Trabecular meshwork cell (TMC) and trabecular meshwork stem cell (TMSC) secretome effects on schlemm's canal endothelial cells (SCEC)

Abstract

Methods of increasing wound healing, proliferation, permeability, ciliogenesis, and / or intercellular pore formation, and / or decreasing cellular stiffness and / or storage modulus of Schlemm's canal endothelial cells (SCEC) in a subject in need thereof using trabecular meshwork cell (TMC) secretome and / or trabecular meshwork stem cell (TMSC) secretome.

Description

TRABECULAR MESHWORK CELL (TMC) AND TRABECULAR MESHWORK STEM CELL (TMSC) SECRETOME EFFECTS ON SCHLEMM'S CANAL ENDOTHELIAL CELLS (SCEC)CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 559,917, filed March 1, 2024, which is incorporated by reference herein in its entirety.GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under Grant No. EY025643 awarded by the National Institutes of Health. The Government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] The sequence listing submitted on March 3, 2025, as an .XML file entitled“ 11001-218WO1-ST26. xml” created on March 3, 2025, and having a file size of 4,721 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52 (e) (5).BACKGROUND

[0004] Glaucoma is a leading cause of irreversible blindness worldwide, characterized by degeneration of retinal ganglion cells (RGCs) and progressive optic neuropathy (Y. C. Tham et al., Global prevalence of glaucoma and projections of glaucoma burden through 2040: a systematic review and meta-analysis. Ophthalmology 121 , 2081 -2090 (2014)). Elevated intraocular pressure (IOP) is the primary risk factor of glaucoma, resulting from increased resistance of aqueous humor (AH) outflow, which is mainly through the conventional outflow pathway. The main resistance is at the trabecular meshwork (TM) and the adjacent Schlemm’s canal (D. L. Epstein, Framing glaucoma questions: what are the opportunities for glaucoma treatment? A personal perspective. Invest Ophthalmol Vis Sci 53, 2462-2463 (2012); T. Carreon, et al., Aqueous outflow - A continuum from trabecular meshwork to episcleral veins. Progress in Retinal and Eye Research 57, 108-133 (2017)). In glaucoma, the TM cellularity is reduced with extracellular matrix changes and both the TM and Schlemm’s canal endothelial cells (SCEC) are stiffer than normal and the resistance increases. SCEC form a unique lymphatic-like endothelial layer that regulates AH outflow through intercellular pores andparacellular pathways and maintains IOP homeostasis together with the TM tissue. Dysfunction of TM cells and SCEC significantly contributes to increased outflow resistance, elevated IOP, and subsequent glaucomatous optic neuropathy (S. Mallick, et al., Cell-Based Therapies for Trabecular Meshwork Regeneration to Treat Glaucoma. Biomolecules 11 (2021); S. J. Coulon et al., A novel glaucoma approach: Stem cell regeneration of the trabecular meshwork. Prog Retin Eye Res 90, 101063 (2022)).

[0005] Thus, there is a need for improved methods of treating glaucoma and SCEC dysfunction. These needs and others are at least partially satisfied by the present disclosure.SUMMARY

[0006] In one aspect, provided is a method of increasing wound healing, proliferation, and / or permeability of Schlemm’s canal endothelial cells (SCEC) in a subject in need thereof, the method including administering a therapeutically effective amount of trabecular meshwork cell (TMC) secretome and / or trabecular meshwork stem cell (TMSC) secretome to a Schlemm’s canal of the subject.

[0007] In another aspect, provided is a method of increasing ciliogenesis and / or intercellular pore formation in Schlemm’s canal endothelial cells (SCEC) in a subject in need thereof, the method including administering a therapeutically effective amount of trabecular meshwork cell (TMC) secretome and / or trabecular meshwork stem cell (TMSC) secretome to a Schlemm’s canal of the subject.

[0008] In yet another aspect, provided is a method of decreasing cellular stiffness and / or storage modulus of Schlemm’s canal endothelial cells (SCEC) in a subject in need thereof, the method including administering a therapeutically effective amount of trabecular meshwork cell (TMC) secretome and / or trabecular meshwork stem cell (TMSC) secretome to a Schlemm’s canal of the subject.

[0009] Other systems, methods, features and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and / or advantages be included within this description and be protected by the accompanying claims.BRIEF DESCRIPTION OF DRAWINGS

[0010] FIGURES 1A-1E depict secretome preparation and toxicity test. FIG. 1A shows a schematic diagram illustrating the workflow for TMSC-Scr and TM-Scr preparation under serum starving conditions. FIG. IB shows immunofluorescent staining to detect cell qualityafter serum starving for secretome. TMSC were stained with Nestin and 0CT4, while TM cells stained with CHI3L1 before and after starvation. Scale bars: 50 |im. FIGS. 1C-1D show MTT assay results showing the viability of GSCEC treated with different concentrations of TMSC- Scr (FIG. 1C) and TM-Scr (FIG. ID) for 48 hours. Data are presented as mean ± SD (n = 5 / group); One-way ANOVA with Tukey’s test. FIG. IE shows representative images of GSCEC stained with calcein-AM (live cells, green) and PI (dead cells, red) after 48-hour treatment with 25% and 50% of TMSC-Scr and TM-Scr. Nuclei were counterstained with DAPI (blue). Scale bars: 50 pm.

[0011] FIGURES 2A-2E depict the effects of TMSC-Scr and TM-Scr on GSCEC wound healing, proliferation, and permeability. FIG. 2A shows representative wound healing images of GSCEC at 0, 24, and 48 hrs with basal medium containing 25% or 50% TMSC-Scr or TM- Scr or basal medium only (GSCEC). FIG. 2B shows quantification of wound closure at 48 hrs with different treatments. Data are presented as mean ± SD. FIG. 2C shows relative mRNA expression of Ki-67 in wounded GSCEC at 48 hrs with different treatments by qPCR. N=3. FIG. 2D shows that TEER measures the permeability of GSCEC and GSCEC treated with 25% TMSC-Scr or 25% TM-Scr over 16 days. Data represent mean ± SD; n=6. Black stars compare TM-Scr and untreated GSCEC, while red stars compare TMSC-Scr and untreated GSCEC. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. FIG. 2E shows FITC-dextran permeability assays at days 2 and 6 post-treatment. Data are presented as mean ± SD; n=6.

[0012] FIGURES 3A-3D depict TMSC-Scr and TM-Scr upregulating GSCEC biomarkers PROXI and FBLN2. FIG. 3A shows representative Western blot (WB) bands showing expression of PROXI and FBLN2 in GSCEC treated with different conditions. Beta-actin was used as a loading control. FIG. 3B shows quantitative analysis of PROXI and FBLN2 expression by WB in GSCEC. FIG. 3C shows immunofluorescent (IF) staining images of PROXI and FBLN2 with and without 25% TMSC-Scr or TM-Scr treatment. DAPI stains nuclei. Scale bars: 50 pm. FIG. 3D shows quantitative analysis of PROXI and FBLN2 expression by IF (MFI: mean fluorescent intensity). Data are presented as mean ± SD; n=5.

[0013] FIGURES 4A-4C depict TMSC-Scr and TM-Scr differentially changing transmembrane proteins N-Cad and OB-Cad. FIG. 4A shows representative Western blot (WB) bands showing expression of N-Cad and OB-Cad in GSCEC treated with different conditions. Beta-actin was used as a loading control. FIG. 4B shows quantitative analysis of N-Cad and OB-Cad expression by WB in GSCEC. FIG. 4C shows immunofluorescent (IF) staining images of N-Cad and OB-Cad with and without 25% TMSC-Scr or TM-Scr treatment. DAPI stains nuclei. Scale bars: 50 pm.

[0014] FIGURES 5A-5F depict TMSC-Scr enhancing cell-cell junction protein expression in GSCEC. FIG. 5A shows representative Western blot bands showing the expression of cell junction markers ZO-1, Claudin-11, and Connexin 43 in GSCEC with different treatments. FIGS. 5B-5D show quantitative analysis of WB bands of ZO-1 (FIG. SB), CLDN11 (FIG. SC), Connexin 43 (FIG. 5D) protein levels with b-actin as loading control. FIG. 5E shows IF staining images of GSCEC treated with 25% TMSC-Scr or 25% TM-Scr or untreated control. Scale bars: 50 pm. FIG. 5F shows quantitative IF analysis of Connexin 43 showing mean fluorescent intensity (MFI). Data are presented as mean ± SD. N=5.

