Human Corneal Stromal Stem Cells Research Articles

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

Human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) therapies are promising alternatives for the treatment of retinal degenerative diseases caused by RPE degeneration. The generation of autologous RPE cells from human adult donors, which has the advantage of avoiding immune rejection and teratoma formation, is an alternative cell resource to gain mechanistic insight into and test potential therapies for RPE degenerative diseases. Here, we found that limbal stem cells (LSCs) from hESCs and adult primary human limbus have the potential to produce RPE cells and corneal stromal stem cells (CSSCs). We showed that hESC-LSC-derived RPE cells (LSC-RPE) expressed RPE markers, had a phagocytic function, and synthesized tropical factors. Furthermore, during differentiation from LSCs to RPE cells, cells became pigmented, accompanied by a decrease in the level of LSC marker KRT15 and an increase in the level of RPE marker MITF. The Wnt signaling pathway plays a role in LSC-RPE fate transition, promotes MITF expression in the nucleus, and encourages RPE fate transition. In addition, we also showed that primary LSCs (pLSCs) from adult human limbus similar to hESC-LSC could generate RPE cells, which was supported by the co-expression of LSC and RPE cell markers (KRT15/OTX2, KRT15/MITF), suggesting the transition from pLSC to RPE cells, and typical polygonal morphology, melanization, RPE cell marker genes expression (TYR, RPE65), tight junction formation by ZO-1 expression, and the most crucial phagocytotic function. On the other hand, both hESC-LSCs and pLSCs also differentiated into CSSCs (LSC-CSSCs) that expressed stem cell markers (PAX6, NESTIN), presented MSC features, including surface marker expression and trilineage differentiation capability, like those in human CSSCs. Furthermore, the capability of pLSC-CSSC to differentiate into cells expressing keratocyte marker genes (ALDH3A1, PTGDS, PDK4) indicated the potential to induce keratocytes. These results suggest that the adult pLSC is an alternative cell resource, and its application provides a novel potential therapeutic avenue for preventing RPE dysfunction-related retinal degenerative diseases and corneal scarring.

Stability and Function of Extracellular Vesicles Derived from Immortalized Human Corneal Stromal Stem Cells: A Proof of Concept Study

With significant advancement and development of extracellular vesicle (EV)-based therapies, there is a growing need to understand how their storage affects their physical and functional characteristics. EVs were isolated from the conditioned medium of a corneal stromal stem cell line (imCSSC) using Total Exosome isolation kit (TEI) and ultracentrifugation (UC) combined protocol. Purified EVs were stored at 4°C, − 80°C, room temperature (RT) after lyophilization with or without trehalose for 4 weeks. EVs stored at − 80°C and RT (lyophilization with trehalose) demonstrated a comparable morphology, while the freeze-dried samples without trehalose showed aggregation and degradation under a transmission electron microscope (TEM). Lyophilized samples without trehalose demonstrated a decreased particle concentration, recovery rate and protein concentration, which was remediated by the addition of trehalose. EVs stored at − 80℃ showed no change in the protein expression of CD9, CD63, and CD81. Regardless of the storage condition, all EV samples investigated reduced inflammation, as well as inhibited expression of fibrotic markers in vitro. Lyophilization of EVs with trehalose was a feasible storage method that retained the physical property and in vitro biological activities of EVs after 4 weeks of storage, while − 80°C offered the best retention of imCSSC-derived EV physical properties. For the first time, this data demonstrated a practical and translatable method for the storage of CSSC-derived EVs for clinical use.Graphical

Generation of Functional Immortalized Human Corneal Stromal Stem Cells.

