3D-Bioprinted Biomimetic Epithelial-Stromal Hydrogel Construct with In Situ Photocrosslinkable Bioadhesive for Suture-Free Corneal Regeneration.

In this issue of Stem Cell Reports, Park et al. (2019) describe real-time in vivo visual monitoring of keratin-14+, Confetti-labeled limbal epithelial stem cells and their progeny as they contribute to central corneal wound-healing. The authors show that corneal wounds initially heal by “basal epithelial cell migration” into the wound-bed.

3D printed biomimetic bilayer limbal implants for regeneration of the corneal structure in limbal stem cell deficiency.

With insufficient options to meet the clinical demand for cornea transplants, one emerging area of emphasis is on cornea tissue engineering. In the present study, the goal was to combine the corneal stroma and epithelium into one coculture system, to monitor both human corneal stromal stem cell (hCSSC) and human corneal epithelial cell (hCE) growth and differentiation into keratocytes and differentiated epithelium in these three-dimensional tissue systems in vitro. Coculture conditions were first optimized, including the medium, air-liquid interface culture, and surface topography and chemistry of biomaterial scaffold films based on silk protein. The silk was used as scaffolding for both stromal and epithelial tissue layers because it is cell compatible, can be surface patterned, and is optically clear. Next, the effects of proliferating and differentiating hCEs and hCSSCs were studied in this in vitro system, including the effects on cell proliferation, matrix formation by immunochemistry, and gene expression by quantitative reverse transcription-polymerase chain reaction. The incorporation of both cell types into the coculture system demonstrated more complete differentiation and growth for both cell types compared to the corneal stromal cells and corneal epithelial cells alone. Silk films for corneal epithelial culture were optimized to combine a 4.0-μm-scale surface pattern with bulk-loaded collagen type IV. Differentiation of each cell type was in evidence based on increased expression of corneal stroma and epithelial proteins and transcript levels after 6weeks in coculture on the optimized silk scaffolds.

The cornea is a transparent, well-organized connective tissue that provides two-thirds of the eye’s total refractive power. The centermost layer of the cornea is the stroma that makes up approximately 90% of the corneal volume. The corneal stromal limbus contains anatomical features, named the palisades of Vogt, that form a niche for both limbal epithelial stem cells (LESCs) and corneal stromal stem cells (CSSC). CSSC are derived from the neural crest as they express genes typical for descendants of the neural ectoderm such as PAX6, Six2, Six3, and Notch1. The adult stem cell marker ABCG2 is well expressed by CSSC. Moreover, CSSC express mesenchymal stem cell (MSC) markers such as CD73, CD90, and CD166. They exhibit clonal growth, self-renewal properties, and a potential for differentiation into multiple distinct tissue types. Upon the incubation in a keratocyte differentiation medium, CSSC showed the potential to differentiate into a keratocyte by producing extracellular matrix components that in adults are uniquely expressed in the corneal stroma such as keratocan, keratan sulfate, and aldehyde dehydrogenase 3A1. Several studies and the anatomical proximity of LESC and CSSC populations in the limbal niche suggest that the principal role of CSSC in vivo is the homeostatic maintenance of the LESC. Human CSSC do not trigger a xenogeneic T-cell-mediated immune response reaction in vivo and suppress T-cell proliferation in vitro. These findings support an immunomodulatory function for CSSC and the potential use of allogeneic CSSC in different cell-based or tissue-engineered therapeutic applications.

Induction of human ESC-derived and adult primary multipotent limbal stem cells into retinal pigment epithelial cells and corneal stromal stem cells

Major advances are currently being made in regenerative medicine for cornea. Stem cell-based therapies represent a novel strategy that may substitute conventional corneal transplantation, albeit there are many challenges ahead given the singularities of each cellular layer of the cornea. This review recapitulates the current data on corneal epithelial stem cells, corneal stromal stem cells and corneal endothelial cell progenitors. Corneal limbal autografts containing epithelial stem cells have been transplanted in humans for more than 20 years with great successful rates, and researchers now focus on ex vivo cultures and other cell lineages to transplant to the ocular surface. A small population of cells in the corneal endothelium was recently reported to have self-renewal capacity, although they do not proliferate in vivo. Two main obstacles have hindered endothelial cell transplantation to date: culture protocols and cell delivery methods to the posterior cornea in vivo. Human corneal stromal stem cells have been identified shortly after the recognition of precursors of endothelial cells. Stromal stem cells may have the potential to provide a direct cell-based therapeutic approach when injected to corneal scars. Furthermore, they exhibit the ability to deposit organized connective tissue in vitro and may be useful in corneal stroma engineering in the future. Recent advances and future perspectives in the field are discussed.