[0015] FIGURES 6A-6E depict TMSC-Scr and TM-Scr differentially regulating GSCEC ultrastructure and cilia. FIGS. 6A-6B show scanning electron microscopy (SEM) images of GSCEC treated with TMSC-Scr and TM-Scr showing Intercellular pores and cilia. FIG. 6B is magnified image of the framed area in FIG. 6A. Scale bars: 1 pm. FIG. 6C shows IF staining of acetylated alpha-tubulin (AC-a-tubulin) in GSCEC to visualize primary cilia. DAPI stains nuclei. Scale bars: 20 pm. FIG. 6D shows quantitative analysis of cilium prevalence (%). FIG. 6E shows quantitative analysis of cilium length (pm). Data are presented as mean ± SD.

[0016] FIGURES 7A-7F depict TMSC-Scr and TM-Scr influence GSCEC mechanical properties by AFM. FIGS. 7A-7B show density distribution and statistical analysis of stiffness in TMSC-Scr-treated, TM-Scr-treated, and untreated GSCEC measured by atomic force microscopy (AFM). FIGS. 7C-7D show storage modulus comparison among GSCEC treated under different conditions, representing the elastic response of the cells. FIGS. 7E-7F show loss modulus analysis, reflecting the energy of cells to dissipate stress. Statistical significance was determined using two-way ANOVA followed by Tukey post-test.

[0017] FIGURE 8 depicts a diagram of an eye and an illustration of how the AH flows through the trabecular meshwork to Schlemm’s canal (and further flows to the collector channels and to the venous system).

[0018] FIGURE 9 depicts a method flow diagram.

[0019] FIGURE 10 depicts SCE cell isolation and identification.

[0020] FIGURE 11 shows that TMSC affects SCEC barrier function / permeability.

[0021] FIGURE 12 shows that VE-Cad may play a very important role in regulating the permeability of SCEC.

[0022] FIGURE 13 depicts SCEC characterization.

[0023] FIGURE 14 further depicts SCEC characterization.

[0024] FIGURES 15A-15B further shows that TMSC affects SCEC permeability.

[0025] FIGURES 16A-16D depict TMSC vs. SCEC DEGs (differentially expressed genes).

[0026] FIGURE 17 depicts another method flow diagram for comparing gene expression between TMC and SCEC using microarray assay and cross-referenced the DEGs with scRNA- Seq data and confirmed by qRT-PCR.

[0027] FIGURES 18A-18P depicts validation of TMC and SCEC identity.

[0028] FIGURES 19A-19F depict that the microarray revealed 341 DEGs, with TMC enriched in metabolic and signaling pathways, and SCEC enriched in adhesion, immune, and morphogenesis-related processes.

[0029] FIGURES 20A-20H depict that cross-referencing with scRNA-Seq data refined the list of candidate biomarkers and qPCR confirmed the significant differences in gene expression between TM and SCE cells. CTTNBP2 and MGARP were highly expressed in TM cells. JAM2, PODXL and IFI27 were significantly upregulated in SCE cells.DETAILED DESCRIPTION

[0030] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.DEFINITIONS

[0031] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0032] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of”and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.

[0033] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” or “a composition” includes, but is not limited to, two or more such compounds, or compositions, and the like.

[0034] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0035] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y ’ as well as the range greater than x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0036] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0037] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0038] As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material. For example, an “effective amount” of a monomer refers to an amount that is sufficient to achieve the desired improvement in the property modulated by the formulation component, e.g. desired antioxidant release rate or viscoelasticity. The specific level in terms of wt% in a composition required as an effective amount will depend upon a variety of factors including the amount and type of monomer, amount and type of polymer, e.g., acrylamide, amount of antioxidant, and desired release kinetics.

[0039] As used herein, the term “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors within the knowledge and expertise of the health practitioner and which maybe well known in the medical arts. In the case of treating a particular disease or condition, in some instances, the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to halt the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.

[0040] For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0041] A response to a therapeutically effective dose of a disclosed drug delivery composition can be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following administration of the treatment or pharmacological agent. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response. The amount of a treatment may be varied for example by increasing or decreasing the amount of a disclosed compound and / or pharmaceutical composition, by changing the disclosed compound and / or pharmaceutical composition administered, by changing the route of administration, by changing the dosage timing and so on. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

[0042] As used herein, the term “prophylactically effective amount” refers to an amount effective for preventing onset or initiation of a disease or condition.

[0043] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially byadvance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0044] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0045] As used interchangeably herein, “subject,” “individual,” or “patient” can refer to a vertebrate organism, such as a mammal (e.g. human). "Subject" can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.

[0046] As used herein, the terms "treating" and "treatment" can refer generally to obtaining a desired pharmacological and / or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term "treatment" as used herein can include any treatment of a disease disorder in a subject, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and / or its symptoms or conditions. The term "treatment" as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with the disorder and / or those in which the disorder is to be prevented. As used herein, the term "treating", can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and / or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0047] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and / or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.

[0048] As used herein, “therapeutic” can refer to treating, healing, and / or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect.

[0049] The term “secretome,” as used herein, refers to an array of secretory cytokines, growth factors, small RNAs, non-coding RNAs, ECM mediators, small molecules, proteins, or any other compositions or moieties secreted by a cell.METHODS

[0050] In one aspect, provided is a method of increasing wound healing, proliferation, and / or permeability of Schlemm’s canal endothelial cells (SCEC) in a subject in need thereof, the method including administering a therapeutically effective amount of trabecular meshwork cell (TMC) secretome and / or trabecular meshwork stem cell (TMSC) secretome to a Schlemm’s canal of the subject.

[0051] In some aspects, SCEC wound closure can reach about 80% or more (e.g., about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 100%) following administration of the TMC secretome and / or TMSC secretome.

[0052] In some aspects, the method can increase Ki-67 expression in the SCEC by about1.5-fold or more (e.g., about 1.6-fold or more, about 1.7-fold or more, about 1.8-fold or more, about 1.9-fold or more, about 2-fold or more, about 2.1 -fold or more, about 2.2-fold or more, about 2.3-fold or more, about 2.4-fold or more, about 2.5-fold or more, about 2.6-fold or more, about 2.7-fold or more, about 2.8-fold or more, about 2.9-fold or more, about 3-fold or more) following administration of the TMC secretome and / or TMSC secretome. In some aspects, the method can increase Ki-67 expression in the SCEC by about 3-fold or less (e.g., about 2.9-fold or less, about 2.8-fold or less, about 2.7-fold or less, about 2.6-fold or less, about 2.5-fold or less, about 2.4-fold or less, about 2.3-fold or less, about 2.2-fold or less, about 2.1-fold or less, about 2-fold or less, about 1.9-fold or less, about 1.8-fold or less, about 1.7-fold or less, about1.6-fold or less, about 1.5-fold or less). The method can increase Kr-67 expression in the SCEC following administration of the TMC secretome and / or TMSC secretome by an amount ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the method can increase Ki-67 expression in the SCEC by from about 1.5-fold to about 3-fold (e.g., from about 1.6-fold to about 2.9-fold, from about1.7-fold to about 2.8-fold, from about 1.8-fold to about 2.7-fold, from about 1.9-fold to about 2.6-fold, from about 2-fold to about 2.5-fold, from about 2.1-fold to about 2.4-fold, from about2.2-fold to about 2.3-fold, from about 1.5-fold to about 2.3-fold, from about 1.6-fold to about2.2-fold, from about 1.7-fold to about 2.1 -fold, from about 1.8-fold to about 2-fold, from about2.2-fold to about 3-fold, from about 2.3-fold to about 2.9-fold, from about 2.4-fold to about2.8-fold, from about 2.5-fold to about 2.7-fold) following administration of the TMC secretome and / or TMSC secretome.

[0053] In some aspects, the method can increase permeability of the SCEC by about 20% or more (e.g., about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 100% or more) following administration of the TMC secretome and / or TMSC secretome. In some aspects, the method can increase permeability of the SCEC by about 100% or less (e.g., about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less) following administration of the TMC secretome and / or TMSC secretome. The method can increase permeability of the SCEC following administration of the TMC secretome and / or TMSC secretome by an amount ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the method can increase permeability of the SCEC by from about 20% to about 100% (e.g., from about 25% to about 95%, from about 30% to about 90%, from about 35% to about 85%, from about 40% to about80%, from about 45% to about 75%, from about 50% to about 70%, from about 55% to about65%, from about 20% to about 60%, from about 25% to about 55%, from about 30% to about50%, from about 35% to about 45%, from about 60% to about 100%, from about 65% to about95%, from about 70% to about 90%, from about 75% to about 85%) following administration of the TMC secretome and / or TMSC secretome.

