Role of Human Corneal Stroma-Derived Mesenchymal-Like Stem Cells in Corneal Immunity and Wound Healing

Abstract

Corneal tissue regeneration is of crucial importance for maintaining normal vision. We aimed to isolate and cultivate human corneal stroma-derived mesenchymal stem-like cells (CSMSCs) from the central part of cadaver corneas and study their phenotype, multipotency, role in immunity and wound healing. The isolated cells grew as monolayers in vitro, expressed mesenchymal- and stemness-related surface markers (CD73, CD90, CD105, CD140b), and were negative for hematopoietic markers as determined by flow cytometry. CSMSCs were able to differentiate in vitro into fat, bone and cartilage. Their gene expression profile was closer to bone marrow-derived MSCs (BMMSCs) than to limbal epithelial stem cells (LESC) as determined by high-throughput screening. The immunosuppressive properties of CSMSCs were confirmed by a mixed lymphocyte reaction (MLR), while they could inhibit proliferation of activated immune cells. Treatment of CSMSCs by pro-inflammatory cytokines and toll-like receptor ligands significantly increased the secreted interleukin-6 (IL-6), interleukin-8 (IL-8) and C-X-C motif chemokine 10 (CXCL-10) levels, as well as the cell surface adhesion molecules. CSMSCs were capable of closing a wound in vitro under different stimuli. These cells thus contribute to corneal tissue homeostasis and play an immunomodulatory and regenerative role with possible implications in future cell therapies for treating sight-threatening corneal diseases.

Figure 1. Morphology of the in vitro CSMSC cultures.

Corneal stroma graft (*) isolated from the central part of the cornea was cultured in a cell culture plate (A), giving rise to elongated cells in the first 14 days of in vitro culture. Cells formed a monolayer by day 28, and showed fibroblastoid morphology according to their cytoskeletal structure. The expression of carbohydrate molecules on the surface of CSMSCs labelled by fluorescein-conjugated lectins is being shown (B). GSL I:Griffonia (Bandeiraea) simplicifolialectin I (Griffoniasimplicifolia); LCA:Lens culinaris agglutinin (Lens culinaris); PHA E:Phaseolus vulgaris erythroagglutinin (Phaseolus vulgaris); PHA L:Phaseolus vulgaris leucoagglutinin (Phaseolus vulgaris); PSA:Pisumsativum agglutinin (Pisumsativum; sWGA: succinylatedWheat germ agglutinin (Triticum vulgaris);DBA: Horse gram lectin/Dolichosbiflorus agglutinin (Dolichosbiflorus); ConA: Concanavalin A (Canavaliaensiformis);PNA: Peanut agglutinin (Arachishypogaea); RCA 120:Ricinus communis agglutinin (Ricinuscommunis);SBA: Soy bean agglutinin (Glycine max); UEA:Ulexeuropaeus agglutinin (Ulexeuropaeus); WGA: Wheat germ agglutinin (Triticum vulgaris); AIL:Jacalin(Artocarpusintegrifolia) (Magnification: 100X A1-3; 200XB1-14, Phalloidin-TRITC staining A-3, 400× Lectin staining B1-14).

Figure 2. Heatmap of the differentially expressed genes in CSMSCs compared to LESCs and BMMSCs.

Different expression levels of the transcripts and functional clustering of the genes expressed in in vitro cultured CSMSCs, LESCs and BMMSCs. Genes related to stemness, HOX, Notch and SOX signalling, differentiation and lineage, cell cycle and oncogenes were selected. The cluster analysis and dendrograms show the difference between the three cell types, and strengthen the finding that CSMSCs are more closely related to BMMSCs than to LESCs. Red and yellow colors indicate high and low expression, respectively.

Figure 3. MSC-related phenotype, differentiation potential and immunosuppressive effects of in vitro cultured CSMSCs.

Hierarchical clustering of cell surface molecules’ expression divided the stem cells of different tissue origin into two upper classes: CSMSCs were more closely related to BMMSC than to LESCs (A) (Color key represents percentage of positive cells in the in vitro cell cultures). CSMSCs were able to differentiate into the canonical mesodermal lines in vitro. Osteogenic differentiation shows the calcium deposits present as red-brown hue in the cell cultures visualized by Alizarin-red staining. Oil Red-O stained lipid droplet accumulations are shown in the adipocytes derived from CSMSCs in vitro. Metachromasia of the extracellular matrix stained by toluidine blue is shown in the cartilaginous section after chondrogenic differentiation (B). CSMSC could inhibit the proliferation of immune cells activated by PHA and ConA even in low cell numbers (left-hand panel) in vitro. Opposite to the mitogen-induced reaction, CSMSCs could block lymphocyte proliferation only at high doses in the mixed lymphocyte reaction (right-hand panel) (Data shown are mean ± SD, N = 3) (C).

Figure 4. Cytokine secretion by activated CSMSCs.

The bar graphs illustrate results of the quantitation of cellular responses of cytokine production by in vitro cultured CSMSCs and BMMSCs of both IL-6 and IL-8 and in response to activation by TRL ligands (LPS, Poly:IC), as well as pro-inflammatory cytokines (TNFα, IFNγ, IL-1β) observed after 12 h and 24 h intervals (A). Involvement of NFκB and IκB in the signalling pathways initiated by the TLR ligands and pro-inflammatory cytokines is being shown (B). (Data shown are mean ± SEM; *p < 0.05 **p < 0.01 ***p < 0.001 N = 6 for the BMMSCs and N = 9 for the CSMSCs, respectively).

Figure 5. The effect of inflammatory stimuli on the CSMSC expression of cell adhesion molecules and wound healing ability.

The percentage of positive cells for CD54/ICAM-1 increased upon pro-inflammatory stimuli within the in vitro CSMSC cell cultures (A). Median fluorescent intensity (MFI) changed just in the case of CD47 molecules (B). Dot plot of the selected surface markers is being shown (C). Impedance-based wound healing assay results investigating the wound healing properties of CSMSCs under normal and inflammatory conditions. Cells were treated after wound creation by pro-inflammatory cytokines and TLR ligands, which prolonged the time of wound closure as shown by the Whiskers boxes (D). (N = 6 donors in triplicates, *p < 0.05 **p < 0.01 ***p < 0.001).