Stem cells can be defined as cells that have the capacity to self-renew and the ability to generate differentiated progeny or multiple cell lineages. True stem cells can turn into any type of cells, while progenitor cells are more or less committed to becoming cell types of a particular tissue. Human corneal epithelial stem cells (CESCs) represent a great example and model of adult stem or progenitor cells. Human CESCs have been identified to locate in the basal epithelial layer of the limbus, and thus also referred as to limbal stem cells. We would like to use the both terms, stem and progenitor cells in this chapter based on previous use in the literature for more than two decades. Although the CESCs have been identified to reside at the limbus and many stem cell markers have been proposed, there is no consensus to date regarding the definitive markers for CESCs, and identification and isolation of these cells are still challenging. Based on evaluation of a variety of proposed markers, we have characterized that the CESCs located in the basal layer of human limbal epithelium are small primitive cells expressing three patterns of molecular markers, which represent a unique phenotype of putative corneal epithelial stem or progenitor cells. Based on adult stem cell criteria and the putative limbal stem cell phenotype, our group has attempted to enrich for human CESCs through novel approaches including cell-sizing, adhering to extracellular matrix collagen type IV, and cell sorting for side population or for expression of ABCG2 or connexin 43 cell surface markers. The 5 clonogenic populations isolated from limbal epithelium and its cultures by different methods show the properties that are characteristics of adult stem/progenitor cells: 1) relatively undifferentiated, 2) high proliferative potential, 3) self-renewal. Expansion and cultivation of corneal epithelial progenitor cells have been achieved using different methods, such as limbal tissue explant culture, and limbal epithelial cell suspension co-culture with mouse 3T3 fibroblast feed layer. To avoid the use of xeno-components, two cell lines of commercial human fibroblasts have been identified that support human corneal epithelial regeneration, and have potential use in replacing mouse 3T3 cells for corneal tissue bioengineering. The concept of CESCs has formed the basis for identifying a class of blinding diseases that display features of corneal epithelial stem cell deficiency or limbal stem cell deficiency (LSCD), where the limbal epithelium is damaged. LSCD is characterized by persistent or recurrent epithelial defects, ulceration, corneal vascularization, chronic inflammation, scarring, and conjunctivalization (conjunctival epithelial ingrowth). Only transplantation of CESCs can restore vision. Due to an increasing shortage of corneal donors, corneal tissue engineering is becoming an important discipline that holds great promise for corneal reconstruction. CESCs and optical substrates are known to be the most important factors for corneal tissue bioengineering in regenerative medicine. Our team has recently explored the utilization of natural donor corneal stroma in corneal tissue engineering. In combination with fresh limbal epithelium containing stem cells, and the donor corneal stroma, a great source of natural optical substrate, we developed a native-like corneal equivalent construct with proliferative potential. This corneal construct provides a new clinical cell therapy for corneal reconstruction.

PurposeMillions worldwide suffer vision impairment or blindness from corneal injury, and there remains an urgent need for a more effective and accessible way to treat corneal defects. We have designed and characterized an in situ-forming semi-interpenetrating polymer network (SIPN) hydrogel using biomaterials widely used in ophthalmology and medicine.MethodsThe SIPN was formed by cross-linking collagen type I with bifunctional polyethylene glycol using N-hydroxysuccinimide ester chemistry in the presence of linear hyaluronic acid (HA). Gelation time and the mechanical, optical, swelling, and degradation properties of the SIPN were assessed. Cytocompatibility with human corneal epithelial cells and corneal stromal stem cells (CSSCs) was determined in vitro, as was the spatial distribution of encapsulated CSSCs within the SIPN. In vivo wound healing was evaluated by multimodal imaging in an anterior lamellar keratectomy injury model in rabbits, followed by immunohistochemical analysis of treated and untreated tissues.ResultsThe collagen-hyaluronate SIPN formed in situ without an external energy source and demonstrated mechanical and optical properties similar to the cornea. It was biocompatible with human corneal cells, enhancing CSSC viability when compared with collagen gel controls and preventing encapsulated CSSC sedimentation. In vivo application of the SIPN significantly reduced stromal defect size compared with controls after 7 days and promoted multilayered epithelial regeneration.ConclusionsThis in situ-forming SIPN hydrogel may be a promising alternative to keratoplasty and represents a step toward expanding treatment options for patients suffering from corneal injury.Translational RelevanceWe detail the synthesis and initial characterization of an SIPN hydrogel as a potential alternative to lamellar keratoplasty and a tunable platform for further development in corneal tissue engineering and therapeutic cell delivery.