[0054] In another aspect, provided is a method of increasing ciliogenesis and / or intercellular pore formation in Schlemm’s canal endothelial cells (SCEC) in a subject in need thereof, the method including administering a therapeutically effective amount of trabecular meshwork cell (TMC) secretome and / or trabecular meshwork stem cell (TMSC) secretome to a Schlemm’s canal of the subject.

[0055] In some aspects, the method can increase the length and / or prevalence of cilia on the SCEC following administration of the TMC secretome and / or TMSC secretome.

[0056] In some aspects, the method can increase the length of cilia on the SCEC by about 5% or more (e.g., about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more). In some aspects, the method can increase the length of cilia on the SCEC by about 75% or less (e.g., about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less). The method can increase the length of cilia on the SCEC by an amount ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the method can increase the length of cilia on the SCEC by from about 5% to about 75% (e.g., from about 10% to about 70%, from about 15% to about 65%, from about 20% to about 60%, from about 25% to about 55%, from about 30% to about 50%, from about 35% to about 45%, from about 5% to about 40%, from about 10% to about 35%, from about 15% to about 30%, from about 20% to about 25%, from about 40% to about 75%, from about 45% to about 70%, from about 50% to about 65%, from about 55% to about 60%).

[0057] In some aspects, the method can increase the prevalence of cilia on the SCEC (e.g., number or percentage of cells having at least one cilium) by about 0.25% or more (e.g., about 0.5% or more, about 0.75% or more, about 1% or more, about 1.5% or more, about 2% or more, about 2.5% or more, about 3% or more, about 3.5% or more, about 4% or more, about 4.5% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 16% or more, about 17% or more, about 18% or more, about 19% or more, about 20% or more, about 21% or more, about 22% or more, about 23% or more, about 24% or more, about 25% or more). In some aspects, the method can increase the prevalence of cilia on the SCEC by about 25% or less (e.g., about 24% or less, about 23% or less, about 22% or less, about 21% or less, about 20% or less, about 19% or less, about 18% or less, about 17% or less, about 16% or less, about 15% or less, about 14% or less, about 13% or less, about 12% or less, about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4.5% or less, about 4% or less, about 3.5% or less, about 3% or less, about 2.5% or less, about 2% or less, about 1.5% or less, about 1% or less, about 0.75% or less, about 0.5% or less, about 0.25% orless). The method can increase the prevalence of cilia on the SCEC by an amount ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the method can increase the prevalence of cilia on the SCEC by from about 0.25% to about 25% (e.g., from about 0.5% to about 24%, from about 0.75% to about 23%, from about 1% to about 22%, from about 1.5% to about 21%, from about 2% to about 20%, from about 2.5% to about 19%, from about 3% to about 18%, from about 3.5% to about 17%, from about 4% to about 16%, from about 4.5% to about 15%, from about 5% to about 14%, from about 6% to about 13%, from about 7% to about 12%, from about 8% to about 11%, from about 9% to about 10%, from about 0.25% to about 10%, from about 0.5% to about 9%, from about 0.75% to about 8%, from about 1% to about 7%, from about 1.5% to about 6%, from about 2% to about 5%, from about 2.5% to about 4.5%, from about 3% to about 4%, from about 9% to about 25%, from about 10% to about 24%, from about 11% to about 23%, from about 12% to about 22%, from about 13% to about 21%, from about 14% to about 20%, from about 15% to about 19%, from about 16% to about 18%).

[0058] In some aspects, the method can increase density of intercellular pores between the SCEC following administration of the TMC secretome and / or TMSC secretome. In some aspects, the method can yield a density of intercellular pores (e.g., number of intercellular pores per number of cells) of about 0.1 pores per cell or more (e.g., about 0.2 pores per cell or more, about 0.3 pores per cell or more, about 0.4 pores per cell or more, about 0.5 pores per cell or more, about 0.75 pores per cell or more, about 1 pores per cell or more, about 1.25 pores per cell or more, about 1.5 pores per cell or more, about 1 .75 pores per cell or more, about 2 pores per cell or more, about 1.25 pores per cell or more, about 2.5 pores per cell or more, about 2.75 pores per cell or more, about 3 pores per cell or more, about 3.25 pores per cell or more, about 3.5 pores per cell or more, about 3.75 pores per cell or more, about 4 pores per cell or more). In some aspects, the method can yield a density of intercellular pores of about 4 pores per cell or less (e.g., about 3.75 pores per cell or less, about 3.5 pores per cell or less, about 3.25 pores per cell or less, about 3 pores per cell or less, about 2.75 pores per cell or less, about 2.5 pores per cell or less, about 1.25 pores per cell or less, about 2 pores per cell or less, about 1.75 pores per cell or less, about 1.5 pores per cell or less, about 1.25 pores per cell or less, about 1 pores per cell or less, about 0.75 pores per cell or less, about 0.5 pores per cell or less, about 0.4 pores per cell or less, about 0.3 pores per cell or less, about 0.2 pores per cell or less, about 0.1 pores per cell or less). The method can yield a density of intercellular pores ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the method can yield a density of intercellular pores of from about 0.1 poresper cell to about 4 pores per cell (e.g., from about 0.2 pores per cell to about 3.75 pores per cell, from about 0.3 pores per cell to about 3.5 pores per cell, from about 0.4 pores per cell to about 3.25 pores per cell, from about 0.5 pores per cell to about 3 pores per cell, from about 0.75 pores per cell to about 2.75 pores per cell, from about 1 pores per cell to about 2.5 pores per cell, from about 1.5 pores per cell to about 2 pores per cell, from about 0.1 pores per cell to about 1.75 pores per cell, from about 0.2 pores per cell to about 1.5 pores per cell, from about 0.3 pores per cell to about 1.25 pores per cell, from about 0.4 pores per cell to about 1 pores per cell, from about 0.5 pores per cell to about 0.75 pores per cell, from about 1 .75 pores per cell to about 4 pores per cell, from about 2 pores per cell to about 3.75 pores per cell, from about 1.25 pores per cell to about 3.5 pores per cell, from about 2.5 pores per cell to about 3.25 pores per cell, from about 2.75 pores per cell to about 3 pores per cell).

[0059] In yet another aspect, provided is a method of decreasing cellular stiffness and / or storage modulus of Schlemm’s canal endothelial cells (SCEC) in a subject in need thereof, the method including administering a therapeutically effective amount of trabecular meshwork cell (TMC) secretome and / or trabecular meshwork stem cell (TMSC) secretome to a Schlemm’s canal of the subject.

[0060] In some aspects, the method can decrease cellular stiffness of the SCEC by about 40% or more (e.g., about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 100% or more) following administration of the TMC secretome and / or TMSC secretome. In some aspects, the method can decrease cellular stiffness of the SCEC by about 100% or less (e.g., about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less) following administration of the TMC secretome and / or TMSC secretome. The method can decrease cellular stiffness of the SCEC following administration of the TMC secretome and / or TMSC secretome by an amount ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the method can decrease cellular stiffness of the SCEC by from about 40% to about 100% (e.g., from about 45% to about 95%, from about 50% to about 90%, from about 55% to about 85%, from about 60% to about 80%, from about 65% to about 75%, from about 40% to about 70%, from about 45% to about 65%, from about 50% to about 60%, from about 70% to about 100%, from about 75% to about 95%, from about 80% to about 90%) following administration of the TMC secretome and / or TMSC secretome.

[0061] In some aspects, the method can decrease storage modulus of the SCEC by about 40% or more (e.g., about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 100% or more) following administration of the TMC secretome and / or TMSC secretome. In some aspects, the method can decrease storage modulus of the SCEC by about 100% or less (e.g., about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less) following administration of the TMC secretome and / or TMSC secretome. The method can decrease storage modulus of the SCEC following administration of the TMC secretome and / or TMSC secretome by an amount ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the method can decrease storage modulus of the SCEC by from about 40% to about 100% (e.g., from about 45% to about 95%, from about 50% to about 90%, from about 55% to about 85%, from about 60% to about 80%, from about 65% to about 75%, from about 40% to about 70%, from about 45% to about 65%, from about 50% to about 60%, from about 70% to about 100%, from about 75% to about 95%, from about 80% to about 90%) following administration of the TMC secretome and / or TMSC secretome.

[0062] In some aspects, the any of the disclosed methods can include: a) determining if the subject has increased intraocular pressure (IOP), a thin retinal nerve fiber layer (RNFL), and / or an enlarged optic nerve cup to disc ratio; and b) administering a therapeutically effective amount of TMC secretome and / or TMSC secretome to the Schlemm’s canal of the subject.

