Regenerative therapy for the Cornea

Abstract

The cornea is the outmost layer of the eye, unique in its transparency and strength. The cornea not only transmits the light essential for vision, also refracts light, giving focus to images. Each of the three layers of the cornea has properties essential for the function of vision. Although the epithelium can often recover from injury quickly by cell division, loss of limbal stem cells can cause severe corneal surface abnormalities leading to corneal blindness. Disruption of the stromal extracellular matrix and loss of cells determining this structure, the keratocytes, leads to corneal opacity. Corneal endothelium is the inner part of the cornea without self-renewal capacity. It is very important to maintain corneal dehydration and transparency. Permanent damage to the corneal stroma or endothelium can be effectively treated by corneal transplantation; however, there are drawbacks to this procedure, including a shortage of donors, the need for continuing treatment to prevent rejection, and limits to the survival of the graft, averaging 10-20 years. There exists a need for new strategies to promote regeneration of the stromal structure and restore vision. This review highlights critical contributions in regenerative medicine with the aim of corneal reconstruction after injury or disease. These approaches include corneal stromal stem cells, corneal limbal stem cells, embryonic stem cells, and other adult stem cells, as well as induced pluripotent stem cells. Stem cell-derived trophic factors in the forms of secretomes or exosomes for corneal regeneration are also discussed. Corneal sensory nerve regeneration promoting corneal transparency is discussed. This article provides description of the up-to-date options for corneal regeneration and presents exciting possible avenues for future studies toward clinical applications for corneal regeneration.

Keywords: Cornea; Exosomes; Regeneration; Secretomes; Sensory nerve; Stem cells.

Figure 1.. Localization and marker expression of human CSSCs.

(A) Transverse frozen sections were obtained from the limbal and central regions of unfixed human corneas as indicated by white lines in the photograph, the central region was indicated by the white circle while the limbal region was between 2 dotted red circles. (B) ABCG2 protein (green) was detected in epithelial and stromal cells of limbal sections. Cells were counterstained with To-Pro- 3 (red). Arrows show stained stromal cells, and triangles indicate stained epithelial cells. Inset shows magnification of two cells designated by arrows. (C) ABCG2 protein (green) was detected in limbal sections. Arrows show stained stromal cells, and triangles indicate stained epithelial cells. (D) ABCG2 was detected in occasional stromal cells in central cornea (arrow). Scale bars = 50 μM. Adapted from Du et al. STEM CELLS, 2009, 23: 1266–1275. License number 5046631145230. (E) Schematics of the CSSC and LSC niche. The section shows the anterior region of the transition zone between cornea and sclera known as the limbus. Epithelium is thickened over regions of undulations in the epithelial basement membrane known as the Palisades of Vogt. LSCs are localized in limbal basal epithelium. Unlike the central cornea, limbal stroma is vascularized, containing melanocytes (black) and mesenchymal keratocytes (blue). CSSCs (green) are located subjacent to the basement membrane near LSCs. Adapted from Pinnamaneni et al. STEM CELLS 2012; 30:1059–1063. License number 5046631345115 (F, G) Cell surface markers on CSSCs. CSSCs, passage 3, were stained for cell surface antigens and analyzed by flow cytometry. 98% of CSSCs stained positive for both CD90 and CD166 (F); 97% of CSSCs stained positive for CD73, but <2% were positive for the hematopoietic stem cell marker CD34 (G). Adapted from Funderburgh et al. Ocul Surf. 2016. 14: 113–120. License number 5046620856317

Figure 2.. CSSC intrastromal transplantation rescues corneal collagen phenotype and corneal transparency in Lum−/− mice.

