Donor age and long-term culture do not negatively influence the stem potential of limbal fibroblast-like stem cells
© The Author(s). 2016
Received: 16 June 2015
Accepted: 16 May 2016
Published: 13 June 2016
The Erratum to this article has been published in Stem Cell Research & Therapy 2016 7:106
In regenerative medicine the maintenance of stem cell properties is of crucial importance. Ageing is considered a cause of reduced stemness capability. The limbus is a stem niche of easy access and harbors two stem cell populations: epithelial stem cells and fibroblast-like stem cells. Our aim was to investigate whether donor age and/or long-term culture have any influence on stem cell marker expression and the profiles in the fibroblast-like stem cell population.
Fibroblast-like stem cells were isolated and digested from 25 limbus samples of normal human corneo-scleral rings and long-term cultures were obtained. SSEA4 expression and sphere-forming capability were evaluated; cytofluorimetric assay was performed to detect the immunophenotypes HLA-DR, CD45, and CD34 and the principle stem cell markers ABCG2, OCT3/4, and NANOG. Molecular expression of the principal mesenchymal stem cell genes was investigated by real-time PCR. Two-dimensional gel electrophoresis and mass spectrometric sequencing were performed and a stable proteomic profile was identified. The proteins detected were explored by gene ontology and STRING analysis. The data were reported as means ± SD, compared by Student’s unpaired t test and considering p < 0.05 as statistically significant.
The isolated cells did not display any hematopoietic surface marker (CD34 and CD45) and HLA-DR and they maintained these features in long-term culture. The expression of the stemness genes and the multilineage differentiation under in-vitro culture conditions proved to be well maintained. Proteomic analysis revealed a fibroblast-like stem cell profile of 164 proteins with higher expression levels. Eighty of these showed stable expression levels and were involved in maintenance of “the stem gene profile”; 84 were differentially expressed and were involved in structural activity.
The fibroblast-like limbal stem cells confirmed that they are a robust source of adult stem cells and that they have good plasticity, good proliferative capability, and long-term maintenance of stem cell properties, independently of donor age and long-term culture conditions. Our findings confirm that limbal fibroblast-like stem cells are highly promising for application in regenerative medicine and that in-vitro culture steps do not influence their stem cell properties. Moreover, the proteomic data enrich our knowledge of fibroblast-like stem cells.
The limbus, located at the junction of the cornea and conjunctiva of the ocular surface, is characterized by stromal invaginations (the palisades of Vogt). These structures give anatomical and functional properties protecting stem cells from insults and allowing constant renewal of the corneal epithelium [1–8].
Two stem cell populations have been described in the limbus: limbal epithelial stem cells (LESCs) and limbal stromal stem cells. Their similar and different features have not been defined unequivocally [9–12]. Polisetty et al.  proposed a role for limbal mesenchymal stromal cells (MSCs) in maintaining support of the stem cell niche. More recent data have validated the hypothesis of “niche stromal cells” based on the capacity of limbal stromal cells to be an efficient feeder layer for ex-vivo limbal epithelial cell expansion [8, 14, 15]. However, given the complexity of the limbal niche structure and its cellular components, which cellular type has the main role in normal tissue maintenance remains to be seen [16–18].
We previously identified a subpopulation of limbal stem cells, which we referred to as fibroblast-like limbal stem cells (f-LSCs) . We indicated a core set of attributes that uniquely characterize f-LSCs and that classify them as mesenchymal stem cells. In support of this, f-LSCs expressed stem cell surface antigens SSEA4 (stage-specific embryonic antigen-4), TRA 1–60, and TRA 1–81 and several nuclear transcription factors, such as OCT4 (octamer-binding transcription factor 4), NANOG (Homeobox protein NANOG), and SOX2 (SRY (sex determining region Y)-box 2), involved in self-renewal and maintenance of pluripotency of both embryonic and adult stem cells [20, 21]. The f-LSCs were positive for the limbal stem cell marker ABCG2 (ATP-binding cassette sub-family G member 2) and were negative for the LESC marker ΔNp63 (a splice variant of p63) [22–26]. More recently it has been hypothesized that age negatively affects stem cell number and potential, also referring to the LESC population [27, 28].