[0063] In some aspects, the SCEC can be glaucomatous and / or the subject can have glaucoma. In some aspects, the subject can have a family history of glaucoma.

[0064] In some aspects, the subject can have high interocular pressure (IOP).

[0065] In some aspects, the any of the disclosed methods can include administering whole trabecular meshwork cells (TMCs) and / or whole trabecular meshwork stem cells (TMSCs) to the Schlemm’s canal of the subject; and the TMCs and / or TMSCs can secrete the therapeutically effective amount of the TMC secretome and / or TMSC secretome in vivo.

[0066] In some aspects, the TMC secretome and / or TMSC secretome can be administered as a composition including about 10% or more total secretome (e.g., about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more,about 90% or more, about 95% or more, about 100% or more). In some aspects, the TMC secretome and / or TMSC secretome can be administered as a composition including about 100% or less total secretome (e.g., about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less). The TMC secretome and / or TMSC secretome can be administered as a composition including an amount of total secretome ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the TMC secretome and / or TMSC secretome can be administered as a composition including from about 10% to about 100% total secretome (e.g., from about 15% to about 95%, from about 20% to about 90%, from about 25% to about 85%, from about 30% to about 80%, from about 35% to about75%, from about 40% to about 70%, from about 45% to about 65%, from about 50% to about60%, from about 10% to about 55%, from about 15% to about 50%, from about 20% to about45%, from about 25% to about 40%, from about 30% to about 35%, from about 55% to about100%, from about 60% to about 95%, from about 65% to about 90%, from about 70% to about 85%, from about 75% to about 80%).

[0067] In some aspects, the TMC secretome and / or TMSC secretome can be administered intraocularly or via eye drops. For example, in some aspects, the TMC secretome and / or TMSC secretome can be administered via subconjunctival injection.

[0068] In some aspects, the TMC secretome and / or TMSC secretome can be loaded into a vehicle and administered to the subject. In some aspects, the vehicle can be administered intraocularly or via eye drops. For example, in some aspects, the vehicle can be administered via subconjunctival injection or placed in the subject’s conjunctival sac under the eyelid. Examples of vehicles are discussed in further detail below.

[0069] In some aspects, the TMC secretome and / or TMSC secretome can be substantially free of cells and / or cellular debris.COMPOSITIONS AND VEHICLES

[0070] In an aspect, provided is a composition including trabecular mesh cell (TMC) secretome and trabecular mesh stem cell (TMSC) secretome.

[0071] In some aspects, the TMC secretome and the TMSC secretome can be present in a ratio of about 0.1:1 or more (e.g., about 0.2:1 or more, about 0.3:1 or more, about 0.4: 1 or more, about 0.5: 1 or more, about 1:1 or more, about 1.5: 1 or more, about 2: 1 or more, about 2: 1 or more, about 2.5:1 or more, about 3: 1 or more, about 3.5: 1 or more, about 4: 1 or more, about4.5:1 or more, about 5:l or more, about 5.5: l or more, about 6:1 or more, about 6.5:1 or more, about 7:1 or more, about 7.5: 1 or more, about 8: 1 or more, about 8.5: 1 or more, about 9: 1 or more, about 9.5:1 or more, about 10:1 or more). In some aspects, the TMC secretome and the TMSC secretome can be present in a ratio of about 10:1 or less (e.g., about 9.5:1 or less, about 9:1 or less, about 8.5:1 or less, about 8:1 or less, about 7.5:1 or less, about 7:1 or less, about 6.5:1 or less, about 6:1 or less, about 5.5:1 or less, about 5: 1 or less, about 4.5:1 or less, about 4: 1 or less, about 3.5: 1 or less, about 3: 1 or less, about 2.5: 1 or less, about 2: 1 or less, about 2:1 or less, about 1.5:1 or less, about 1 :1 or less, about 0.5:1 or less, about 0.4: 1 or less, about 0.3:1 or less, about 0.2:1 or less, about 0.1:1 or less). The TMC secretome and the TMSC secretome can be present in a ratio ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the TMC secretome and the TMSC secretome can be present in a ratio of from about 0.1 : 1 to about 10: 1 (e.g., from about 0.2:1 to about 9.5: 1, from about 0.3:1 to about 9: 1 , from about 0.4:1 to about 8.5:1, from about 0.5:1 to about 8: 1, from about 1 :1 to about 7.5:1, from about 1.5:1 to about 7:1, from about 2: 1 to about 6.5: 1, from about 2: 1 to about 6: 1 , from about 2.5: 1 to about 5.5: 1, from about 3:1 to about 5: 1, from about 3.5: 1 to about 4.5:1, from about 0.1: 1 to about 4: 1, from about 0.2:1 to about 3.5:1, from about 0.3:1 to about 3:1 , from about 0.4:l to about 2.5:1, from about 0.5:1 to about 2:1, from about 1 :1 to about 2:1, from about 4:1 to about 10:1, from about 4.5:1 to about 9.5:1, from about 5:1 to about 9:1, from about 5.5:1 to about 8.5:1, from about 6: 1 to about 8:1, from about 6.5: 1 to about 7.5: 1).

[0072] In some aspects, the composition can include about 10% or more total secretome (e.g., about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 100% or more). In some aspects, the composition can include about 100% or less total secretome (e.g., about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about20% or less, about 15% or less, about 10% or less). The composition can include an amount of total secretome ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the composition can include from about 10% to about 100% total secretome (e.g., from about 15% to about 95%, from about 20% to about 90%, from about 25% to about 85%, from about 30% to about 80%, from about35% to about 75%, from about 40% to about 70%, from about 45% to about 65%, from about50% to about 60%, from about 10% to about 55%, from about 15% to about 50%, from about20% to about 45%, from about 25% to about 40%, from about 30% to about 35%, from about55% to about 100%, from about 60% to about 95%, from about 65% to about 90%, from about70% to about 85%, from about 75% to about 80%).

[0073] In another aspect, provided is a vehicle comprising trabecular mesh cell (TMC) secretome and / or trabecular mesh stem cell (TMSC) secretome. As used herein, the terms “vehicle,” “delivery vehicle,” “transfer vehicle,” “nanoparticle” or grammatical equivalent, are used interchangeably.

[0074] In some aspects, the TMC secretome and / or TMSC secretome may be delivered via a single delivery vehicle. In some aspects, the TMC secretome and / or TMSC secretome may be delivered via one or more delivery vehicles each of a different composition. According to various aspects, suitable delivery vehicles include, but are not limited to polymer based carriers, such as polyethyleneimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, both natural and synthetically-derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, calcium phosphor-silicate nanoparticulates, calcium phosphate nanoparticulates, silicon dioxide nanoparticulates, nanocrystalline particulates, semiconductor nanoparticulates, poly(D- arginine), sol-gels, nanodendrimers, starch-based delivery systems, micelles, emulsions, niosomes, multi-domain-block polymers (vinyl polymers, polypropyl acrylic acid polymers, dynamic polyconjugates), cell- or platelet-derived exosomes, ethosomes, or transfersomes.

[0075] In some aspects, a suitable delivery vehicle is a lipid nanoparticle. As used herein, “lipid nanoparticles” refer to particles having at least one dimension on the order of nanometers (e.g., 1-1000 nm) and including one or more lipids. In the context of the present invention, a lipid nanoparticle typically serves to transport a desired TMC secretome and / or TMSC secretome to a target cell or tissue. Accordingly, in some aspects, a suitable delivery vehicle is capable of enhancing the stability of the TMC secretome and / or TMSC secretome contained therein and / or facilitate the delivery of the TMC secretome and / or TMSC secretome to the target cell or tissue.EXAMPLESExample 1

[0076] Current treatment strategies for glaucoma primarily focus on lowering IOP using topical eye drops, laser therapies, and surgical interventions to increase outflow or reduce inflow. However, these approaches have limitations, including variable efficacy, potential sideeffects, and an inability to prevent the progressive loss of vision in all patients. Consequently, there is an urgent need for therapies that offer long-term efficacy and neuroprotection to effectively manage glaucoma. Stem cell-based therapies have emerged as promising candidates for glaucoma treatment. Various stem cell types, including trabecular meshwork stem cells (TMSCs) (Y. Du, et al., Stem cells from trabecular meshwork home to TM tissue in vivo. Invest Ophthalmol Vis Sci 54, 1450-1459 (2013); H. Yun et al., Human stem cells home to and repair laser-damaged trabecular meshwork in a mouse model. Commun Biol 1, 216 (2018)), bone- marrow mesenchymal stem cells (R. Manuguerra-Gagne et al., Transplantation of mesenchymal stem cells promotes tissue regeneration in a glaucoma model through laser- induced paracrine factor secretion and progenitor cell recruitment. Stem Cells 31, 1136-1148 (2013); C. Roubeix et al., Intraocular pres sure reduction and neuroprotection conferred by bone marrow-derived mesenchymal stem cells in an animal model of glaucoma. Stem Cell Res Ther 6, 177 (2015)), induced pluripotent stem cells (W. Zhu et al., Transplantation of iPSC-derived TM cells rescues glaucoma phenotypes in vivo. Proc Natl Acad Sci U S A 113, E3492-3500 (2016)), and adipose-derived stem cells (Y. Zhou et al., Adipose-derived stem cells integrate into trabecular meshwork with glaucoma treatment potential. FASEB J 34, 7160-7177 (2020)), have shown potential in regenerating TM tissue, reducing IOP, preserving RGCs, and ultimately preventing vision loss. Among these, TMSCs are particularly intriguing due to their native role in maintaining TM homeostasis and their potential to influence adjacent ocular structures.