Mesenchymal stem cells from corneal stromal stem cells (CSSC) prevent fibrotic scarring and stimulate regeneration of transparent stromal tissue after corneal wounding in mice. These effects rely on the ability of CSSC to block neutrophil infiltration into the damaged cornea. The current study investigated the hypothesis that tissue regeneration by CSSC is mediated by secreted extracellular vesicles (EVs). CSSC produced EVs 130–150 nm in diameter with surface proteins that include CD63, CD81, and CD9. EVs from CSSC reduced visual scarring in murine corneal wounds as effectively as did live cells, but EVs from human embryonic kidney (HEK)293T cells had no regenerative properties. CSSC EV treatment of wounds decreased expression of fibrotic genes Col3a1 and Acta2, blocked neutrophil infiltration, and restored normal tissue morphology. CSSC EVs labeled with carboxyfluorescein succinimidyl ester dye, rapidly fused with corneal epithelial and stromal cells in culture, transferring microRNA (miRNA) to the target cells. Knockdown of mRNA for Alix, a component of the endosomal sorting complex required for transport, using siRNA, resulted in an 85% reduction of miRNA in the secreted EVs. The EVs with reduced miRNA were ineffective at blocking corneal scarring. Furthermore, CSSC with reduced Alix expression also lost their regenerative function, suggesting EVs as an obligate component in the delivery of miRNA. The results of these studies support an essential role for extracellular vesicles in the process by which CSSC cells block scarring and initiate regeneration of transparent corneal tissue after wounding. EVs appear to serve as a delivery vehicle for miRNA, which affects the regenerative action. stem cells translational medicine2019;8:1192–1201

The discovery of corneal epithelial stem cells in the limbal basal epithelium, and their contribution to the homeostasis and renewal of the corneal epithelium, has supposed a tremendous breakthrough in ocular surface reconstruction, with limbal tissue graft transplantation providing for the first time a successful alternative to the transplantation of donor’s cornea in patients with limbal stem cell deficiency. More recent discoveries in the peripheral corneal stroma and endothelium layers of cells with stem/progenitor cells characteristics have opened up the promise of obtaining a supply of adequately differentiated cells for tissue engineering of corneal stromal and endothelial equivalents. Much applications based on the use of these corneal stem/progenitor cells are however still at the experimental stage, and their development will require to solve different challenges, such as optimizing their isolation and purification, improving their in vitro expansion, preserving their stem/progenitor phenotype in culture, and also defining the factors and signaling pathways which are required for their adequate differentiation. In this chapter, we recover past and current findings on the biological and molecular hallmarks of corneal layer-specific stem/progenitor cells, limbal epithelial stem cells, corneal stromal stem cells, and corneal endothelial progenitor cells and make emphasis on which are the different methods used for their isolation and ex vivo expansion.

Human corneal fibroblasts (HCF) and corneal stromal stem cells (CSSC) each secrete and organize a thick stroma-like extracellular matrix in response to different substrata, but neither cell type organizes matrix on tissue-culture polystyrene. This study compared cell differentiation and extracellular matrix secreted by these two cell types when they were cultured on identical substrata, polycarbonate Transwell filters. After 4 weeks in culture, both cell types upregulated expression of genes marking differentiated keratocytes (KERA, CHST6, AQP1, B3GNT7). Absolute expression levels of these genes and secretion of keratan sulfate proteoglycans were significantly greater in CSSC than HCF. Both cultures produced extensive extracellular matrix of aligned collagen fibrils types I and V, exhibiting cornea-like lamellar structure. Unlike HCF, CSSC produced little matrix in the presence of serum. Construct thickness and collagen organization was enhanced by TGF-ß3. Scanning electron microscopic examination of the polycarbonate membrane revealed shallow parallel grooves with spacing of 200–300 nm, similar to the topography of aligned nanofiber substratum which we previously showed to induce matrix organization by CSSC. These results demonstrate that both corneal fibroblasts and stromal stem cells respond to a specific pattern of topographical cues by secreting highly organized extracellular matrix typical of corneal stroma. The data also suggest that the potential for matrix secretion and organization may not be directly related to the expression of molecular markers used to identify differentiated keratocytes.

