Good manufacturing practice production of human corneal limbus-derived stromal stem cells and in vitro quality screening for therapeutic inhibition of corneal scarring.

BACKGROUND: Mesenchymal stem cells in the adult corneal stroma (named corneal stromal stem cells, CSSCs) inhibit corneal inflammation and scarring and restore corneal clarity in pre-clinical corneal injury models. This cell therapy could alleviate the heavy reliance on donor materials for corneal transplantation to treat corneal opacities. Herein, we established Good Manufacturing Practice (GMP) protocols for CSSC isolation, propagation, and cryostorage, and developed in vitro quality control (QC) metric for in vivo anti-scarring potency of CSSCs in treating corneal opacities. METHODS: A total of 24 donor corneal rims with informed consent were used-18 were processed for the GMP optimization of CSSC culture and QC assay development, while CSSCs from the remaining 6 were raised under GMP-optimized conditions and used for QC validation. The cell viability, growth, substrate adhesion, stem cell phenotypes, and differentiation into stromal keratocytes were assayed by monitoring the electric impedance changes using xCELLigence real-time cell analyzer, quantitative PCR, and immunofluorescence. CSSC's conditioned media were tested for the anti-inflammatory activity using an osteoclastogenesis assay with mouse macrophage RAW264.7 cells. In vivo scar inhibitory outcomes were verified using a mouse model of anterior stromal injury caused by mechanical ablation using an Algerbrush burring. RESULTS: By comparatively assessing various GMP-compliant reagents with the corresponding non-GMP research-grade chemicals used in the laboratory-based protocols, we finalized GMP protocols covering donor limbal stromal tissue processing, enzymatic digestion, primary CSSC culture, and cryopreservation. In establishing the in vitro QC metric, two parameters-stemness stability of ABCG2 and nestin and anti-inflammatory ability (rate of inflammation)-were factored into a novel formula to calculate a Scarring Index (SI) for each CSSC batch. Correlating with the in vivo scar inhibitory outcomes, the CSSC batches with SI < 10 had a predicted 50% scar reduction potency, whereas cells with SI > 10 were ineffective to inhibit scarring. CONCLUSIONS: We established a full GMP-compliant protocol for donor CSSC cultivation, which is essential toward clinical-grade cell manufacturing. A novel in vitro QC-in vivo potency correlation was developed to predict the anti-scarring efficacy of donor CSSCs in treating corneal opacities. This method is applicable to other cell-based therapies and pharmacological treatments.

Molecular nature of ocular surface barrier function, diseases that affect it, and its relevance for ocular drug delivery.

The structural and functional integrity of the ocular surface, a continuous epithelial structure comprised of the cornea, the conjunctiva, and the ductal surface of the lacrimal as well as meibomian glands, is crucial for proper vision. The ocular surface barrier function (OSBF), sum of the different types of protective mechanisms that exist at the ocular surface, is essential to protect the rest of the eye from vision-threatening physical, chemical, and biological insults. OSBF helps maintain the immune privileged nature of the cornea and the aqueous humor by preventing entry of infectious agents, allergens, and noxious chemicals. Disruption of OSBF exposes the dense nerve endings of the cornea to these stimuli, resulting in discomfort and pain. This review summarizes the status of our knowledge related to the molecular nature of OSBF, describes the effect of different ocular surface disorders on OSBF, and examines the relevance of this knowledge for ocular drug delivery.

Posterior corneoscleral limbus: Architecture, stem cells, and clinical implications.

The limbus is a transition from the cornea to conjunctiva and sclera. In human eyes, this thin strip has a rich variation of tissue structures and composition, typifying a change from scleral irregularity and opacity to corneal regularity and transparency; a variation from richly vascularized conjunctiva and sclera to avascular cornea; the neural passage and drainage of aqueous humor. The limbal stroma is enriched with circular fibres running parallel to the corneal circumference, giving its unique role in absorbing small pressure changes to maintain corneal curvature and refractivity. It contains specific niches housing different types of stem cells for the corneal epithelium, stromal keratocytes, corneal endothelium, and trabecular meshwork. This truly reflects the important roles of the limbus in ocular physiology, and the limbal functionality is crucial for corneal health and the entire visual system. Since the anterior limbus containing epithelial structures and limbal epithelial stem cells has been extensively reviewed, this article is focused on the posterior limbus. We have discussed the structural organization and cellular components of the region beneath the limbal epithelium, the characteristics of stem cell types: namely corneal stromal stem cells, endothelial progenitors and trabecular meshwork stem cells, and recent advances leading to the emergence of potential cell therapy options to replenish their respective mature cell types and to correct defects causing corneal abnormalities. We have reviewed different clinical disorders associated with defects of the posterior limbus and summarized the available preclinical and clinical evidence about the developing topic of cell-based therapy for corneal disorders.