[0077] Secretome refers to the repertoire of bioactive molecules secreted by cells, encompassing cytokines, growth factors, extracellular matrix proteins, microRNAs, noncoding RNAs, and mRNAs. Stem cell-derived secretomes have demonstrated therapeutic potential across a range of diseases, including cardiovascular and ocular disorders (C. R. Harrell et al., Therapeutic Potential of Mesenchymal Stem Cells and Their Secretome in the Treatment of Glaucoma. Stem Cells Int 2019, 7869130 (2019)). Notably, paracrine factors from stem cells have been shown to promote RGC survival and expand the progenitor cell pool within the eye (F. Sanie-Iahromi, et al., A Review on the Application of Stem Cell Secretome in the Protection and Regeneration of Retinal Ganglion Cells; a Clinical Prospect in the Treatment ofOptic Neuropathies. Curr Eye Res 47, 1463-1471 (2022)). Secretome from TMSC can prevent and restore TM cell function treated by dexamethasone and promote TM cell regeneration and reduce IOP in vivo (X. S. Ajay Kumar, et al., Stem cell-free therapy for glaucoma to preserve vision. Biorxiv (2021), A. Kumar, et al., Trabecular MeshworkRegeneration for Glaucoma Treatment Using Stem Cell-Derived Trophic Factors. Methods Mol Biol 2848, 59-71 (2025)).

[0078] This study aims to investigate the effects of the secretomes from TMSC (TMSC- Scr) and TM cells (TM-Scr) on SCEC. The study evaluated 1) if TMSC-Scr and TM-Scr are toxic, 2) their impact on SCEC proliferation, migration, and permeability, and 3) their underlying mechanisms of action. By elucidating the interplay between TMSC-Scr, TM-Scr and SCEC, this study can provide insights into the pathophysiology of glaucoma and potentially uncover new therapeutic targets for modulating aqueous humor outflow and IOP.Materials and Methods

[0079] Primary cell culture and secretome preparation: Primary cell cultures were established as described previously (Y. Du et al., Multipotent stem cells from trabecular meshwork become phagocytic TM cells. Invest Ophthalmol Vis Sci 53, 1566-1575 (2012)). TMSC were cultured in Opti-MEM (Invitrogen) with supplements including 5% fetal bovine serum, 0.08% chondroitin sulfate, 100 ug / ml bovine pituitary extract (ThermoFisher), 20 pg / ml ascorbic acid, 10 ng / ml epidermal growth factor, 200 pg / ml calcium chloride (Sigma-Aldrich), 50 mg / ml gentamicin, 100 mg / ml streptomycin, and 100 lU / ml penicillin (ThermoFisher). TM cells and SCE cells were cultivated in DMEM: HAM’s F12 (1: 1) medium with 10% FBS. For the preparation of secretome, cells were cultured in complete media to 60-70% confluency in a T175 flask. The cells were gently washed with phosphate-buffered saline, the media were changed to basal media (without FBS or any additive supplements), and the cells were cultured in a humidified cell culture incubator with 5% CO2 for 3 days. Then the culture supernatant was collected and centrifuged (10 min at 500 g, 20 min at 2000 g and 60 min at 4000 g) to remove cell debris, and aliquoted into 2 mL vials. Samples were stored at -80°C until use. For secretome concentration, each 2 mL secretome aliquot was concentrated to a final volume of '■“400 pL using an Amicon Ultra centrifugal filter unit with 10 kDa MWCO (Merck). FIG. 1A illustrates the schematic workflow for secretome preparation. After the conditioned media were harvested, the cells were subjected to immunofluorescent staining to confirm the expression of cell surface markers.

[0080] MTT assay: To determine the proliferative capacity of different concentration of secretome, SCE (1 x 104cells / well) were seeded in 96-well plates. After 24 hr of incubation, the cells were treated with stem cell secretome for 48 hr. MTT reagent (Millipore) was used to assess the formation of formazan crystals at end point. The optical density was measured viamicroplate readers (Tecan) using 570 nm wavelength and considering 600 nm as reference to eliminate any background noise.

[0081] Calcein AM / PI staining: Post secretome treatment for 48 hr, GSCE were stained for 15 minutes in dark with the viability dyes Calcein- AM (2 pM. Invitrogen), Propidium Iodide (PI, 1 pg / mL, Invitrogen), and Hoechst 33342 (1 pg / mL, Invitrogen) at 37°C for 20-30 minutes in the dark. After incubation, cells were gently washed with PBS and scanned using a Keyence fluorescence microscope to capture images under appropriate filters.

[0082] Cell Migration Analyses: Cell migration was evaluated by the scratch wound assay. GSCE cells were seeded in 6-well plates and cultured until they reached 100% confluence. Once full confluence was achieved, the cells were serum-starved overnight in basal medium. A sterile 100 pL pipette tip was used to create a straight scratch (wound) across the cell monolayer. After gently washing the wells with phosphate-buffered saline (PBS) to remove detached cells, fresh medium containing 50% or 25% concentrations of TMSC / TM-Scr conditioned medium, or basal medium as a control, was added to each well. The wound closure was monitored using a Keyence microscope in phase contrast mode, and images were captured every 30 minutes for 48 hours. The wound area was measured by ImageJ (version 1.54). The wound area was quantified using the Imagel plugin Wound Healing Size Tool (A. Suarez- Amedo et al., An image J plugin for the high throughput image analysis of in vitro scratch wound healing assays. PLoS One 15, e0232565 (2020)). The migration percentage was calculated based on the average area reduction every 6 hours, compared to time 0. The experiments were performed in triplicate, and the means + SD from the three independent experiments was calculated.

[0083] Differentiation of SC Cell Monolayers: SCE cells were seeded onto 24-well Transwell inserts (pore size 0.4 pm, Costar) and maintained in complete medium until the net transendothelial electrical resistance (TEER) exceeded 10 Q-cm2. Once this threshold was reached, the cells were subjected to overnight serum starvation. Subsequently, the cells were treated with TMSC-Scr, TM-Scr, or basal medium. TEER measurements were recorded every two days for 16 days using the Millicell™ ERS 3.0 Digital Voltohmmeter (Millipore).

[0084] Permeability of FITC-dextran: The paracellular permeability of SCE cell monolayers was evaluated using FITC-Dextran (4 kDa, Sigma-Aldrich) on day 2 and day 6 following treatment with TMSC-Scr, TM-Scr, or basal medium. The culture medium in the apical chamber of the 24-well Transwell inserts was replaced with FITC-Dextran solution (1 mg / mL, Sigma-Aldrich) dissolved in Live Cell Imaging Solution (Invitrogen), while the basolateral chamber contained the same solution without FITC-Dextran. After incubation at37°C for 2 hours, samples were collected from the basolateral chamber, and fluorescence intensity was measured using a microplate reader (excitation: 485 nm, emission: 535 nm, Tecan) to assess paracellular permeability.

[0085] Western Blottin g: Cells were lysed using RIPA buffer (SantaCruz Biotechnology). Protein concentration was measured using the BCA Protein Assay Kit (Pierce Biotechnology). Protein samples were loaded for each group in each well and run on 4-20% sodium dodecyl sulfate-polyacrylamide (SDS) gel (ThermoFisher) for electrophoresis. After gel run, samples were transferred to the PVDF membrane. Non-specific binding was blocked using blocking buffer (LI-COR Biosciences). After blocking, membranes were incubated overnight with primary antibodies: PROXI, Fibulin 2 (R & D Systems), ZO-1 (Invitrogen), Claudin 11 (Invitrogen), N-Cadherin, Connexin 43 (Cell Signaling Technology), and OB-Cadherin overnight at 4°C with 0-actin (Invitrogen) as normalization control. Corresponding secondary antibodies (Alexa Fluor™ Plus 488, IRDye 680LT and IRDye 800CW, Invitrogen, LI-COR Biosciences) were incubated after three washes of 0.1% Tween 20 in Tris-buffered saline. Detection and capture of fluorescent signals were performed using iBright imaging system (ThermoFisher). ImageJ was used for the quantification and analysis of protein expression with P-actin as internal control.