The current study was undertaken to explore whether ex-vivo expansion of f-LSCs could modify their stem molecular features. Our study aimed to increase knowledge in the field of limbal mesenchymal stem cell research regarding three facets: firstly, we aimed to discover whether long-term culture affects the gene expression and proteomic profile of f-LSCs; secondly, we proposed a preserved “proteomic stem cell pattern” in ageing and long-term culture conditions; and finally, we evaluated the maintenance of multilineage differentiation capability in vitro.
Establishment of limbal cell cultures
Human tissues were used in accordance with the Declaration of Helsinki and informed written consent was given by each patient. The study was approved by the Ethical Committee of the AOUP, University of Palermo (No. 09/2009).
Normal human corneo-scleral rings from donors aged between 24 and 74 years were obtained 2–3 hours post surgery in the Ophthalmology Department (AOUP, University of Palermo, Italy). The rings were kept in Hank’s Balanced Salt Solution (HBSS; PAA, Pashing, Austria) and then cut into small segments to facilitate isolation of the limbus from the sclera.
Enzymatic digestion and culture
The limbal segments were incubated with collagenase I (5 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) overnight at 37 °C in a shaking bath. The following day, the digest was placed in a p60 dish culture (Corning, New York, USA) with the fibroblastic-maintenance medium (DMEM/F12 supplemented with 10 % embryonic stem cell-tested fetal bovine serum (EC-FBS; PAA), 1× ITS (5 μg/ml insulin, 5 μg/ml transferrin, 5 μg/ml selenium; PAA), and 20 ng/ml basic fibroblast growth factor (b-FGF; Preprotech, London, UK)) until cells reached confluence, changing the medium when necessary.
Subsequently, the f-LSC subculture was kept in the expansion medium (f-EM: DMEM/F12 supplemented with 5 % EC-FBS (PAA), 1× ITS (PAA), and 4 ng/ml b-FGF (Preprotech)).
The f-LSCs were subcultured and seeded at a density of 4 × 103 cells/cm2 and cell counts were performed at 24, 48, 72, 96, and 120 hours by optical microscope observation after staining with vital trypan blue. The doubling time was calculated online (http://www.doubling-time.com/compute.php; Roth V. 2006). For each sample of donor limbus, three sets of experiments were used for calculations.
The f-LSCs were placed in ultralow-attachment six-well plates (Corning) at a density of no more than 1 × 102 cells/cm2, and were cultured in f-EM without serum. Sphere formation was assessed by counting the number of spheres (cells > 3) under an optical microscope.
The spheres were transferred into a cell-culture chamber slide (Labtek II; Nunc, Waltham, MA, USA) and incubated in f-EM, under adhesion-condition culture at 37 °C in 5 % CO2 to allow their attachment. After 2 hours, cells were washed with phosphate-buffered saline (PBS) and fixed for 30 minutes in 2 % (wt/vol.) paraformaldehyde in PBS at room temperature, then incubated with 1 μg/ml PE-conjugated anti-human SSEA-4 monoclonal antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) in PBS/bovine serum albumin (BSA; PAA) for 30 minutes, at room temperature. After incubation the cells were washed three times with PBS, counterstained with DAPI (Sigma Aldrich), and observed under a Zeiss Axiophot fluorescence microscope equipped with a Nikon DS-FI1 CCD camera.
Human bone marrow-derived MSCs (BM-MSCs; Lonza, Walkersville, MD, USA) were used as a stem marker positive control, HeLa (kindly obtained from Dr Salvatore Feo at University of Palermo) were used as an LESC marker positive control, and unstained f-LSCs were used as a negative control.
Sorting assay through magnetic isolation
The f-LSCs were magnetically separated for SSEA4 expression by MACS MicroBead Technology (Miltenyi Biotec) according to the manufacturer’s instructions.
Flow cytometry analysis
The cells were harvested and filtered through a 40-μm filter mesh and suspended at a concentration of 1 × 106 cells/ml. Then 100 μl of cell suspension containing 5 × 105 cells was used for each flow cytometric test.