(A) Lumican (red) is largely localized in the posterior stroma of WT mice. (B) Lumican is absent in Lum−/− mice. (C) After injection of CSSCs, lumican was distributed throughout the posterior cornea of Lum−/− mice. DAPI stains nuclei, shown as green (A–C). (D) Electron microscopy of the posterior stroma of WT mice revealed aligned collagen fibrils with highly regular diameters. (E) Lum−/− posterior stroma collagen had numerous aggregated fibrils (green arrows). (F-G) Twelve weeks after CSSC injection, the posterior stroma of Lum−/− mice exhibited regular collagen architecture, lacking fibril aggregates (G) and indistinguishable from stromas of noninjected littermate heterozygote controls (F). (H) Stromal thickness was significantly less in Lum−/− mice than in WT (p < .01) but was significantly increased (p < .01) in Lum−/− eyes 12 weeks after injection of CSSCs (Lum−/−SC). (I) Stromal light scatter was significantly greater in Lum−/− mice (p < .001) but was reduced to normal range after CSSC injection (p < .001). Modified from Du et al. STEM CELLS, 2009, 27: 1635–1642. License number 5046640043756

Figure 3.. CSSCs from limbal biopsy (LBSCs) synthesize stromal matrix and influence light transmission in mouse corneas in vivo.

Fluorescent DiO-labeled human LBSCs were transferred to a superficially debrided mouse cornea in a fibrin gel. (A) At 1 week, whole-mount staining showed persistence of the human LBSCs (green) in the central corneal region. (B) At 1-month, histological sections immunostained with human-specific antibodies showed human keratocan and collagen type I. The control was also used without the primary antibody for keratocan and collagen type I to omit nonspecific staining. Nuclei are stained with DAPI (blue). Anterior of the eye is oriented up in each image in (B), and the corneal epithelium is visible as a dense layer of cells near the top of each image. (C) Transmission electron micrographs taken 4 weeks after debridement show the ablated region of the anterior stroma. epi, epithelial cells; bm, basement membrane; k, keratocyte processes; L, amorphous matrix deposit (lake). Insets show magnification of a box (1 μm × 1 μm) from the indicated region containing orthogonal views of collagen fibrils. Scale bar, 2 μm. Images are representative of n = 3 animals. (D) Light scatter in 3D optical coherence tomography (OCT) scans at 2 and 4 weeks was compared with the values in preoperative eyes. Light scatter is shown as mean ± SD. The number of eyes is indicated in the graph. P values were determined with unpaired t tests at each time point compared to respective Pre-op values. Modified from Basu et al. Sci Transl Med 2014;6:266ra172. License number 5046640220452

Figure 4.. Prevention of corneal scarring by CSSC embedded in compressed collagen gel (CCG).

Sections from mouse corneas 14 days after wounding were stained with H&E (A, B) or immunostained with antibody to collagen type III (C, D). (A, C) Corneas treated with CSSC in CCG compared to (B, D) corneas treated with CCG only. Scale bars=50 μm. (E) Scarring at 14 days after wounding was evident in 5/6 corneas treated with CCG only, but 0/10 corneas showed visible scarring at 14 days when treated with CCG + CSSC. Contingency analysis (Fisher’s Exact Test) of these data indicated a high level of significance (p = .0014). Modified from Shojaati et al. Stem Cells Transl Med, 2018, 7: 487–494. License number 5046640517457

Figure 5.. Dental pulp cells (DPCs) produce human corneal stromal matrix in vivo.

Cryosections of mouse corneas, 2 weeks after intrastromal injection of DPCs, were immunostained with human-specific antibodies and imaged using confocal microscopy. (A, B) Collagen type I (red) localized to regions containing DiO-labeled DPCs (green) in the injected cornea. (C, D) In contrast, no human collagen was seen in the corneas without injected DPCs. (E, F) Human keratocan (red) was localized near DiO-labeled DPCs (green) but was not detected in uninjected control corneas (G, H). In each case, higher magnification of the boxed region is shown in the image on the right. Nuclei were stained with DAPI (blue). Scale bars = 250 μm (A, C, E, G) and 100 μm (B, D, F, H). Adapted from Syed-Picard et al. Stem Cells Transl Med. 2015, 4:276–85. License number 5046640755442

Figure 6.. Human CSSC secrete extracellular matrix (ECM) on aligned PEUU fibrous substrates.