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.

INTRODUCTION: Corneal 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. OBJECTIVES: To 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. METHODS: Small 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. RESULTS: Twenty-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 ß1-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. CONCLUSION: We 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.

A novel glaucoma approach: Stem cell regeneration of the trabecular meshwork.

Glaucoma is the leading cause of global irreversible blindness, necessitating research for new, more efficacious treatment options than currently exist. Trabecular meshwork (TM) cells play an important role in the maintenance and function of the aqueous outflow pathway, and studies have found that there is decreased cellularity of the TM in glaucoma. Regeneration of the TM with stem cells has been proposed as a novel therapeutic option by several reports over the last few decades. Stem cells have the capacity for self-renewal and the potential to differentiate into adult functional cells. Several types of stem cells have been investigated in ocular regenerative medicine: tissue specific stem cells, embryonic stem cells, induced pluripotent stem cells, and adult mesenchymal stem cells. These cells have been used in various glaucoma animal models and ex vivo models and have shown success in IOP homeostasis and TM cellularity restoration. They have also demonstrated stability without serious side effects for a significant period of time. Based on current knowledge of TM pathology in glaucoma and existing literature regarding stem cell regeneration of this tissue, we propose a human clinical study as the next step in understanding this potentially revolutionary treatment paradigm. The ability to protect and replace TM cells in glaucomatous eyes could change the field forever.

Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.

Regenerative therapy for the Cornea.

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.

Cell-Based Therapies for Trabecular Meshwork Regeneration to Treat Glaucoma.

Glaucoma is clinically characterized by elevated intraocular pressure (IOP) that leads to retinal ganglion cell (RGC) and optic nerve damage, and eventually blindness if left untreated. Even in normal pressure glaucoma patients, a reduction of IOP is currently the only effective way to prevent blindness, by either increasing aqueous humor outflow or decreasing aqueous humor production. The trabecular meshwork (TM) and the adjacent Schlemm's canal inner wall play a key role in regulating IOP by providing resistance when aqueous humor drains through the tissue. TM dysfunction seen in glaucoma, through reduced cellularity, abnormal extracellular matrix accumulation, and increased stiffness, contributes to elevated IOP, but current therapies do not target the TM tissue. Stem cell transplantation for regeneration and re-functionalization of damaged TM has shown promise in providing a more direct and effective therapy for glaucoma. In this review, we describe the use of different types of stem cells for TM regeneration in glaucoma models, the mechanisms of regeneration, and the potential for glaucoma treatment using autologous stem cell transplantation.

Diffusion Tensor Imaging of Visual Pathway Abnormalities in Five Glaucoma Animal Models.

Purpose: To characterize the visual pathway integrity of five glaucoma animal models using diffusion tensor imaging (DTI). Methods: Two experimentally induced and three genetically determined models of glaucoma were evaluated. For inducible models, chronic IOP elevation was achieved via intracameral injection of microbeads or laser photocoagulation of the trabecular meshwork in adult rodent eyes. For genetic models, the DBA/2J mouse model of pigmentary glaucoma, the LTBP2 mutant feline model of congenital glaucoma, and the transgenic TBK1 mouse model of normotensive glaucoma were compared with their respective genetically matched healthy controls. DTI parameters, including fractional anisotropy, axial diffusivity, and radial diffusivity, were evaluated along the optic nerve and optic tract. Results: Significantly elevated IOP relative to controls was observed in each animal model except for the transgenic TBK1 mice. Significantly lower fractional anisotropy and higher radial diffusivity were observed along the visual pathways of the microbead- and laser-induced rodent models, the DBA/2J mice, and the LTBP2-mutant cats compared with their respective healthy controls. The DBA/2J mice also exhibited lower axial diffusivity, which was not observed in the other models examined. No apparent DTI change was observed in the transgenic TBK1 mice compared with controls. Conclusions: Chronic IOP elevation was accompanied by decreased fractional anisotropy and increased radial diffusivity along the optic nerve or optic tract, suggestive of disrupted microstructural integrity in both inducible and genetic glaucoma animal models. The effects on axial diffusivity differed between models, indicating that this DTI metric may represent different aspects of pathological changes over time and with severity.