Monoclonal antibodies used for the characterization of stem cell markers and cell phenotypes
Primary antibody/localization marker
Santa Cruz, sc-18875
Santa Cruz, sc-19621
Santa Cruz, sc-28369
Miltenyi Biotec, 130-98-371
30 minutes, r.t.
Santa Cruz, sc-293121
Santa Cruz, sc-5279
Life Technologies, Z25402
20 minutes, r.t.
Life Technologies, Z25007
20 minutes, r.t.
Stem cell phenotypes
The cells were double-stained with human anti-SSEA4 and human anti-ABCG2 monoclonal antibody, both surface MSC markers. The f-LSCs were tested for SSEA4 and for the human nuclear markers ΔNp63 or OCT4 or NANOG monoclonal antibody, after permeabilization with PBS supplemented with 0.1 % saponin and 1 % BSA for 20 minutes.
The antibody dilution, incubation, and detection conditions are presented in Table 1.
All reaction mixtures were then acquired with a FACS Calibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) and analyzed with the CellQuest Pro software. BM-MSCs were used as a positive control for SSEA4, NANOG, ABCG2, and OCT4, and HeLa cells were used as a positive control for ΔNp63.
Analysis of cell cycle status of MSCs
Single cell suspensions of f-LSCs were obtained from two different culture passages: P4 and P30. For DNA content analysis, Nicoletti’s protocol was performed. Briefly, 1 × 106 cells were fixed in 70 % ethanol, rehydrated in PBS, and then resuspended in a DNA extraction buffer (with 0.2 M NaHPO4, 0.1 % Tritonx-100, pH 7.8). After staining with 1 μg/ml of propidium iodide (PI) for 5 minutes, the fluorescence intensity was determined by analysis on a FACS Calibur flow cytometer (Becton-Dickinson). Data acquisition was performed with CellQuest software (Becton Dickinson), and the percentages of phase G1, S, and G2 cells were calculated with the MODFIT-LT software program (Verity Software House, Inc. Augusta, Topsham, ME, USA).
RNA extraction, quantification, and retrotranscription
Total RNA was extracted and purified using E.Z.N.A. Total RNA Kit I (Omega Bio-Tek Inc., Norcross, GA, USA) according to the manufacturer’s instructions. RNA quantity and quality were assessed by Nano Drop 2000 (Thermo Scientific, Waltham, MA, USA), and 2 μg of f-LSC total RNA was reverse-transcribed to cDNA in a volume of 20 μl with Oligo dT primers (Applied Biosystems, Carlsbad, CA, USA) and the Reverse Transcriptase Rnase kit (Improm II; Promega, Madison, WI, USA).
Real-time quantitative PCR
Real-time quantitative PCR primers used for gene expression investigation
The f-LSCs were scraped and incubated on ice for 30 minutes with M-RIPA buffer (50 mM Tris, pH 7.5, 0.1 % Nonidet P-40, 0.1 % deoxycholate, 150 mM NaCl, 4 mM EDTA) and a mixture of protease inhibitors (0.01 % aprotinin, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM PMSF). The whole cellular lysate was centrifuged at 12,000 rcf for 8 minutes to clear cell debris and the supernatant was dialyzed against ultrapure distilled water, lyophilized, and stored at –80 °C, as described previously [30, 31]. The protein concentration in the cellular extracts was determined using the Quick Start™ Bradford Protein Assay (BIO RAD, Segrate, Milan, Italy) according to the manufacturer’s instructions.
Two-dimensional gel electrophoresis (2D-IPG) was performed as described previously [30, 31]. Briefly, protein samples of f-LSCs were solubilized, and aliquots of 45 μg (analytical gels) or 1.5 mg (preparative gels) of total proteins were separately applied for isoelectrofocusing (IEF) using commercial sigmoidal IPG strips, 18 cm long with pH range 3.0–10; BIO RAD). The focused proteins were then separated on 9–16 % linear gradient polyacrylamide gels (SDS-PAGE) and were visualized by means of ammoniacal silver staining.
