The Impact of Limbal Mesenchymal Stromal Cells on Healing of Acute Ocular Surface Wounds Is Improved by Pre-cultivation and Implantation in the Presence of Limbal Epithelial Cells

Abstract

While limbal epithelial cells are used for treating ocular surface wounds, the therapeutic potential of mesenchymal cells cultivated from the limbal stroma (LMSC) is less clear. We have therefore examined the effects of LMSC when applied to acute ocular surface wounds. LMSC derived from male rabbits (RLMSC) were applied to the ocular surface of female rabbits immediately following removal of the corneal and limbal epithelium. Human amniotic membrane (HAM) was used as the vehicle for implanting the RLMSC. The effects of RLMSC were examined when applied alone (n = 3) and in conjunction with a stratified culture of human limbal epithelial cells (HLE) grown on the opposing surface of the HAM (n = 3). Outcomes were monitored over 3 months in comparison with animals receiving no treatment (n = 3) or treatment with HLE alone on HAM (n = 3). Animals treated with RLMSC (n = 6) displayed faster re-epithelialization (∼90% versus 70% healing after 12 weeks), with best results being observed when RLMSC were pre-cultivated and implanted in the presence of HLE (p < 0.01; 90% healing by 7 weeks). While all animals displayed conjunctival cells on the corneal surface (by presence of goblet cells and/or keratin 13 expression) and corneal neovascularization, evidence of corneal epithelial regeneration was observed in animals that received RLMSC in the presence of HLE (by staining for keratin 3 and the absence of goblet cells). Conversely, corneal neovascularization was significantly greater when RLMSC were applied in the absence of HLE (<0.05; 90% of cornea compared with 20-30% in other cohorts). Nevertheless, neither human nuclear antigen nor rabbit Y chromosome were detected within the regenerated epithelium. Our results demonstrate that while cultured LMSC encourage corneal re-epithelialization, healing is improved by the pre-cultivation and implantation of these mesenchymal cells in the presence of limbal epithelial cells.

Keywords: Algerbrush II; amniotic membrane; corneal neovascularization; limbal mesenchymal stromal cells; ocular surface; wound healing.

Figure 1.

Confirmation of HLE and/or RLMSC presence in prepared cultures. Representative images of histological sections (H&E stained) obtained from spare cultures of human limbal epithelial (HLE) cells and/or rabbit limbal mesenchymal stromal cells (RLMSC) attached to human amniotic membrane (HAM). Notably, the RLMSC culture was more stratified when grown in the presence of HLE.

Figure 2.

Graphical summary of clinical data. (A) Time course of re-epithelialization as measured under cobalt lamp illumination after fluorescein staining. Analysis of data using a two-way ANOVA (followed by Tukey’s multiple comparisons test) revealed significant differences between each pair of treatment groups (at p < 0.05 or less). Asterisks (* = p < 0.5; ** = p < 0.0001) indicate significant differences between each treatment cohort compared with the non-treated control group (No Tx). (B) Comparison of serum CRP levels. Line graphs indicate the mean +/- SEM of values (mg/L) for each cohort of three rabbits. Single asterisk indicates significant (p < 0.005) difference to animals wounded without treatment (No Tx). Double asterisk indicates a significant difference (p < 0.0001) between animals treated with co-cultures (HLE-HAM-RLMSC) compared with all other cohorts. (C) Comparison of corneal neovascularization observed between animals after 12 weeks. Line and error bars indicate the mean +/- SEM for each treatment cohort. The % corneal neovascularization (CNV) for each animal (A through L) was calculated based upon estimated measures of corneal area with blood vessels using ImageJ. Asterisk indicates a significant difference in CNV for animals receiving HAM with RLMSC cultured on the underlying surface, compared with animals that had been wounded without subsequent treatment.

Figure 3.

Healing patterns (re-epithelialization) of rabbit eyes after 12 weeks, as viewed under cobalt lamp illumination after fluorescein staining. Labels “A” through “L” indicate identity of each rabbit as summarized in Table 1. Treatment groups as described above consisted of controls (No Tx; rabbits A, B, and C), human limbal epithelial cells grown on human amniotic membrane (HLE-HAM; rabbits D, E, and F), HLE and rabbit mesenchymal stromal cells grown on HAM (HLE-HAM-RLMSC; rabbits G, H, and I), or HAM with RLMSC alone (HAM-RLMSC; rabbits J, K, and L).

Figure 4.

Gross appearance of rabbit eyes displaying varying degrees of corneal vascularization at 12 weeks. Labels “A” through “L” indicate identity of each rabbit as summarized in Table 1. Treatment groups as described above consisted of controls (No Tx; rabbits A, B, and C), human limbal epithelial cells grown on human amniotic membrane (HLE-HAM; rabbits D, E, and F), HLE and rabbit limbal mesenchymal stromal cells grown on HAM (HLE-HAM-RLMSC; rabbits G, H, and I), or HAM with RLMSC alone (HAM-RLMSC; rabbits J, K, and L).

Figure 5.

Basic histology of rabbit corneas at 12 weeks as revealed by staining of sections with hematoxylin and eosin (H&E) and periodic acid–Schiff stain (PAS). Labels “A” through “L” indicate identity of each rabbit as summarized in Table 1. Treatment groups consisted of controls (No Tx), human limbal epithelial cells grown on human amniotic membrane (HLE-HAM), HLE and rabbit mesenchymal stromal cells grown on HAM (HLE-HAM-RLMSC), or HAM with RLMSC alone (HAM-RLMSC). Arrows highlight the location of goblet cells (GC) and blood vessels (BV).

Figure 6.

LMSC affect the phenotype of healed epithelium. Immunohistochemical staining of rabbit corneas at 12 weeks to demonstrate typical presence of corneal (K3) and conjunctival (K13) epithelium. Labels “A” through “L” indicate identity of each rabbit as summarized in Table 1. Treatment groups as described above consisted of controls (No Tx), human limbal epithelial cells grown on human amniotic membrane (HLE-HAM), HLE and rabbit mesenchymal stromal cells grown on HAM (HLE-HAM-RLMSC), or HAM with RLMSC alone (HAM-RLMSC). Notably, the best healing outcomes were achieved for two animals receiving RLMSC in the presence of HLE (parts G and H).

Figure 7.

The regenerated epithelium does not contain HLE. Upper panel: Confirmation of immunoreactivity of control human tissue toward antibody to human nuclear antigen (mab 235 -1). The terminal end of Bowman’s layer (BL) is visible by phase contrast images (left) and via background fluorescence in stained sections (right) indicating that the images are acquired at the corneal limbus. Lower panel: Demonstrates example of staining outcomes when sections of non-wounded control (No Tx eye) and treated rabbit tissue (Rabbit “G”) are stained for human nuclear antigen. The absence of staining suggests that no human epithelial cells (HLE) have been retained by 12 weeks. NB: BL is not present in the rabbit cornea.

Figure 8.

The regenerated epithelium does not contain RLMSC. Results of FISH staining for the rabbit Y chromosome within sections of control male corneal epithelium (A; with arrow denoting positive labeling and magnified 3-fold within insert) compared with negative reactivity with the regenerated epithelium observed in rabbit “G.”