[0086] Immunofluorescent staining: Cells were fixed in 4% paraformaldehyde for 15 minutes, permeabilized with 0.5% Triton X-100, and blocked with 1% bovine serum albumin (BSA) for 1 hour at room temperature. Cells were incubated with primary antibodies: Nestin (S. Xiong et al., cc5[31 Integrin Promotes Anchoring and Integration of Transplanted Stem Cells to the Trabecular Meshwork in the Eye for Regeneration. Stem Cells and Development 29, 290-300 (2019)), OCT4 (Id.), CHI3L1 (S. Xiong et al., Stem cell transplantation rescued a primary open-angle glaucoma mouse model. Elife 10 (2021)), PROXI, Fibulin 2 (R & D Systems), ZO-1 (Invitrogen), Claudin 11 (Invitrogen), N-Cadherin, Connexin 43 (Cell Signaling Technology), OB-Cadherin, and Acetyl-a-Tubulin (Lys40) (Cell Signaling Technology) overnight at 4°C. After three washes with PBS, corresponding fluorescent secondary antibodies and 4’,6-diamidino-2-phenylindole (DAPI) were applied to the sections for 2 hrs. After five washes, slides were mounted, imaged and analyzed using a fluorescence microscope (Keyence BZ-X800).

[0087] Quantitative Real-Time PCR (qPCR): Cells were lysed with RLT buffer and RNAs were isolated using RNeasy Kit, the same as above-mentioned Microarray assay. cDNAs were synthesized from the RNAs using reverse transcriptase (SuperScript III; Invitrogen). qPCR was conducted using SYBR Green dye (Thermo Fisher, Pittsburgh, PA).Primers for target genes were either newly designed using the NIH Primer-BLAST tool or previously used and published with references cited in TABLE 1, where the sequences were provided. Amplification of 18S rRNA was used as a normalization control, and negative controls without cDNA were included in each assay. Relative mRNA abundance was calculated using the AACt method as previously described (Y. Du, et al., Multipotent stem cells in human corneal stroma. Stem Cells 23, 1266-1275 (2005); Y. Wang et al., Endoplasmic Reticulum Stress Response of Trabecular Meshwork Stem Cells and Trabecular Meshwork Cells and Protective Effects of Activated PERK Pathway. Invest Ophthalmol Vis Sci 60, 265- 273 (2019)). Three independent biological replicates from three different donors of cells and isolated cDNAs were analyzed, with each reaction performed in triplicates as technical repeats.TABLE 1. Primer sequences used for qPCR in the study.*H. Yun et al., Human stem cells home to and repair laser-damaged trabecular meshwork in a mouse model. Commun Biol 1, 216 (2018).

[0088] Atomic force microscopy: A JPK Nanowizard PURE atomic force microscope (AFM) (Broker Nano, Berlin, Germany), mounted on an inverted Zeiss Axiovert 200 M epifluorescence microscope (Carl Zeiss Microscopy, Gottingen, Germany), was utilized to image and characterize the mechanical and viscoelastic properties of cells. For mechanical probing, Biosphere Au Reflex (CONT-Au) cantilevers (Nanotools USA LLC, Henderson, NV) were used, featuring a nominal spring constant of 0.2 N / m, a length of 450 pm, and a nominal resonance frequency of 13 kHz in air, with a spherical tip radius of 5 pm (±10%) (M. Asgari, et al., Revealing Layer - Specific Ultrastructure and Nanomechanics of Fibrillar Collagen in Human Aorta via Atomic Force Microscopy Testing: Implications on Tissue Mechanics at Macroscopic Scale. Advanced NanoBiomed Research 2, 2100159 (2022)). Cells were cultured in petri dishes submerged in culture media, and the AFM probe was guided over the samples using light microscopy to precisely target regions of interest for indentation. Prior to each experiment, the deflection sensitivity of the AFM cantilever was calibrated by bringing it into contact with a clean microscope slide (Id.). The precise spring constant was determined viathermal noise analysis in air, where the primary resonance peak was fitted to a simple harmonic oscillator model using JPK software. Indentation experiments were conducted in a hydrated environment at an indentation rate of 2 pm / s (Id.). The elastic modulus (E) was calculated using the Hertz contact model applied to force-displacement curves (Id.), and AFM data analysis was performed with JPK data processing software. Viscoelastic properties, including the storage modulus (E’) and loss modulus (E”), were measured using the nano-rheometry module, following the methodology described in Asgari et al. (2024) (M. Asgari, et al., Ultrastructural viscoelastic behavior of collagen identified by AFM nano-dynamic mechanical analysis. bioRxiv, 2024.2010. 2019.619231 (2024)). Multiple biological replicates were tested, with each replicate including more than 250 cells subjected to repeated indentations to ensure no permanent deformation occurred.

[0089] Scanning Electron Microscopy (SEM) Methodology: To assess the surface morphology and ultrastructural changes of GSCEs, scanning electron microscopy (SEM) was performed. Cells treated with TMSC-Scr, TM-Scr, or basal medium were fixed in 2.5% glutaraldehyde at 4°C for 2 hours, followed by post- fixation in 1% osmium tetroxide (OsOr) for 1 hour. Samples were then dehydrated through a graded ethanol series (30%— 100%) and critical point dried using liquid CO2. The dried samples were mounted on SEM stubs, sputter- coated with a thin layer of gold or platinum, and imaged using a scanning electron microscope at an accelerating voltage of 5-15 kV. Images were analyzed to evaluate cell surface features, microvilli structure, and intercellular junctions.

[0090] Characterization of the secretome: The secretome was subjected to cytokine array profiling for the initial screening and identification of secretome factors using an 80- target spotted membrane-based human cytokine antibody array (abl33998, Abeam). All control and test dot blots were performed using this cytokine array according to the manufacturer’s protocol. The membranes were developed using chemiluminescence, and images were acquired using iBright imaging system (ThermoFisher). The identified positive signals were quantified using ImageJ. Protein-protein interactions were evaluated, and GO- biological process and Reactome pathways were identified utilizing the STRING database to evaluate interactions between secretomes.

[0091] Statistical Analysis: Results were expressed as mean ± standard deviation (SD). The statistical differences were analyzed by one-way ANOVA followed by Tukey posttest using PRISM. p< 0.05 was considered statistically significant.Results

[0092] TMSC-Scr and TM-Scr Effect on SCEC Viability: To investigate the paracrine effects of TMSC and TM cells on SCEC, the secretomes from TMSC and TM cells were prepared under serum starving conditions (FIG. 1A). To confirm the phenotypic stability of TMSC and TM cells under starvation, the study performed immunofluorescent staining for specific biomarkers. As shown in FIG. IB, TMSCs retained high expression levels of stem cell markers Nestin and OCT4 before and after starving, indicating the maintenance of their stem cell characteristics. TM cells showed consistent expression of the biomarker CHI3L1 , confirming their phenotypic stability under starvation.

[0093] Next, the study evaluated the impact of different concentrations of TMSC-Scr and TM-Scr on the viability of glaucomatous SCEC (GSCEC) using MTT assay to detect if the secretomes are toxic to GSCEC. As depicted in FIGS. 1C-1D, both TMSC-Scr and TM-Scr significantly enhanced GSCEC viability after 48-hr treatment. Notably, the 25% and 50% concentrations showed a significant increase in cell viability, with the 50% concentration exhibiting the most pronounced effect. Specifically, TMSC-Scr increased GSCEC viability by 1.27-fold at 25% and 1.32-fold at 50% concentrations, while TM-Scr resulted in 1.11-fold and 1 .13-fold increases at 25% and 50% concentrations, respectively. This effect may be attributed to the optimal balance of bioactive factors present at these concentrations. Moreover, GSCEC viability was consistently higher in the TMSC-Scr-treated groups compared to the TM-Scr- treated groups, suggesting that TMSC-Scr contains more potent bioactive factors. Based on these findings, 25% and 50% concentrations were selected for subsequent experiments.