For image acquisition and data analysis, silver-stained gels were digitized using a computing densitometer and analyzed with ImageMaster 2D PLATINUM software (Amersham, Little Chalfont, Buckinghamshire, UK). Gel calibration was carried out using an internal standard and the support of the ExPaSy molecular biology server; the quantitative analysis of protein spots was performed in duplicate maps, and normalized as vol. % (integration of optical density over the spot area). The differential expression of proteins was evaluated when the difference in their values was ≥ 3 % volume. The labels correspond to the access number of the Swiss-Prot/TrEMBL database.
Mass spectrometric sequencing was performed with the Voyager DE-PRO (Applied Biosystems, Carlsbad, CA USA) mass spectrometer . Peptide mass fingerprinting was compared with the theoretical masses from the Swiss-Prot or NCBI sequence databases using Mascot (http://www.matrixscience.com/).
Gene ontology and network analysis
The genes expressing the invariant and variant proteins were analyzed for their enrichment in specific gene ontology annotation using the Gene Ontology Consortium website (http://geneontology.org/) (Additional file 1: Table S2) .
Network analysis was performed on the genes expressing the invariant and variant proteins using the STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) website (http://string-db.org/) .
Multilineage potential differentiation assays
Multilineage differentiation capability of f-LSCs at early and late passages (P4 and P20) was assessed. The following differentiation protocols were performed.
For osteogenic differentiation, 5 × 103/cm2 f-LSC cells were cultured in home-made differentiation medium consisting of DMEM supplemented with 15 % FBS, 10–4 mM dexamethasone (Sigma-Aldrich), 10 mM glycerophosphate (Sigma-Aldrich), and 0.05 mM ascorbic acid (Sigma-Aldrich) . After 21 days of culture in the differentiation medium, cells were stained with Alizarin red S (Sigma-Aldrich) to detect the calcium deposits. Briefly, the medium was removed and the cells were fixed with 4 % formaldehyde solution for 30 minutes, and after fixation were rinsed twice with distilled water and stained with 2 % Alizarin red S (pH 4.2) for 3 minutes. After incubation the cells were observed under a light optical microscope at 20–40× magnification.
Adipose differentiation was induced by seeding 5 × 103/cm2 of f-LSCs and culturing in home-made differentiation medium consisting of DMEM medium containing 10 % FBS, 0.5 nM 1-methyl-3-isobutylxanthine (IBMX; Sigma-Aldrich), 10–4 mM dexamethasone, 10 μg/ml insulin, and 100 μM indomethacin (Sigma-Aldrich) . To detect the presence of lipid droplets, cells were fixed with 4 % formaldehyde solution, rinsed twice in distilled water, stained with Oil Red O (Sigma-Aldrich), and observed under a light microscope at 20–40× magnification.
Chondrogenic differentiation was induced by culturing cell mass in serum-free DMEM (PAA) with 10 ng/ml TGFβ-3 (Preprotech), 0.05 mM ascorbic acid (Sigma-Aldrich), 2 mM sodium pyruvate (PAA), and 10–7 mM dexamethasone (Sigma-Aldrich) in a six-well culture plate. Briefly, micromass cultures were generated by seeding 5-μl droplets of a cell solution of 1.6 × 107 viable cells/ml in f-EM. After 2 hours of incubation at 37 °C, culture medium was replaced with chondrogenesis differentiation medium. After 21 days of culture the cell mass was evaluated morphologically and stained with Alcian blue to search for sulfated proteoglycan deposits.
All assays were performed in triplicate. The data were reported as means ± SD and compared using the appropriate version of Student’s unpaired t test. Test results were reported as two-tailed p values, where p < 0.05 was considered statistically significant.
Isolation of f-LSCs
Within 10 days the cells, kept in a low-adhesion culture condition, gave rise to floating spherical cell bodies (considered a hallmark of the stemness feature) with a clear and well-delineated border, which we referred to as “limbospheres” (Fig. 1A.b). When transferred to adhesion conditions the limbospheres spread out and a monolayer culture of fibroblast-like cells formed (Fig. 1A.c,d).