Stem cell transplantation rescued a primary open-angle glaucoma mouse model.

Glaucoma is a leading cause of irreversible blindness. In this study, we investigated if transplanted stem cells are able to rescue a glaucoma mouse model with transgenic myocilin Y437H mutation and explored the possible mechanisms. Human trabecular meshwork stem cells (TMSCs) were intracamerally transplanted which reduced mouse intraocular pressure, increased outflow facility, protected the retinal ganglion cells and preserved their function. TMSC transplantation also significantly increased the TM cellularity, promoted myocilin secretion from TM cells into the aqueous humor to reduce endoplasmic reticulum stress, repaired the TM tissue with extracellular matrix modulation and ultrastructural restoration. Co-culturing TMSCs with myocilin mutant TM cells in vitro promoted TMSCs differentiating into phagocytic functional TM cells. RNA sequencing revealed that TMSCs had upregulated genes related to TM regeneration and neuroprotection. Our results uncovered therapeutic potential of TMSCs for curing glaucoma and elucidated possible mechanisms by which TMSCs achieve the treatment effect.

The anti-scarring effect of corneal stromal stem cell therapy is mediated by transforming growth factor ß3.

BACKGROUND: Corneal stromal stem cells (CSSC) reduce corneal inflammation, prevent fibrotic scarring, and regenerate transparent stromal tissue in injured corneas. These effects rely on factors produced by CSSC to block the fibrotic gene expression. This study investigated the mechanism of the scar-free regeneration effect. METHODS: Primary human CSSC (hCSSC) from donor corneal rims were cultivated to passage 3 and co-cultured with mouse macrophage RAW264.7 cells induced to M1 pro-inflammatory phenotype by treatment with interferon-γ and lipopolysaccharides, or to M2 anti-inflammatory phenotype by interleukin-4, in a Transwell system. The time-course expression of human transforming growth factor ß3 (hTGFß3) and hTGFß1 were examined by immunofluorescence and qPCR. TGFß3 knockdown for > 70% in hCSSC [hCSSC-TGFß3(si)] was achieved by small interfering RNA transfection. Naïve CSSC and hCSSC-TGFß3(si) were transplanted in a fibrin gel to mouse corneas, respectively, after wounding by stromal ablation. Corneal clarity and the expression of mouse inflammatory and fibrosis genes were examined. RESULTS: hTGFß3 was upregulated by hCSSC when co-cultured with RAW cells under M1 condition. Transplantation of hCSSC to wounded mouse corneas showed significant upregulation of hTGFß3 at days 1 and 3 post-injury, along with the reduced expression of mouse inflammatory genes (CD80, C-X-C motif chemokine ligand 5, lipocalin 2, plasminogen activator urokinase receptor, pro-platelet basic protein, and secreted phosphoprotein 1). By day 14, hCSSC treatment significantly reduced the expression of fibrotic and scar tissue genes (fibronectin, hyaluronan synthase 2, Secreted protein acidic and cysteine rich, tenascin C, collagen 3a1 and α-smooth muscle actin), and the injured corneas remained clear. However, hCSSC-TGFß3(si) lost these anti-inflammatory and anti-scarring functions, and the wounded corneas showed intense scarring. CONCLUSION: This study has demonstrated that the corneal regenerative effect of hCSSC is mediated by TGFß3, inducing a scar-free tissue response.

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.

A Biomimetic, Stem Cell-Derived In Vitro Ocular Outflow Model.