In addition to their therapeutic potential in regenerative medicine, human corneal stromal stem cells (CSSCs) could serve as a powerful tool for drug discovery and development. Variations from different donors, their isolation method, and their limited life span in culture hinder the utility of primary human CSSCs. To address these limitations, this study aims to establish and characterize immortalized CSSC lines (imCSSC) generated from primary human CSSCs. Primary CSSCs (pCSSC), isolated from human adult corneoscleral tissue, were transduced with ectopic expression of hTERT, c-MYC, or the large T antigen of the Simian virus 40 (SV40T) to generate imCSSC. Cellular morphology, proliferation capacity, and expression of CSSCs specific surface markers were investigated in all cell lines, including TNFAIP6 gene expression levels in vitro, a known biomarker of in vivo anti-inflammatory efficacy. SV40T-overexpressing imCSSC successfully extended the lifespan of pCSSC while retaining a similar morphology, proliferative capacity, multilineage differentiation potential, and anti-inflammatory properties. The current study serves as a proof-of-concept that immortalization of CSSCs could enable a large-scale source of CSSC for use in regenerative medicine.

In Situ-Forming Collagen-Hyaluronate Semi-Interpenetrating Network Hydrogel Enhances Corneal Defect Repair.

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.

Combined Therapy Using Human Corneal Stromal Stem Cells and Quiescent Keratocytes to Prevent Corneal Scarring after Injury.

Corneal blindness due to scarring is conventionally treated by corneal transplantation, but the shortage of donor materials has been a major issue affecting the global success of treatment. Pre-clinical and clinical studies have shown that cell-based therapies using either corneal stromal stem cells (CSSC) or corneal stromal keratocytes (CSK) suppress corneal scarring at lower levels. Further treatments or strategies are required to improve the treatment efficacy. This study examined a combined cell-based treatment using CSSC and CSK in a mouse model of anterior stromal injury. We hypothesize that the immuno-regulatory nature of CSSC is effective to control tissue inflammation and delay the onset of fibrosis, and a subsequent intrastromal CSK treatment deposited collagens and stromal specific proteoglycans to recover a native stromal matrix. Using optimized cell doses, our results showed that the effect of CSSC treatment for suppressing corneal opacities was augmented by an additional intrastromal CSK injection, resulting in better corneal clarity. These in vivo effects were substantiated by a further downregulated expression of stromal fibrosis genes and the restoration of stromal fibrillar organization and regularity. Hence, a combined treatment of CSSC and CSK could achieve a higher clinical efficacy and restore corneal transparency, when compared to a single CSSC treatment.

Human corneal stromal stem cells express anti-fibrotic microRNA-29a and 381-5p – A robust cell selection tool for stem cell therapy of corneal scarring

IntroductionCorneal blindness due to scarring is treated with corneal transplantation. However, a global problem is the donor material shortage. Preclinical and clinical studies have shown that cell-based therapy using corneal stromal stem cells (CSSCs) suppresses corneal scarring, potentially mediated by specific microRNAs transported in extracellular vesicles (EVs). However, not every CSSC batch from donors achieves similar anti-scarring effects. ObjectivesTo examine miRNA profiles in EVs from human CSSCs showing “healing” versus “non-healing” effects on corneal scarring and to design a tool to select CSSCs with strong healing potency for clinical applications. MethodsSmall RNAs from CSSC-EVs were extracted for Nanostring nCounter Human miRNA v3 assay. MicroRNAs expressed > 20 folds in “healing” EVs (P < 0.05) were subject to enriched gene ontology (GO) term analysis. MiRNA groups with predictive regulation on inflammatory and fibrotic signalling were studied by mimic transfection to (1) mouse macrophages (RAW264.7) for M1 phenotype assay; (2) human corneal keratocytes for cytokine-induced fibrosis, and (3) human CSSCs for corneal scar prevention in vivo. The expression of miR-29a was screened in additional CSSC batches and the anti-scarring effect of cells was validated in mouse corneal wounds. ResultsTwenty-one miRNAs were significantly expressed in “healing” CSSC-EVs and 9 miRNA groups were predicted to associate with inflammatory and fibrotic responses, and tissue regeneration (P <10−6). Overexpression of miR-29a and 381-5p significantly prevented M1 phenotype transition in RAW264.7 cells after lipopolysaccharide treatment, suppressed transforming growth factor β1-induced fibrosis marker expression in keratocytes, and reduced scarring after corneal injury. High miR-29a expression in EV fractions distinguished human CSSCs with strong healing potency, which inhibited corneal scarring in vivo. ConclusionWe characterized the anti-inflammatory and fibrotic roles of miR-29a and 381-5p in CSSCs, contributing to scar prevention. MiR-29a expression in EVs distinguished CSSCs with anti-scarring quality, identifying good quality cells for a scarless corneal healing.