SSEA4 expression was investigated by immunofluorescence analysis both in f-LSC spheres (Fig. 1B.a–c) and in monolayer f-LSCs (Fig. 1B.f–h). BM-MSCs and HeLa cells were respectively used as positive control (Fig. 1B.d–i) and negative control (Fig. 1B.e–l). As reported in the literature, it did not seem possible to obtain spheres from cells that have no SSEA4 expression.
Flow cytometry demonstrated that SSEA4 was highly expressed (88.95 ± 7.8 %) in f-LSC monolayer cultures and did not significantly increase after sorting (90.76 ± 5.6 %). Immunophenotypes were analyzed in the presorting (total) population and the postsorting SSEA4+ population. Both populations showed almost no expression of CD34 (0.32 ± 0.01 % vs. 0.19 ± 0.02 %, respectively), CD45 (1.35 ± 0.7 % vs. 0.65 ± 0.2 %, respectively), and HLA-DR (0.25 ± 0.04 % vs. 0.16 ± 0.07 %, respectively) (Fig. 1C).
Since neither population displayed any significant differences in marker expression, the subsequent cell analyses were only performed on the total monolayer f-LSC population.
Proliferation in long-term culture
Flow cytometry stem cell phenotype characterization in long-term culture
Phenotype characterization was performed at two different culture passages (P3 and P30). A collection of double-positive populations (dpp) was obtained for several stem markers. The data expressions detected with flow cytometry analysis are the following (P3 vs. P30): ABCG2+/SSEA4+ = 98.6 ± 7.1 % vs. 96.6 ± 4.7 % (p = NS); OCT4+/SSEA4+ = 92.1 ± 6.4 % vs. 88.3 ± 2.6 % (p = NS); and NANOG+/SSEA4+ = 95.42 ± 6.8 % vs. 80.52 ± 4.2 % (p = NS). The f-LSCs did not express Δnp63 (Δnp63+/SSEA4+ < 0.5 %). Figure 2b shows one representative experiment out of a total of 25 sets of experiments.
The positive controls are shown in Additional file 2: Figure S1.
Stem-related gene expression is not influenced by age and by long-term cultures
The f-LSC proteomic profile is not influenced by age and by long-term cultures
The protein-interaction networks (PIN) for the unvaried protein groups revealed strong interactions between structural proteins (Fascin-1, cytoskeletal keratin 19), chaperones, including two heat shock proteins (HSP70, HSP90), DNA binding protein (i.e., elongation factors EEF1A, EEF1B2) and remodeling and proteosome complex (PSMA5,4,6). This profile is consistent with a stem phenotype that requires plasticity and good proliferative ability [47, 48].
Interestingly, the hypoxia-inducible factor-1 (HIF-1) pathway is a central node of differential protein group PIN with close correlation between superoxide dismutase 2 (SOD2), thioredoxin (TXN), peroxiredoxin 6 (PRDX6), and catepsin D (CTSD). It has been shown that HIF-1 is involved in self-renewal and maintenance of pluripotency, by promoting the expression of the putative stemness genes (OCT4, SOX2, NANOG, Klf4) [49–51].
Multilineage capability of f-LSCs
Moreover, oil red positivity confirmed the presence of neutral triglyceride and lipid vacuoles, suggesting adipogenic differentiation after 28 days of culture (Fig. 6c).
Ease of access and immune-privileged status make the eye an ideal organ for its potential application in regenerative medicine. In our study, we confirmed the ease of isolation of the subpopulation of f-LSCs, which make them ideal candidates for adult stem cell therapy. In spite of the great interest in clinical applications, no exhaustive information is yet available on f-LSCs, especially on their exact characteristics. The International Society for Cellular Therapy has defined criteria to identify MSCs . We adopted these criteria to characterize f-LSCs from the limbus in our study. In addition, this work provides convincing evidence that limbal stroma contains f-LSCs. The latter subpopulation resembles stem cells which reside in the stem cell niche, where they are maintained in an undifferentiated state. These observations were supported by Notara and Daniels  who showed that LESCs are discretely located in the basal layer of the corneal limbal epithelium, at the junction between the transparent cornea and the opaque sclera. The limbal palisades of Vogt have been proposed as the site of the LESC niche whereas f-LSCs seem to reside in the extracellular matrix [3, 4]. Corneal stromal stem cells have been located in the anterior stroma subadjacent to the basal side of the palisades of Vogt [54–60]. In our study we did not aim to define the exact localization of f-LSCs but confirmed that they are completely different from LESCs, as demonstrated by ΔNp63 and CK12 and CK19 negativity, and they can be easily isolated and grown.