(A) Two-photon images of hCSSC-secreted ECM compare (a) aligned fibrous, (b) random fibrous, and (c) cast film PEUU. The second harmonic generation signal for collagens when excited at λ = 830 nm is red. Nuclei are stained green. Adapted from Wu et al. Biomaterials. 2012, 33: 1343–1352. License number 5046640895799 (B) Transmission electron micrographs of CSSC-secreted ECM on the aligned PEUU fibrous substrates varied with growth factor treatment. (a) 10 ng/mL FGF-2, (b) 0.1 ng/mL TGF-β3, and (c) 10 ng/mL FGF-2 + 0.1 ng/mL TGF-β3. The thickness of the ECM secreted by CSSCs as a function of the growth factor is summarized in (d). *p < 0.05 was considered significant. Adapted from Wu et al. Tissue Eng Part A, 2013, 19: 2063–2075. under the Creative Commons Attribution 4.0 (CC BY) license

Figure 7.. Scaffold- free tissue sheet with parallel cell and matrix organization maintains transparency after transplantation into the mouse cornea.

(A) Light micrographs of (a) top view and (b) cross- sectional view of the PDMS substrate show grooves approximately 10 μm wide, 10 μm apart and 5 μm deep. (c) Phase contrast image shows CSSC cultured on the grooved substrate. (d) For better visualization, CSSC were labelled with DiI (red) and cultured on grooved substrate. (e) Two- photon micrograph of 10- day cultures of CSSC on grooved substrates in keratocyte differentiation medium (KDM) shows deposition of parallel organized collagenous matrix (green). Nuclei (blue) were stained by SYTOX- green (blue). (f) After 10 days of culture a robust tissue sheet is formed that can be separated from the substrate using forceps. Scale bars: (a) and (b) = 50 μm, (c–e) = 100 μm. (B) Cross- sectional projection image of untreated control mouse cornea (a), mouse cornea at 1 week after tissue sheet implantation (arrow) (b), mouse cornea at 5 weeks after tissue sheet implantation (c). Quantitation of light scatter from OCT scans of the stroma shows light scatter by implanted tissue sheet 1-week post implantation (d) and 5 weeks post implantation (e), corneas without (−) cell sheet were as normal control (d, e), *p < 0.05. Adapted from Syed-Picard et al. J Tissue Eng Regen Med. 2018, 12:59–69. License number 5046641397700

Figure 8.. CSSC treatment reduces corneal neutrophil filtration after wounding and knockdown of TSG-6 in CSSCs restores neutrophil infiltration after wounding.

Cells pooled from 6 mouse corneas were isolated 24 hr after wounding and separated by flow cytometry using antibodies to antigens present on the surface of neutrophils: CD45, Ly6G and CD11b. (A) Cells from wounded corneas treated with fibrinogen only. (B) Cells from wounded corneas treated with CSSC in fibrinogen. Graph (C) shows TSG-6 in culture media detected by ELISA after 72 hr culture in stem cell growth media (SGM). Untreated CSSC (black); CSSC cultured in 20 ng/ml TNFα and 10 ng/ml IFN-γ (blue); CSSC transfected with siRNA against TSG-6 mRNA and incubated in TNFα+IFN-γ (red); CSSC transfected with a scrambled siRNA and incubated in TNFα+ IFN-γ (green). Error bars show standard deviation (SD) of quadruplicate assays. Graph (D) shows MPO assay of extracts of individual corneas. Corneas were: non-wounded (No-Wnd, black); wounded-untreated (Wnd, green); wounded and treated with CSSC (CSSC-siCtrl, blue); wounded and treated with CSSC with TSG-6 knocked down (CSSCsiTSG6, red). Error bars show SD and p values from t-test comparing individual pairs of samples. Modified from Hertsenberg et al. PLoS One. 2017,12:e0171712, under the Creative Commons Attribution (CC BY) license.

Figure 9.. Transplantation of cultured limbal cells onto LSCD rabbit corneas.

One month after ablation of corneal epithelial and limbal cells, covering conjunctival cellular material was removed by scraping from a region 5 mm beyond the limbus. Human amniotic membrane (HAM) carrying cultured human limbal cells on the basement membrane surface (colored) was sutured on the denuded region and covered by a second HAM with the basement membrane surface oriented toward the cornea. Orientation of the HAM basement membrane is shown by arrows. Adapted from Du et al. Mol Vis. 2003, 9: 635–643, under CC BY-NC-ND 3.0 license.