Therapeutic Potential of Extracellular Vesicles for the Treatment of Corneal Injuries and Scars.

Infection, trauma, and chemical exposure of the ocular surface can severely damage the cornea, resulting in visually significant stromal scars. Current medical treatments are ineffective in mitigating corneal scarring, and corneal transplantation is the only therapy able to restore vision in these eyes. However, because of a severe shortage of corneal tissues, risks of blinding complications associated with corneal transplants, and a higher rate of graft failure in these eyes, an effective and deliverable alternative therapy for the prevention and treatment of corneal scarring remains a significant unmet medical need globally. In recent years, the therapeutic potential of extracellular vesicles (EVs) secreted by cells to mediate cell-cell communication has been a topic of increasing interest. EVs derived from mesenchymal stem cells, in particular human corneal stromal stem cells, have antifibrotic, anti-inflammatory, and regenerative effects in injured corneas. The exact mechanism of action of these functional EVs are largely unknown. Therapeutic development of EVs is at an early stage and warrants further preclinical studies.

A novel transgenic mouse model for corneal scar visualization

Corneal opacities affect vision for millions of individuals worldwide. Fibrotic scar tissues accumulate in reaction to inflammatory responses and remain permanently in corneal stroma, and conventionally correctable only by donor corneal transplantation. Numerous studies have explored innovative approaches to reverse corneal scarring through non-surgical means; however, existing mouse models limit these studies, due to the lack of visibility of scar tissue in mouse corneas with steep curvature. Here, we reported that corneal scarring was modelled using a transgenic mouse line, Tg(Col3a1-EGFP)DJ124Gsat, in which enhanced green fluorescence protein (EGFP) reporter expression was driven by the promoter of collagen 3a1 (COL3a1), a stromal fibrosis gene. Similar to wildtype, Col3a1-EGFP transgenic corneas developed opacities after wounding by alkali burn and mechanical ablation, respectively, as examined under stereomicroscopy and Spectral Domain optical coherent tomography. The time course induction of EGFP was aligned with Col3a1 upregulation and matched with the elevated expression of other fibrosis genes (α-smooth muscle actin, fibronectin and tenascin C). Measured by flow cytometry and enzyme-linked immunosorbent assay, increased number of EGFP expressing cells and fluorescent intensities were correlated to corneal thickening and scar volume. After treatment with human corneal stromal stem cells or their exosomes, EGFP expression was downregulated together with the reduction of scar volume and fibrosis gene expression. These results have demonstrated that the transgenic mouse line, Tg(Col3a1-EGFP)DJ124Gsat, can be a valuable tool for the detection of corneal fibrosis and scarring in vivo, and will be useful in monitoring the changes of corneal fibrosis over time.

Corneal stromal stem cells restore transparency after N2 injury in mice.

Corneal scarring associated with various corneal conditions is a leading cause of blindness worldwide. The present study aimed to test the hypothesis that corneal stromal stem cells have a therapeutic effect and are able to restore the extracellular matrix organization and corneal transparency in vivo. We first developed a mouse model of corneal stromal scar induced by liquid nitrogen (N2) application. We then reversed stromal scarring by injecting mouse or human corneal stromal stem cells in injured cornea. To characterize the mouse model developed in this study and the therapeutic effect of corneal stromal stem cells, we used a combination of in vivo (slit lamp, optical coherence tomography, in vivo confocal microscopy, optical coherence tomography shear wave elastography, and optokinetic tracking response) and ex vivo (full field optical coherence microscopy, flow cytometry, transmission electron microscopy, and histology) techniques. The mouse model obtained features early inflammation, keratocyte apoptosis, keratocyte transformation into myofibroblasts, collagen type III synthesis, impaired stromal ultrastructure, corneal stromal haze formation, increased corneal rigidity, and impaired visual acuity. Injection of stromal stem cells in N2‐injured cornea resulted in improved corneal transparency associated with corneal stromal stem cell migration and growth in the recipient stroma, absence of inflammatory response, recipient corneal epithelial cell growth, decreased collagen type III stromal content, restored stromal ultrastructure, decreased stromal haze, decreased corneal rigidity, and improved vision. Our study demonstrates the ability of corneal stromal stem cells to promote regeneration of transparent stromal tissue after corneal scarring induced by liquid nitrogen.