[0094] The study also assessed GSCEC viability using Calcein-AM and propidium iodide (PI) staining after 48-hr secretome treatment. As shown in FIG. IE, GSCEC treated with 25% and 50% concentrations of TMSC-Scr and TM-Scr exhibited similar levels of Calcein-AM- positive (viable) and Pl-positive (dead) cells across all groups. DAPI staining was used to visualize nuclei. These findings suggest that secretomes are not toxic to GSCEC, rather, they promote GSCEC proliferation.

[0095] TMSC-Scr and TM-Scr Promote GSCEC Wound Healing, Proliferation, and Permeability: To further evaluate the regenerative potential of TMSC-Scr and TM-Scr, the study performed wound healing assays on GSCEC. FIG. 2A shows representative images of wound closure at 0, 24, and 48 hours after treatments immediately following a scratch wound. Quantitative analysis of the wounded area (FIG. 2B) revealed that TMSC-Scr significantly accelerated wound closure compared to TM-Scr and control groups. After 48 hours, wound closure reached 98.68 ± 0.60% in the 50% TMSC-Scr group and 91.68 ± 2.80% in the 25%TMSC-Scr group. In contrast, wound closure was significantly lower in the GSCEC control group (57.86 ± 18.85%), the 25% TM-Scr group (53.56% ± 5.83%), and the 50% TM-Scr group (41.71 ± 14.83%).

[0096] The study next assessed GSCEC proliferation by quantifying Ki-67 mRNA expression using qPCR. As shown in FIG. 2C, GSCEC treated with TMSC-Scr exhibited significantly higher Ki-67 expression compared to TM-Scr-treated and control cells. The Ki- 67 expression levels were 1.33-fold in wounded GSCEC, 2.2-fold in 50% TMSC-Scr group, 1.46-fold in 25% TMSC-Scr group, 0.75-fold in 50% TM-Scr group, and 0.81 -fold in 25% TM-Scr group, compared to a baseline level as 1 in untreated GSCEC. These results suggest enhanced proliferative activity with TMSC-Scr treatment, while TM-Scr showed a limited effect on proliferation.

[0097] To determine the impact of secretomes on GSCEC permeability, the study measured TEER over a 16-day period. From the wound healing and Ki-67 results (FIGS. 2B- 2C), no significant difference between 50% and 25% TMSC-Scr and TM-Scr groups was observed. Thus, 25% TMSC-Scr and 25% TM-Scr were selected for the permeability experiments. As shown in FIG. 2D, TEER results showed significant differences from day 6 onward between the TMSC-Scr-treated and TM-Scr-treated with untreated GSCEC, indicating increased permeability after secretome treatment.

[0098] To further confirm the results, FITC-dextran permeability assays conducted at days 2 and 6 revealed a similar trend. There was no difference between filtered FITC-dextran on day 2. On day 6, FITC-dextran permeability was significantly increased in the TMSC-Scr- treated GSCEC (141.36 ± 7.03%) and the TM-Scr-treated GSCEC (127.44 ± 4.04%) compared to the untreated GSCE group (100 + 4.12%), indicating enhanced permeability consistent with the TEER results.

[0099] TMSC-Scr and TM-Scr Upregulate GSCEC Biomarkers PROXI and FBLN2: To further elucidate the molecular effects of TMSC-Scr and TM-Scr on GSCEC, the study assessed the expression of key GSCEC biomarkers, PROXI and Fibulin-2 (FBLN2), using Western blotting (WB) and immunofluorescent staining (IF). As shown in FIGS. 3A-3D, both TMSC-Scr and TM-Scr significantly upregulated PROXI and FBLN2 protein levels compared to untreated GSCEC. The upregulation was more pronounced in the TMSC-Scr-treated group than the TM-Scr-treated group, indicating a stronger effect of TMSC-Scr than TM-Scr. There were no significant differences between the 50% and 25% concentrations for either TMSC-Scr TM-Scr (FIGS. 3A-3B), indicating that even lower concentration of secretomes effectivelyenhance the expression of these critical endothelial markers. The IF results (FIGS. 3C-3D) represented 25% secretome treated cells.

[0100] TMSC-Scr and TM-Scr Differentially Changed Transmembrane Proteins N- Cad and OB-Cad: Changes of transmembrane proteins N-Cadherin (N-Cad) and OB- Cadherin (OB-Cad) were also examined using WB and IF. As shown in FIGS. 4A-4C, both 25% and 50% TM-Scr and 25% TMSC-Scr significantly upregulated N-Cad expression but not 50% TMSC-Scr, compared to untreated GSCEC. In contrast, all the secretome treated GSCEC groups significantly reduced OB-Cad expression. The IF results (FIG. 4C) represented 25% secretome treated cells. The results suggest differential regulation of the transmembrane proteins that function as cell adhesion molecules.

[0101] TMSC-Scr Enhances Tight Junction and Communication Proteins in GSCEC: To investigate the effects of TMSC-Scr and TM-Scr on cell junction integrity, the study evaluated the expression of key junctional proteins, ZO-1, Claudin-11 (CLDN11), and Connexin 43, using both WB and IF. As shown in FIGS. 5A-5D, WB results demonstrated that TMSC-Scr significantly upregulated the expression of ZO-1, CLDN11, and Connexin 43 in GSCEC compared to TM-Scr and untreated controls.

[0102] Consistent with WB results, IF results (FIGS. 5E-5F) revealed stronger fluorescent staining signals for ZO-1, CLND11, and Connexin 43 in the TMSC-Scr-treated GSCEC, indicating enhanced tight junction integrity and intercellular communication.

[0103] TMSC-Scr and TM-Scr Differentially Regulate GSCEC Ultrastructure and Cilium Features: To explore the ultrastructural changes in GSCEC, the study performed SEM. As shown in FIGS. 6A-6B, TMSC-Scr-treated GSCEC exhibited a higher density of intercellular pores compared to those treated with TM-Scr. The magnified region in FIG. 6B highlights the pores, suggesting enhanced intercellular communication and permeability regulation mechanism by TMSC-Scr.

[0104] TM-Scr-treated GSCEC displayed a significant increase in primary cilia compared to the TMSC-Scr-treated and untreated groups. To further confirm this observation, the study performed IF staining for acetylated alpha-tubulin (AC-a-tubulin), a marker of cilia (FIG. 6C). Quantitative analysis of cilium prevalence (percentage of cells containing cilium, FIG. 6D) and cilium length (FIG. 6E) demonstrated that TM-Scr significantly increased both the percentage of ciliated cells and the average cilium length compared to TMSC-Scr-treated and control groups. These findings indicate that TM-Scr promotes ciliogenesis, while TMSC-Scr enhances intercellular pore formation.

[0105] TMSC-Scr and TM-Scr Influence GSCE Mechanical Properties: To evaluate the mechanical properties of GSCEC following secretome treatments, the study performed atomic force microscopy (AFM) to measure cellular stiffness and storage modulus. As shown in FIG. 7A, AFM imaging revealed differences in cellular stiffness between TMSC-Scr- and TM-Scr-treated GSCEC. Quantitative analysis (FIG. 7B) demonstrated that TMSC-Scr and TM-Scr significantly reduced cellular stiffness in GSCEC compared to untreated control, with TMSC-Scr reduced the most, indicating increased cellular flexibility after TMSC-Scr and TM- Scr treatment and TMSC-Scr effect is stronger than TM-Scr.

[0106] In addition to stiffness, the study assessed the storage modulus, which reflects the elastic behavior of cells. FIG. 7C shows the storage modulus measurements, with statistical analysis presented in FIG. 7D. The results indicated that TMSC-Scr and TM-Scr led to significant reduction in storage modulus compared to untreated control with more effect in TMSC-Scr-treated GSCEC, suggesting enhanced viscoelastic properties and reduced rigidity after secretome treatment.

[0107] Loss modulus, also known as viscous modulus, indicates how much a material dissipates energy. As shown in FIG. 7E shows the loss modulus measurements and FIG. 7F is the statistical analysis. The results indicated that both TMSC-Scr and TM-Scr significantly increased the loss modulus compared to untreated control with more effect in TMSC-Scr- treated GSCEC, suggesting increased ability to dissipate stress.Example 2

[0108] Glaucoma is a leading cause of irreversible blindness worldwide. The resistance of trabecular meshwork (TM) and Schlemm’s canal endothelial cells (SCE) plays very important roles in regulation of aqueous humor outflow and intraocular pressure (IOP). TM cellularity is reduced in glaucoma and transplantation of TM stem cell (TMSC) can increase TM cellularity and reduce IOP in animal models. This study aims to explore if secretomes from TMSC and TM cells (TMSC-Scr and TM-Scr) can regulate SCE proliferation, barrier function, permeability, and mechanotransduction, which can uncover potential therapeutic strategies for glaucoma.