Figure 10.. Expression of CD45, interleukin 2 (IL-2), and matrix metalloproteinase- 2 (MMP- 2) in rat corneas after transplantation.

Rat CD45 and IL-2 were not detected in rat corneal surface transplanted with MSCs on human amnion membrane (HAM, A, G) and LSCs on HAM (B, H), but was highly expressed in rat corneas treated with fibroblast cells (C, I), HAM alone (D, J), dexamethasone (Dex) (E, K), and gentamicin (Gen) (F, L). Green (A–F) indicates CD45 staining, and red (G–L) indicates interleukin 2 staining. MMP- 2 was not detected in rat corneas transplanted with MSCs on HAM (M) or with LSCs on HAM (N). Meanwhile, MMP-2 was highly expressed in rat corneas treated with fibroblast cells (O), HAM alone (P), Dex (Q), and Gen (R). Modified from Ma et al. Stem Cells. 2006, 24:315–21. License number 5046651433070

Figure 11.. Corneal wound healing by stem cell secretome.

(A) Phase-contrast images for temporal live-cell assessment of effect of secretome obtained from various CSSCs on wound-healing capacity of corneal fibroblasts (magnification ×10). (B) Bar diagram showing the average percentage area covered by cells in wound after different time intervals. (C) qPCR analysis for fibrotic markers in corneal fibroblasts at control and post secretome treatment level. *P < 0.05, **P < 0.001. Adapted with permission from Kumar et al. Invest. Ophthalmol. Vis. Sci. 2018;59:3728–3738.

Figure 12.. EV treatment prevents scar formation after corneal wounding.

(A) Corneas were wounded by surface debridement and immediately treated with CSSC or with extracellular vesicles (EVs) from CSSC in a fibrin gel. Two weeks after wounding, scar area was assessed by image analysis of the corneas. Each point represents the scar area of one cornea. (B) In a separate experiment, the therapeutic effect of EVs from CSSC and HEK293T cells was compared by measuring scar area. (C) Expression of mRNA for collagen 3a1 (Col3a1) was compared in corneas from experiment in (A) using qPCR. (D) Expression of mRNA for smooth muscle actin (SMA) was compared using qPCR. Error bars represent standard deviation. In (C) and (D), n = 3. In (A) and (B), n = 8 (*, p < .05; ***, p < .001) using t-test. Adapted from Shojaati et al. Stem Cells Transl Med. 2019, 8:1192–1201. License number 5046660684782

Figure 13.. Superior cervical ganglionectomy eliminates sympathetic nerves, allows regrowth of sensory nerves, and prevents development of severe persistent HSK in BALB/c mice.

BALB/c mice were infected with 13105 pfu of the HSV-1 KOS strain. Groups of infected mice received mock SCGx (superior cervical ganglionectomy) or SCGx at 10 dpi (days post infection) and were followed to 28 dpi, whereas other groups received mock SCGx or SCGx at 54 dpi and were followed to 94 dpi. Opacity scores were recorded on the day mice were sacrificed. Whole mounted corneas were stained for βIII tubulin (green), TH (red), and SP (gray). (A) Superior cervical ganglionectomy administered at 10 dpi effectively eliminated the hyperinnervation at 28 dpi and permitted regrowth of a low density of SP-positive sensory nerves. Administering SCGx after hyperinnervation when sympathetic nerves had established (54 dpi) did not eliminate the hyperinnervating fibers, but the nerves did not express the sympathetic nerve marker TH. Note the hyperinnervation of the corneal stroma of mice that received mock SCGx at 10 or 54 dpi, with nerve fibers expressing the sympathetic nerve marker TH but not the sensory nerve marker SP. (B) Nerve density in corneal stromas of mock or SCGx-treated mice at 28 and 94 dpi was quantitated as the cumulative length of nerve fibers in 5003500-lm areas of the corneal stroma. (C) Opacity of infected corneas was recorded just prior to death at 28 and 94 dpi. **P <0.01, ***P < 0.001, ****P < 0.0001. Adapted with permission from Yun et al. Invest Ophthalmol Vis Sci. 2016, 57:1749–1756.