Silk-Based Therapeutics Targeting Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) infections may lead to severe damage of the cornea, mucosa, and skin. The highly aggressive nature of P. aeruginosa and the rise in multi-drug resistance, particularly in nosocomial settings, lead to an increased risk for permanent tissue damage and potentially death. Thus, a growing need exists to develop alternative treatments to reduce both the occurrence of bacterial infection and biofilm development, as well as pathological progression post-infection. Silk derived from Bombyx mori silkworms serves as a unique biomaterial that is biocompatible with low immunogenicity and high versatility, and thereby ideal for stabilizing therapeutics. In this study, we assessed the cytotoxicity of P. aeruginosa on human corneal stromal stem cells and two mucosal cell lines (Caco-2 and HT29-MTX). To determine whether antibiotic-immobilized scaffolds can serve as alternative therapeutics to free, diffuse forms, we developed novel gentamicin-conjugated silk films as functional scaffolds and compared antimicrobial effects and free gentamicin. The advantages of generating a surface coating with a covalently-bound antibiotic may reduce potential side-effects associated with free gentamicin, as well as limit the diffusion of the drug. Our results suggest that gentamicin conjugated to native silk and carboxyl-enriched silk inhibits P. aeruginosa growth. Development of stabilized antibiotic treatments with surface toxicity selective against bacteria may serve as an alternative approach to treat active infections, as well as potential prophylactic use as coatings in high-risk cases, such as post-surgical complications or prolonged hospitalization.

A thermoresponsive three-dimensional fibrous cell culture platform for enzyme-free expansion of mammalian cells

A three-dimensional thermoresponsive fibrous scaffold system for the subsequent extended culture and enzyme-free passaging of a range of mammalian cell types is presented. Poly(PEGMA188) was incorporated with poly(ethylene terephthalate) (PET) via blend-electrospinning to render the fibre thermoresponsive. Using primary human corneal stromal stem cells as an therapeutically relevant exemplar, cell adhesion, viability, proliferation and phenotype on this fibrous culture system over numerous thermal enzyme-free passages is described. We also illustrate the versatility of this system with respect to fabricating thermoresponsive fibres from biodegradable polymers and for the culture of diverse mammalian cell types including mesenchymal stem cells, colon adenocarcinoma cells and NIH-3T3 fibroblasts. This thermoresponsive scaffold system combines the advantages of providing a physiologically relevant environment to maintain a desirable cell phenotype, allowing routine enzyme-free passaging and expansion of cultured cells, whilst offering mechanical support for cell growth. The system described in this study presents a versatile platform for biomedical applications and more specifically for the expansion of mammalian cells destined for the clinic. Statement of SignificanceThe lack of three-dimensional (3D) cell culture environments significantly impacts mammalian cell morphology, proliferation and phenotype in vitro. A versatile, 3D fibrous scaffold system for the extended culture and passaging of a range of clinically-relevant cell types is presented herein. This methodology can be used to fabricate thermoresponsive fibres from polymer blends of any polymer amenable to electrospinning and with a thermoresponsive component. A variety of mammalian cells cultured on the thermoresponsive system were detached from the surface solely by lowering the temperature whilst retaining high viability, a desirable cell phenotype, and supported long‐term cell proliferation over numerous thermal enzyme-free passages. This is a significant advance for in vitro expansion of diverse cell types destined for the clinic.

Controlling human corneal stromal stem cell contraction to mediate rapid cell and matrix organization of real architecture for 3-dimensional tissue equivalents.

