could serve to maintain LSC cultures for cell therapy in humans, we tested two xenofree media and two feeder layer approaches. We compared a defined commercial medium, designed to sustain keratinocyte growth, with a supplemented hormonal epithelial medium complemented with human serum (XSHEM). Then, we performed a comparison between the gold standard murine 3T3 fibroblasts  and human processed lipoaspirate cells (PLA) as a feeder layer for LSC culture growth. Our results propose a clinical grade alternative free of xenobiotics to sustain optimal LSC culture growth with direct clinical application for advanced therapy.
Materials and methods
Cell culture of feeder layers
Murine 3T3 Swiss Albino fibroblasts were obtained from Kerafast (3T3-J2, EF3003). Cells were cultured with DMEM 4.5 g/l (Thermo Scientific, MA, USA) supplemented with 10% FCS and 1% antibiotics. Human processed lipoaspirate cells (PLA) from fresh human lipoaspirates were collected from healthy donors, during plastic liposuction procedures, in planned lipoaspiration surgeries. PLA were obtained by stromal vascular fraction isolation, and cultured and characterized as previously described . PLA cells accomplished the criteria for mesenchymal stem cell characterization (data not shown) . Then, both 3T3 and PLA cells were inactivated by irradiation with 6000 rads. After this, cells were plated onto culture dishes at 2 × 104 cells/cm2 for feeder-layer use or downstream experiments.
Human LSC and corneal epithelial cells
Cadaveric adult human limbal tissues from six different donors were obtained from the Barcelona Tissue Bank (BTB-BST, Barcelona, Spain; http://www.bancsang.net/en_index/). LSC were isolated as previously described [26, 27]. LSC from each donor were equally divided and cultured until sub-confluence with xenofree supplemented hormonal epithelial media (XSHEM) or CnT07 medium (CellnTec, Bern, Switzerland) on 3T3 or PLA feeder layers that were seeded 24 h before. XSHEM composition consisted of the following: Dulbecco’s modified Eagle’s medium/Ham’s and F-12 (2:1 vol:vol) (DMEM/F12; Invitrogen, Carlsbad, CA) supplemented with 2 mM l-glutamine (Lonza, Verviers, Belgium), 5 μg/ml human insulin (Sigma Aldrich, Munich, Germany), 10 ng/ml human epidermal growth factor (hEGF, Sigma-Aldrich), 0.5% dimethyl sulfoxide (DMSO, Sigma-Aldrich), 0.4 μg/ml hydrocortisone (Sigma-Aldrich), 2 nM triiodothyronine (Sigma-Aldrich), 0.18 mM adenine (Sigma-Aldrich), and 10% Human AB Serum (Corning, Manassas, VA). After the isolation passage, cells were used for downstream applications. Human corneal epithelial cells (CO) were obtained by mechanical scrapping of the central corneal epithelium of five different donors, avoiding the perilimbal region, and used as control for qPCR experiments.
Colony-forming assay (CFA) and doubling population time (DPT)
For CFA determination, LSC that were previously cultured on 3T3 or PLA feeder layers were seeded in 35-mm-diameter plates and cultured for 14 days  with 3T3 feeder-layer support. Colonies were fixed and stained with 0.5% crystal violet in methanol. Analysis was performed according to previous criteria and presented as a percentage after applying the previously described formula . The diameter of each colony was measured using ImageJ software . DPT was calculated as described elsewhere .
Cells of each experimental group (5 × 105) were added to ThinPrep® PreservCyt solution (Hologic Iberia SL, Barcelona, Spain) for fixation and preservation. Cells were then transferred to slides using ThinPrep 3000 processor (Hologic), which allowed the cells to be seeded in a single plane without forming clumps. Slides were preserved in methanol until use, permeabilized, blocked, and then incubated with primary antibodies. After several washes in 100 mM PBS solution, proper secondary antibody was added for 60 min at 37 °C in a humidified chamber. The antibodies and concentrations used are detailed in Additional file 2: Table S1. Cells were observed in an epifluorescence microscope (BX61; Olympus R-FTL-T; Olympus America Inc., Center Valley, PA), coupled with a program for digital image acquisition (Olympus DP Controller Program). Images were processed with ImageJ software .
mRNA extraction and quantitative polymerase chain reaction (qPCR) analysis
Total RNA was extracted from co-cultures of LSC with either 3T3 or PLA feeder layers or from monocultures of the feeder layers at the last day of LSC culture. The extraction was performed using RNA Purelink Mini Kit (Ambion, Invitrogen), following the manufacturer’s instructions. The RNA concentration was measured using NanoDrop lite spectrophotometer (Thermo Scientific). RNA (1 μg) was reverse-transcribed using Superscript III (Invitrogen) according to the manufacturer’s instructions. Then, cDNA (1 μl) was used for qPCR in a final volume of 18 μl with Lightcycler 480 Sybr Green I Master (Roche, Barcelona, Spain) and a 0.2-μM primer concentration. The qPCR was performed using Lightcycler 480 II (Roche) hardware and software. The expression level of target genes was normalized to internal 18s (rrn18s, TATAA Biocenter, Sweden) and represented as relative expression using 2-ΔΔCt formula. The sequences and annealing temperatures of PCR primers are listed in Additional file 2: Table S2.