Age-related human trabecular meshwork (HTM) cell loss is suggested to affect its ability to regulate aqueous humor outflow in the eye. In addition, disease-related HTM cell loss is suggested to lead to elevated intraocular pressure in glaucoma. Induced pluripotent stem cell (iPSC)-derived trabecular meshwork (TM) cells are promising autologous cell sources that can be used to restore the declining TM cell population and function. Previously, an in vitro HTM model is bioengineered for understanding HTM cell biology and screening of pharmacological or biological agents that affect trabecular outflow facility. In this study, it is demonstrated that human iPSC-derived TM cells cultured on SU-8 scaffolds exhibit HTM-like cell morphology, extracellular matrix deposition, and drug responsiveness to dexamethasone treatment. These findings suggest that iPSC-derived TM cells behave like primary HTM cells and can thus serve as reproducible and scalable cell sources when using this in vitro system for glaucoma drug screening and further understanding of outflow pathway physiology, leading to personalized medicine.

Two-step induction of trabecular meshwork cells from induced pluripotent stem cells for glaucoma.

Glaucoma is a leading cause of irreversible blindness worldwide. Reducing intraocular pressure is currently the only effective treatment. Elevated intraocular pressure is associated with increased resistance of the outflow pathway, mainly the trabecular meshwork (TM). Despite great progress in the field, the development of novel and effective treatment for glaucoma is still challenging. In this study, we reported that human induced pluripotent stem cells (iPSCs) can be cultured as colonies and monolayer cells expressing OCT4, alkaline phosphatase, SSEA4 and SSEA1. After induction to neural crest cells (NCCs) positive to NGFR and HNK1, the iPSCs can differentiate into TM cells. The induced iPSC-TM cells expressed TM cell marker CHI3L1, were responsive to dexamethasone treatment with increased expression of myocilin, ANGPTL7, and formed CLANs, comparable to primary TM cells. To the best of our knowledge, this is the first study that induces iPSCs to TM cells through a middle neural crest stage, which ensures a stable NCC pool and ensures the high output of the same TM cells. This system can be used to develop personalized treatments using patient-derived iPSCs, explore high throughput screening of new drugs focusing on TM response for controlling intraocular pressure, and investigate stem cell-based therapy for TM regeneration.

Adipose-derived stem cells integrate into trabecular meshwork with glaucoma treatment potential.

The trabecular meshwork (TM) is an ocular tissue that maintains intraocular pressure (IOP) within a physiologic range. Glaucoma patients have reduced TM cellularity and, frequently, elevated IOP. To establish a stem cell-based approach to restoring TM function and normalizing IOP, human adipose-derived stem cells (ADSCs) were induced to differentiate to TM cells in vitro. These ADSC-TM cells displayed a TM cell-like genotypic profile, became phagocytic, and responded to dexamethasone stimulation, characteristic of TM cells. After transplantation into naive mouse eyes, ADSCs and ADSC-TM cells integrated into the TM tissue, expressed TM cell markers, and maintained normal IOP, outflow facility, and extracellular matrix. Cell migration and affinity results indicated that the chemokine pair CXCR4/SDF1 may play an important role in ADSC-TM cell homing. Our study demonstrates the possibility of applying autologous or allogeneic ADSCs and ADSC-TM cells as a potential treatment to restore TM structure and function in glaucoma.

A bioengineering approach to Schlemm's canal-like stem cell differentiation for in vitro glaucoma drug screening.

Human Schlemm's canal (HSC) cells are critical for understanding outflow physiology and glaucoma etiology. However, primary donor cells frequently used in research are difficult to isolate. HSC cells exhibit both vascular and lymphatic markers. Human adipose-derived stem cells (ADSCs) represent a potential source of HSC due to their capacity to differentiate into both vascular and lymphatic endothelial cells, via VEGF-A and VEGF-C. Shear stress plays a critical role in maintaining HSC integrity, function, and PROX1 expression. Additionally, the human trabecular meshwork (HTM) microenvironment could provide cues for HSC-like differentiation. We hypothesize that subjecting ADSCs to VEGF-A or VEGF-C, shear stress, and co-culture with HTM cells could provide biological, mechanical, and cellular cues necessary for HSC-like differentiation. To test this hypothesis, effects of VEGF-A, VEGF-C, and shear stress on ADSC differentiation were examined and compared to primary HSC cells in terms of cell morphology, and HSC marker expression using qPCR, immunoblotting, and immunocytochemistry analysis. Furthermore, the effect of co-culture with HTM cells on porous scaffolds on ADSC differentiation was studied. Treatment with VEGF-C under shear stress is effective in differentiating ADSCs into PROX1-expressing HSC-like cells. Co-culture with HTM cells on porous scaffolds leads to HTM/ADSC-derived HSC-like constructs that regulate through-flow and respond as expected to dexamethasone. STATEMENT OF SIGNIFICANCE: We successfully generated human Schlemm's canal (HSC) like cells from adipocyte-derived stem cells induced by biochemical and biomechanical cues as well as bioengineered human trabecular meshwork (HTM) on micropatterned, porous SU8 scaffolds. These stem cell-derived HSC-like cells co-cultured with HTM cells on SU8 scaffolds can regulate through-flow, and in particular, are responsive to steroid treatment as expected. These findings show that ADSC-derived HSC-like cells have the potential to recreate the ocular outflow pathway for in vitro glaucoma drug screening. To the best of our knowledge, it is the very first time to demonstrate derivation of Schlemm's canal-like cells from stem cells. It provides an important alternative source to primary Schlemm's canal cells that are very difficult to be isolated and cultured from human donors.