For the first time, we have demonstrated that human CSSC exhibit the phenomenon of cellular self-alignment in tethered collagen gels. We found this mediated rapid co-alignment of collagen fibrils and thus subsequently exploited this property in vitro to improve the architecture of engineered RAFT tissue equivalents of the corneal stroma. Existing techniques are extremely lengthy and carry significant risk and cost for GMP manufacture. This rapid and tunable technique takes just 8 h of culture and is therefore ideal for clinical manufacture, creating biomimetic tissue equivalents with both cellular and ECM organization. Thus, cellular self-alignment can be a useful bioengineering tool for the development of organized tissue equivalents in a variety of applications.

Multi-layered silk film coculture system for human corneal epithelial and stromal stem cells.

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.

Scaffold-free tissue engineering of functional corneal stromal tissue.

Blinding corneal scarring is predominately treated with allogeneic graft tissue; however, there is a worldwide shortage of donor tissue leaving millions in need of therapy. Human corneal stromal stem cells (CSSC) have been shown produce corneal tissue when cultured on nanofibre scaffolding, but this tissue cannot be readily separated from the scaffold. In this study, scaffold-free tissue engineering methods were used to generate biomimetic corneal stromal tissue constructs that can be transplanted in vivo without introducing the additional variables associated with exogenous scaffolding. CSSC were cultured on substrates with aligned microgrooves, which directed parallel cell alignment and matrix organization, similar to the organization of native corneal stromal lamella. CSSC produced sufficient matrix to allow manual separation of a tissue sheet from the grooved substrate. These constructs were cellular and collagenous tissue sheets, approximately 4μm thick and contained extracellular matrix molecules typical of corneal tissue including collagen types I and V and keratocan. Similar to the native corneal stroma, the engineered corneal tissues contained long parallel collagen fibrils with uniform diameter. After being transplanted into mouse corneal stromal pockets, the engineered corneal stromal tissues became transparent, and the human CSSCs continued to express human corneal stromal matrix molecules. Both in vitro and in vivo, these scaffold-free engineered constructs emulated stromal lamellae of native corneal stromal tissues. Scaffold-free engineered corneal stromal constructs represent a novel, potentially autologous, cell-generated, biomaterial with the potential for treating corneal blindness. Copyright © 2016 John Wiley & Sons, Ltd.

3D Functional Corneal Stromal Tissue Equivalent Based on Corneal Stromal Stem Cells and Multi-Layered Silk Film Architecture.

The worldwide need for human cornea equivalents continues to grow. Few clinical options are limited to allogenic and synthetic material replacements. We hypothesized that tissue engineered human cornea systems based on mechanically robust, patterned, porous, thin, optically clear silk protein films, in combination with human corneal stromal stem cells (hCSSCs), would generate 3D functional corneal stroma tissue equivalents, in comparison to previously developed 2D approaches. Silk film contact guidance was used to control the alignment and distribution of hCSSCs on RGD-treated single porous silk films, which were then stacked in an orthogonally, multi-layered architecture and cultured for 9 weeks. These systems were compared similar systems generated with human corneal fibroblasts (hCFs). Both cell types were viable and preferentially aligned along the biomaterial patterns for up to 9 weeks in culture. H&E histological sections showed that the systems seeded with the hCSSCs displayed ECM production throughout the entire thickness of the constructs. In addition, the ECM proteins tested positive for keratocyte-specific tissue markers, including keratan sulfate, lumican, and keratocan. The quantification of hCSSC gene expression of keratocyte-tissue markers, including keratocan, lumican, human aldehyde dehydrogenase 3A1 (ALDH3A1), prostaglandin D2 synthase (PTDGS), and pyruvate dehydrogenase kinase, isozyme 4 (PDK4), within the 3D tissue systems demonstrated upregulation when compared to 2D single silk films and to the systems generated with the hCFs. Furthermore, the production of ECM from the hCSSC seeded systems and subsequent remodeling of the initial matrix significantly improved cohesiveness and mechanical performance of the constructs, while maintaining transparency after 9 weeks.