Cell culture medium was recovered at every change of medium and was centrifuged at 13,000 rpm during 5 min. Supernatants were stored at − 80 °C until analysis. ELISA assay for interleukin-6 (IL-6) was performed with a specific human ELISA kit for IL-6 (Biosource Europe, Medgenix, Nivelles, Belgic) according to the manufacturer’s instructions.
Cell growth was tested using WST-1 assay (Abcam, Cambridge, UK) attending the manufacturer’s recommendations at every medium change. Plates were read at 450 nm with a reference wavelength of 680 nm in an absorbance plate reader (Biotek).
Viability was tested using live/dead assay (Invitrogen) before and after detachment of the cultures following the manufacturer’s instructions. Moreover, viability calculation was performed using trypan blue exclusion assay on a Neubauer chamber after detachment of the cells with TrypLE Select® (Sigma-Aldrich).
Experiments were performed in triplicate. A two-tailed Student’s t test was run, and p values < 0.05 were considered statistically significant (PRISM, version 6.0 GraphPad Software, San Diego, CA). Results are presented as the mean ± standard error (MD ± SE) or, in the case of the qPCR analysis, mean ± standard deviation (MD ± SD).
XSHEM produced cells with LSC morphology and higher viability
LSC in XSHEM and in CnT07 were positive for p63 and negative for corneal differentiation markers
PLA increased the clonogenicity and growth of LSC
LSC-PLA expressed more IL-6 and SDF-1 than LSC-3T3
When assayed by qPCR, monocultures of PLA showed higher expression of SDF-1 than monocultures of 3T3 cells (Fig. 3c). Accordingly, the co-cultures of LSC and 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
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 intervention 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 smaller, with higher nucleus-to-cytoplasm ratio, and the cultures showed stratifications. Although stratification in LSC cultures is prevented in cell therapy because they can induce cell differentiation by cell confluence, this is a well-known trait of LSC cultures and an indication of the quality of the culture . The cultures of CnT07 grew in monolayers, had an impaired viability after detachment, and had lower expression of progenitor markers Bmi1 and ABCG2. This pointed out that XSHEM maintains better LSC culture characteristics than CnT07, although there were no differences in the expression of the putative stemness marker p63 by qPCR and immunofluorescence. In addition, both medium conditions prevented the expression of corneal differentiation epithelial markers CK3 and CK12. Our results also support previous data showing that LSC express lower levels of PAX6 when compared to more differentiated progenies . Moreover, our results are consistent with previous research showing that serum supplemented media maintain better survival and enrichment of LSC than commercial defined keratinocyte media .
The use of non-defined media has drawbacks, such as batch to batch serum variations ; however, “in house” formulations allow independence of commercial companies overcoming the need of further GMP validations if the product became discontinued. Another drawback of a non-defined medium is the potential risk of disease transmission . However, this risk can also be managed effectively by the application of the European regulatory controls [17, 18]. Thus, microbiologic purity of the media can be tested and further validated to avoid disease transmission and assure an aseptic manufacturing process .
LSC growth under an undifferentiated state is proved with defined media only when supplemented with either human serum or synthetic supplements [20, 38]. Here, we showed that our defined medium without serum supplementation hinders the viability of LSC, making the culture less cost-effective. One explanation for the viability loss could be that the recombinant protease used for cell detachment was not completely inhibited by defined media, which usually contain low concentration of proteins and other elements such as calcium. Then, the cell viability loss in CLET could impair the outcomes of the transplantation by decreasing the survival of undifferentiated progenies . For all these reasons, XSHEM medium represented a better alternative to culture LSC for human application and was chosen to test different feeder layers.