α5ß1 Integrin Promotes Anchoring and Integration of Transplanted Stem Cells to the Trabecular Meshwork in the Eye for Regeneration.

Stem cell-based therapy to restore the function of abnormal trabecular meshwork (TM) and decrease intraocular pressure (IOP) provides a novel approach to treat open-angle glaucoma. However, molecular mechanism for stem cells homing and anchoring to the TM remains unclear. This study aimed to discover the function of integrins in homing and integration of exogenous TM stem cells (TMSCs) to the TM. Integrin expression in TMSCs and fibroblasts was evaluated by quantitative real-time PCR (qPCR), flow cytometry, immunofluorescent staining, and western blotting. Expression of integrin ligand fibronectin was detected in cultured TM cells and murine TM tissue by immunostaining. Cell affinity to TM cells or fibronectin matrix was examined to compare TMSCs with TMSCs functionally blocked with an α5ß1 integrin antibody. TMSCs and TMSCs with α5ß1 integrin-blocking were intracamerally injected into wild-type mice. Wholemounts and cryosections were analyzed to discover cell distribution and integration at 3 days and 1 month. IOP was measured to detect possible changes. We discovered that human TMSCs expressed a higher level of α5ß1 integrin than fibroblasts, but similar levels of αvß3 and αvß5 integrin. Upregulation of fibronectin was found in both TM cells treated with dexamethasone for 14 days and murine TM tissues damaged by laser photocoagulation. TMSCs were able to attach to the TM cells and fibronectin matrix in vitro. When the surface α5ß1 integrin was blocked, the attached cell numbers were significantly reduced. Both TMSCs and TMSCs incubated with an α5ß1 integrin-blocking antibody could home to the mouse TM after injection. TMSCs blocked with the α5ß1 integrin-blocking antibody were not retained in the TM tissue at 1 month. The injected cells did not affect mouse IOP. In conclusion, highly expressed α5ß1 integrin participates in maintaining TMSCs anchored and integrated to the TM, which would be crucial for stem cell-based therapy for glaucoma.

Stem Cells from Human Trabecular Meshwork Hold the Potential to Develop into Ocular and Non-Ocular Lineages After Long-Term Storage.

Stem cells from the eye hold a great potential for vision restoration and can also be used for regeneration in other tissues. In this study, we characterized the stem cell properties of Trabecular meshwork stem cells (TMSCs) after long-term cryopreservation (∼8 years). TMSCs derived from four donors were examined for their viability and proliferation, as well as stem cell marker expression. Spheroid formation, colony formation, and multipotency were investigated. We observed that TMSCs were fully viable with variable proliferation ability. They expressed the stem cell markers CD90, CD166, CD105, CD73, OCT4, SSEA4, Notch1, KLF4, ABCG2, Nestin, and HNK1 detected by flow cytometry, quantitative polymerase chain reaction, or immunofluorescent staining. They could form spheroids and colonies after thawing. All TMSCs were able to differentiate into osteocytes, neural cells, and trabecular meshwork (TM) cells, but not adipocytes. Differentiated TM cells responded to dexamethasone treatment with increased expression of myocilin and angiopoietin-like 7 (ANGPTL7). In a nutshell, our study demonstrated that TMSCs retain their stem cell properties after long-term cryopreservation and hence can be an effective cell therapy source for various clinical applications.