To assess the suitability of human donor corneas maintained in long-term organ culture for the isolation and expansion of viable and functional corneal stromal stem cells (CSSCs). These cells display properties similar to mesenchymal stem cells and demonstrate the ability to reproduce an organized matrix in vitro. Therefore, CSSCs have great potential for the development of cell-based therapies for corneal blindness or stromal tissue bioengineering. Human donor corneas that had been stored either in organ-culture medium (OC) up to 4 weeks (n = 3) or in Optisol medium (OS) up to 6 days (n = 3) were used for isolation of CSSCs and maintained in culture until passage 4. Cell phenotype of isolated CSSCs was assessed with light microscopy and immunocytochemistry (PAX6, CD73, and CD90). PAX6 protein expression was further confirmed with immunoblot analysis. A comparison of CSSCs isolated from corneas stored under OC and OS conditions revealed no obvious differences in their morphology. Immunocytochemistry revealed CSSCs from both OC and OS corneas maintained positive staining for PAX6 and mesenchymal stem cell markers CD73 and CD90. Immunoblotting confirmed protein expression of PAX6 in cells from both tissue types. Human CSSCs exhibit survival capacity by retaining their phenotype following isolation from long storage, OC corneas. This advantageous property enables the retrieval of CSSCs from OC corneas that are more abundantly available for research than OS-stored corneas. Organ-culture corneas are also often discarded for retrieval of other cell types, such as corneal epithelial and endothelial cells, which require high tissue quality for their preservation.

Corneal stem/progenitor cells are typical adult stem/progenitor cells. The human cornea covers the front of the eyeball, which protects the eye from the outside environment while allowing vision. The location and function demand the cornea to maintain its transparency and to continuously renew its epithelial surface by replacing injured or aged cells through a rapid turnover process in which corneal stem/progenitor cells play an important role. Corneal stem/progenitor cells include mainly corneal epithelial stem cells, corneal endothelial cell progenitors and corneal stromal stem cells. Since the discovery of corneal epithelial stem cells (also known as limbal stem cells) in 1971, an increasing number of markers for corneal stem/progenitor cells have been proposed, but there is no consensus regarding the definitive markers for them. Therefore, the identification, isolation and cultivation of these cells remain challenging without a unified approach. In this review, we systematically introduce the profile of biological characterizations, such as anatomy, characteristics, isolation, cultivation and molecular markers, and clinical applications of the three categories of corneal stem/progenitor cells.

Chondroitin sulfate (CS) and hyaluronic acid (HA) methacrylate (MA) hydrogels are under investigation for biomedical applications. Here, the hydrolytic (in)stability of the MA esters in these polysaccharides and hydrogels is investigated. Hydrogels made with glycidyl methacrylate-derivatized CS (CSGMA) or methacrylic anhydride (CSMA) degraded after 2–25 days in a cross-linking density-dependent manner (pH 7.4, 37 °C). HA methacrylate (HAMA) hydrogels were stable over 50 days under the same conditions. CS(G)MA hydrogel degradation rates increased with pH, due to hydroxide-driven ester hydrolysis. Desulfated chondroitin MA hydrogels also degrade, indicating that sulfate groups are not responsible for CS(G)MA’s hydrolytic sensitivity (pH 7.0–8.0, 37 °C). This sensitivity is likely because CS(G)MA’s N-acetyl-galactosamines do not form hydrogen bonds with adjacent glucuronic acid oxygens, whereas HAMA’s N-acetyl-glucosamines do. This bond absence allows CS(G)MA higher chain flexibility and hydration and could increase ester hydrolysis sensitivity in CS(G)MA networks. This report helps in biodegradable hydrogel development based on endogenous polysaccharides for clinical applications.

The cornea is a major protective shield of the interior of the eye and represents two thirds of its refractive power. It is made up of three tissue layers that have different developmental origins: the outer, epithelial layer develops from the ectoderm overlying the lens vesicle, whereas the stroma and the endothelium have mesenchymal origin. In the adult organism, the outermost corneal epithelium is the most exposed to environmental damage, and its constant renewal is assured by the epithelial stem cells that reside in the limbus, the circular border of the cornea. Cell turnover in the stromal layer is very slow and the endothelial cells probably do not reproduce in the adult organism. However, recent experimental evidence indicates that stem cells may be found in these layers. Damage to any of the corneal layers leads to loss of transparency and low vision. Corneal limbal stem cell deficiency results in severe ocular surface disease and its treatment by transplantating ex vivo expanded limbal epithelial cells is becoming widely accepted today. Stromal and endothelial stem cells are potential tools of tissue engineering and regenerative therapies of corneal ulcers and endothelial cell loss. In the past few years, intensive research has focused on corneal stem cells aiming to improve the outcomes of the current corneal stem cell transplantation techniques. This review summarizes the current state of knowledge on corneal epithelial, stromal and endothelial stem cells. Special emphasis is placed on the molecular markers that may help to identify these cells, and the recently revealed mechanisms that could maintain their "stemness" or drive their differentiation. The techniques for isolating and culturing/expanding these cells are also described.