LSC cultures obtained in suspension systems directly on amniotic membrane without feeder layers in xenofree conditions produce grafts enriched in differentiated cells, positive for CK3 and CK12, indicating the loss of stem cell progenies . This highlights the importance of the feeder layer to culture LSC. Although 3T3 feeder cells are necessary to maintain better cumulative LSC cell numbers even with xenofree medium , the risk of murine fibroblast use is the potential inflammatory responses against the graft generated by the presence of xeno-antigens in LSC transplant . This would impair the results of the transplantation since any inflammatory reaction could lead to detrimental outcomes after grafting [40, 41, 42, 43]. Avoiding this risk, one study changed the 3T3 feeder cells by human embryonic fibroblast cell line , demonstrating the same feasibility to support LSC cell growth. Since the use of human embryonic cells entails ethical implications and is related to teratogenic risks , somatic stem cell lines, such as PLA [45, 46], may be more advantageous for clinical applications. Human PLA are easy to obtain and isolate  and could be used for allogenic or autologous feeder layer purposes. Allogenic PLA feeder cells could be well characterized following international recommendations , screened for paracrine secretion, optimized to sustain LSC growth, and GMP-banked [46, 47, 48]. Moreover, PLA cells do not express HLA DRII  being invisible to the immune system and avoiding rejection in allogenic clinic application .
Successful clinical outcomes with CLET approach are related to the presence of undifferentiated progenies . Since our culture system with XSHEM medium and PLA feeder layer generates undifferentiated LSC with higher clonogenic potential, expressing more Bmi1, highly positive for p63 and negative for the expression of CK3 and CK12, it is likely to provide better outcomes in clinical transplantation. Moreover, we demonstrated that PLA feeders induced faster proliferation and cell growth on LSC, so the cultures were more cost-effective. Moreover, the viability of LSC cultured on both feeders after detachment was similar. This fact makes sense, since both cultures used the same medium and the protease could be inhibited with the same efficacy.
Finally, there is a close interaction between LSC and their microenvironment, such as their neighboring cells and extracellular matrix, which regulate their proliferation and differentiation in the limbal niche . When LSC are co-isolated with limbal stromal niche cells, the LSC proliferate faster and form more clones through the expression of SDF-1 , highlighting the importance of the communication between LSC and the stromal cells for their proliferation and maintenance. Moreover, IL-6 is secreted by limbal and stromal cells which act as a mediator between both cell populations, increasing the clonogenic potential and maintaining an undifferentiated state of LSC in in vitro culture systems . Here, we also demonstrated that co-cultures with PLA induced more secretion of IL-6 and SDF-1, than co-cultures with 3T3, explaining the higher clonogenic capability, and the faster proliferation and cell growth. Human PLA secreted high amounts of IL-6 in the first 24 h of seeding, and its secretion could possibly influence the fate of LSC culture. In addition, it is well known that murine IL-6 does not have effect on human cells due to conformational differences within the IL-6 molecules . This fact also supports the reasoning of using feeder layers from human origin to avoid species-specific effects.
In summary, we demonstrated that the substitution of xenobiotics by human-derived alternatives is a feasible clinical grade xenofree option for LSC cultures in advanced therapy for ocular surface regeneration. The use of XSHEM medium combined with PLA feeder layers not only maintained LSC characteristics but also improved the LSC potential. This approach will have a direct clinical application in cell therapy for LSCD treatment.
The authors thank Mrs. Engracia Pineda, Mrs. Judit Vela, and Mrs. Adriana Alias from the Cytology Laboratory (Anatomopathology Service, Hospital Clinic de Barcelona, Barcelona, Spain) for their expert assistance. They also thank Ms. Nausica Otero, Mrs. Elba Agustí (Barcelona Tissue Bank, BST, Barcelona, Spain), and all technicians working in the Barcelona Tissue Bank.
EMC and NNN designed and performed the experiments, analyzed the data, and wrote the manuscript in consultation with RCM. AVG and CAR performed the experiments and analyzed the data. AV contributed to the critical revision of the manuscript. RCM devised the project, designed the experiments, was involved in the final approval of the manuscript to be published, and funded the research. All authors read and approved the final manuscript.
This work was funded in part by a grant from Fondos de Investigaciones Sanitarias del Instituto Carlos III (FIS14-PI00196 and FIS18-PI00355) and Fundació Marató TV3 (20120630-30-31). Research project was co-financed by the European Regional Development Fund (FEDER) of European Union.
Ethics approval and consent to participate
This study followed the ethical precepts of the Declaration of Helsinki (Fortaleza, Brazil, Oct 2013) and was approved by our local ethics committee (CEI, Hospital Clinic de Barcelona; Ref 7365-12). Human samples were obtained, processed, and analyzed according to current guidance in relation 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 accordance with the protocol and legal requirements for the use of biological samples and biomedical research in Spain (Law 14/2007 and RD 1716/2011) and following the agreement on the use of human donated tissue for ocular transplantation, research, and future technologies (The 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 Organ Transplants (RD 9/2014). All the information provided before donation, together with informed consent, stressed that the samples obtained were to be used for clinical application and/or applied research. The use, protection, communication, and transfer of personal data complied with local regulations (Law 03/2018).
Consent for publication
Not applicable. This manuscript does not contain data from any individual person.
The authors declare that they have no competing interests.
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