Early wound healing of laser in situ keratomileusis–like flaps after treatment with human corneal stromal stem cells

PurposeTo use a well-established organ culture model to investigate the effects of corneal stromal stem cells on the optical and biomechanical properties of corneal wounds after laser in situ keratomileusis (LASIK)–like flap creation.SettingSchool of Optometry and Vision Sciences, Cardiff University, Cardiff, Wales, United Kingdom.DesignExperimental study.MethodsThe LASIK-like flaps were produced in sheep corneas. The flap beds were treated with corneal stromal stem cells and were then replaced and allowed to heal for different periods of up to 3 weeks in organ culture. The optical transmission of the cornea, the force required to detach the flap, and the presence of myofibroblasts near the flap bed were measured.ResultsCorneal stromal stem cell–treated flap beds were statistically significantly more transparent after 3 weeks in culture than the untreated controls. At 3 weeks, the mean force necessary to detach the flap was more than twice the force required for the respective control samples. Concurrently, there were 44% activated cells immediately below the flap margin of the controls compared with 29% in the same region of the corneal stromal stem cell–treated flaps.ConclusionsIn this system, the presence of corneal stromal stem cells at the wound margin significantly increased the adherence of LASIK-like flaps while maintaining corneal transparency. It is postulated that this is achieved by the deposition of extracellular connective tissue similar to that found in the normal cornea and by the paucity of activated keratocytes (myofibroblasts), which are known to scatter a significant amount of the incident light.Financial DisclosureNo author has a financial or proprietary interest in any material or method mentioned.

Human corneal stromal stem cells support limbal epithelial cells cultured on RAFT tissue equivalents.

Human limbal epithelial cells (HLE) and corneal stromal stem cells (CSSC) reside in close proximity in vivo in the corneal limbal stem cell niche. However, HLE are typically cultured in vitro without supporting niche cells. Here, we re-create the cell-cell juxtaposition of the native environment in vitro, to provide a tool for investigation of epithelial-stromal cell interactions and to optimize HLE culture conditions for potential therapeutic application. RAFT (Real Architecture For 3D Tissue) tissue equivalents (TEs) were used as a 3-dimensional substrate for co-culturing HLE and CSSC. Our results demonstrate that a monolayer of HLE that maintained expression of p63α, ABCB5, CK8 and CK15 (HLE markers), formed on the surface of RAFT TEs within 13 days of culture. CSSC remained in close proximity to HLE and maintained expression of mesenchymal stem cell markers. This simple technique has a short preparation time of only 15 days with the onset of HLE layering and differentiation observed. Furthermore, co-cultivation of HLE with another niche cell type (CSSC) directly on RAFT TEs, eliminates the requirement for animal-derived feeder cells. RAFT TEs may be useful for future therapeutic delivery of multiple cell types to restore the limbal niche following ocular surface injury or disease.

Coculture of dorsal root ganglion neurons and differentiated human corneal stromal stem cells on silk-based scaffolds.

Corneal tissue displays the highest peripheral nerve density in the human body. Engineering of biomaterials to promote interactions between neurons and corneal tissue could provide tissue models for nerve/cornea development, platforms for drug screening, as well as innovative opportunities to regenerate cornea tissue. The focus of this study was to develop a coculture system for differentiated human corneal stromal stem cells (dhCSSCs) and dorsal root ganglion neurons (DRG) to mimic the human cornea tissue interactions. Axon extension, connectivity, and neuron cell viability were studied. DRG neurons developed longer axons when cocultured with dhCSSCs in comparison to neuron cultures alone. To assess the mechanism involved in the coculture response, nerve growth factors (NGF) secreted by dhCSSCs including NGF, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and neurotrophin-3 were characterized with greater focus on BDNF secretion. DhCSSCs also secreted collagen type I, an extracellular matrix molecule favorable for neuronal outgrowth. This coculture system provides a slowly degrading silk matrix to study neuronal responses in concert with hCSSCs related to innervation of corneal tissue with utility toward human corneal nerve regeneration and associated diseases.

Corneal stem cells and tissue engineering: Current advances and future perspectives.

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.

Human corneal stromal stem cells exhibit survival capacity following isolation from stored organ-culture corneas.

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.