In our research we showed that f-LSCs fulfilled the criteria for multipotency established today.
In particular, our group investigated the possible effects of long-term culture conditions and donor age on the f-LSC phenotype. The cells isolated did not display surface expression of any hematopoietic marker (CD34 and CD45) and HLA-DR. By contrast, they expressed a variety of stemness markers (SSEA4, ABCG2, OCT3/4, and NANOG).
They showed low adhesion growth capability as limbo-spheres and they should proliferate in vitro as adherent cells for long-term culture, maintaining positivity for ABCG2, OCT3/4, NANOG, SOX2, THY-1, and SSEA4 and negativity for CD34, CD45, and HLD-DR; and they have multilineage differentiation potential under in-vitro culture conditions. By comparing the cell cycle distribution and the proliferation curve in the early and late passages there was a weak increase in the percentage of cells in phase G1 with a concomitant decrease in the percentage of cells in phase S. This event is reflected as a slight elongation in the doubling time in late passage culture but was not analyzed in more detail.
The evaluation of molecular expression of f-LSCs isolated from different donors (with the same or different age) and at different culture passages revealed a strong stability of the “limbal stem molecular pattern”, not affected by long-term culture and donor age. No significant differences were found regarding specific osteogenic, adipogenic, or chondrogenic staining.
For the first time, we constructed a two-dimensional electrophoresis (2-DE) proteomic pattern of cultured f-LSCs. The derived pattern of resolved protein spots was highly consistent; we identified 164 proteins stably expressed in the different conditions, defining profiles enriched in proteins linked to cell plasticity and proliferative and self-renewal capability. Furthermore, overlapping protein expression profiles confirm the stability of the stem cell phenotype with higher expression of structural proteins and proteins involved in the stem molecular pathway.
f-LSCs represent a robust source of MSCs independently of donor age and in-vitro conditions; the stability of their proteomic pattern could be very promising, suggesting their potential use in regenerative medicine.
ABCG2, ATP-binding cassette sub-family G member 2; BM-MSC, bone marrow-derived mesenchymal stromal cell; f-LSC, fibroblast-like limbal stem cell; LESC, limbal epithelial stem cell; MSC, mesenchymal stromal cell; NANOG, Homeobox protein NANOG; OCT4, octamer-binding transcription factor 4; P, passage of culture; PBS, phosphate-buffered saline; PIN, protein-interaction networks; SOX2, SRY (sex determining region Y)-box 2; SSEA4, stage-specific embryonic antigen-4; ΔNp63, a splice variant of p63
The authors thank A Criscimanna and G Zito for their efforts in the first part of the limbus research area.
This work is dedicated to our former director Prof. Aldo Galluzzo, who prematurely died in 2011 when the Laboratory of Regenerative Medicine was created and research in this field started.
LT was responsible for conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing. RM was responsible for collection and assembly of data and revision of the manuscript. GC and SC were responsible for conception and design, provision of study material or patients, and revision of the manuscript. GP, MP, AC, and WA were responsible for data analysis and interpretation and drafting the manuscript. GDC was responsible for acquisition of data and revision of the manuscript. IP-M was responsible for manuscript writing and revision of the manuscript. CG was responsible for conception and design, data analysis and interpretation, manuscript writing and final approval of manuscript, manuscript drafting, revising critically for important intellectual content, and financial support. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manuscript.
Financial competing interests
The work was partially funded by PON 01_00829 2007/2013 with European Community funds administered by the Italian Ministry for the University and RIMEDRI PO FESR 2007/2013 (CG was Scientific Director for both).
The authors declare that they have applied for a patent concerning the possible use of f-LSCs in type 1 diabetes.
Nonfinancial competing interests
The authors declare that they have no nonfinancial competing interests in relation to this manuscript.
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