[0109] Methods: Human TMSC and TM cells were characterized and secretomes from both cell types (TMSC-Scr and TM-Scr) were prepared under serum starvation conditions. Cultured SCE from glaucoma donors (GSCE) were treated with 50% and 25% secretomes and compared with untreated GSCE in basal medium. Secretome toxicity was assessed by comparing cell viability using MTT assays and calcein-AM / PI staining. Secretome effects on GSCE were evaluated on wound healing assays, cell proliferation by Ki67 expression, barrierfunction by transepithelial electrical resistance (TEER) and FITC-dextran permeability assays. Western blotting (WB) and immunofluorescent staining (IF) assessed biomarker expression. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) measuring Young’s modulus, Storage modulus, and Loss modulus to assess GSCE ultrastructural features and mechanotransduction properties. One-Way or two-way ANOVA was used for statistical analyses followed by Tukey’s multiple comparisons test using Graph-pad Prism 10. P<0.05 was considered statistically significant.

[0110] Results: Both TMSC and TM cells maintained their characteristics with similar cell marker expression after serum starving for secretome collection. Neither TMSC-Scr nor TM- Scr was toxic to GSCE without inducing cell death. TMSC-Scr significantly enhanced GSCE viability by MTT assay, wound healing, proliferation with increased Ki67 expression, and reduced barrier function by TEER and dextran assays compared to untreated GSCE. TM-Scr significantly reduced GSCE barrier function without much changes on wound healing and proliferation. TMSC-Scr-treated GSCEs showed upregulated expression of PROXI, FBLN2, ZO-1, Claudin-11, and Connexin 43, with reduced N-Cadherin and OB-Cadherin expression. SEM revealed increased intercellular pores in TMSC-Scr-treated GSCE, while TM-Scr-treated GSCE exhibited more and longer primary cilia, confirmed by acetylated (AC)-a-tubulin expression.

[0111] TMSC-Scr promotes GSCE proliferation, cell integrity, and mechanical adaptability. It partially elucidates the mechanisms of TMSC effects in glaucoma treatment and unveil a new avenue of stem cell-free therapies for glaucoma. On the other hand, TM-Scr supports structural stability and mechanosensation. These findings highlight the complementary roles of TMSC- and TM-secretomes in regulating SCE functions and the potential to develop therapies for glaucoma.Example 3

[0112] The trabecular meshwork (TM) and Schlemm's canal endothelial (SCE) cells are integral to the proximal aqueous humor outflow pathway, and their dysfunction is a major contributor to elevated intraocular pressure in glaucoma. This study aims to identify and validate key biomarkers in TM and SCE cells to better understand their roles in glaucoma pathophysiology.

[0113] Methods: Primary cultured human TM and SCE cells from 4 different donors, respectively, between passages 3-5 were identified by responsiveness to dexamethasone treatment with increased myocilin expression (TM) and expression of fibulin 2 (SCE). Microarray was performed to analyze their transcriptome profiling. Differentially expressedgenes (DEGs) were identified and cross-referenced with published single-cell RNA sequencing (scRNA-seq) datasets to ensure cell-specific relevance. Validation of candidate biomarkers was performed using quantitative PCR (qPCR) on independently cultured three strains of TM and SCE cells to confirm expression trends observed in the microarray and scRNA-seq analyses. Prism one-way ANOVA was used for statistical analysis and p<0.05 was considered significant.

[0114] Results: Microarray analysis identified numerous DEGs in TM and SCE cells, with distinct functional implications. In TM cells, DEGs were enriched in pathways related to cell cycle regulation, purine metabolism, and carbon metabolism. In contrast, DEGs in SCE cells were associated with cell adhesion molecules, cytokine-cytokine receptor interactions, and cytoskeleton-related regulation. Cross-referencing with scRNA-seq data refined the list of candidate biomarkers by confirming their specific expression in TM and SCE cells. qPCR results revealed significant differences in gene expression between TM and SCE cells. CTTNBP2 and MGARP were highly expressed in TM cells as compared to SCE cells, with expression levels 37.16-fold and 28.17-fold higher, respectively. Conversely, in SCE cells, specific markers exhibited markedly higher expression than TM cells. JAM2, PODXL, and IFI7 were upregulated by 4.98-fold, 233.49-fold, and 11.11 -fold, respectively. These genes are implicated in pathways critical to aqueous humor outflow regulation and glaucoma pathogenesis.

[0115] The validated biomarkers offer insights into glaucoma mechanisms and lay the groundwork for targeted therapies. Future studies will explore their functions and translational potential in preclinical models.Example 4

[0116] Transplanted trabecular meshwork stem cells (TMSC) can reduce intraocular pressure (IOP) in a mouse glaucoma model. In this study, it was hypothesized that TMSC and TM cells (TMC) can regulate the Schlemm’s canal endothelial cell (SCEC) permeability which contributes for IOP regulation.

[0117] Methods: Cultured human TMSC, TM and SCEC were verified using flow cytometry, immunostaining, qPCR and Western blotting. TMSC or TMC were co-cultured with SCEC using culture inserts in 6- well or 24-well plates without contacting each other. The effects of condition media (CM) from TMSC or TMC were also examined for SCEC culture. Transendothelial electrical resistance (TEER) was measured daily for 7 days to assess SCEC permeability. SCEC was treated with Rho kinase inhibitor Y27632 and without any treatments served as controls. At least 3 SCEC strains from different donors were used as biologicalrepeats. Images were analyzed using ImageJ and Prism. P<0.05 was considered statistically significant.

[0118] Results: TMSC expressing more than 90% of CD73, CD90, CD 105 by flow cytometry and positive to OCT4 by staining were used for the experiments. TMC were confirmed by increased myocilin expression after dexamethasone treatment. SCEC were stained positive to Prox-1, VE-cadherin (VE-Cad), and fibulin-2. SCEC had significantly increased expression of Prox-1 (10-15 times), CD31 (5-6 times), VE-Cad (60-80 times), ZO-1 (5-6 times), fibulin-2 (>1000 times), and similar or reduced expression of connexin 43 and N- Cad, as compared to TMC. SCEC co-cultured with TMSC cells or with TMSC-CM started to show reduced TEER from day 2 after co-culture. TEER reduced to about 30% of that of SCEC cultured along at day 7, comparable to the effect of Y27632. SCEC co-cultured with TMC or TM-CM showed similar TEER to SCEC cultured alone. Immunostaining showed that VE-Cad expression in the SCEC with TMC was similar to that of SCEC cultured alone, which was significantly reduced in the SCEC with TMSC or with Y27632. The result indicates that VE- Cad plays a very important role in regulating the permeability of SCEC.

[0119] Human TMSC and TMSC-CM were able to reduce the permeability of co-cultured SCEC, while TMC had little effect on the SCEC permeability. It reveals that TMSC might secret important factors that can regulate SCEC permeability while TMC might need to be in contact with SCEC to regulate SCEC function.Example 5

[0120] Glaucoma is the second leading cause of irreversible blindness, which is associated with reduction of aqueous humor outflow, a drainage system of the eye. The sponge-like tissue, trabecular mesh work, and the adjacent Schlemm’s canal, a tube- like tissue draining the aqueous humor, are considered as the main resistant sites which play important roles in regulation of the pressure in the eye, the intraocular pressure (IOP). A diagram of the eye is shown in FIG. 8. Stem cells from the trabecular meshwork can reconstruct the trabecular mesh work and reduce IOP in mouse glaucoma models. This study aims to explore if the stem cells can further affect the Schlemm’s canal’s structure and function for glaucoma treatment.

[0121] Methods: Schlemm’s canal endothelial cells (SCEC) were co-cultured with trabecular meshwork stem cells (TMSC) or trabecular meshwork cells (TMC) using transwells in 6- well plates. Transendothelial electrical resistance (TEER) was measured daily for 2 weeks to assess SCEC permeability. At least 3 SCEC strains from different donors were used as biological repeats. A flowchart of this method is shown in FIG. 9.

[0122] Results: SCEC had increased expression of Prox-1, VE-Cad, ZO-1, and fibulin-2. First, the gelatin goated sutures were inserted into the Schlemm’s canal lumen under microscope, and the tissues were cultured in a 60 mm dish for ~3 weeks. Medium was changed every 3 days. SCE cells grew along the suture. The sutures were removed from the SC and cells were seeded onto culture plates. These results are shown in FIG. 10. SCEC co-cultured with TMSC started to show reduced TEER from day 7 after co-culture (P < 0.05). SCEC cocultured with TMC showed similar TEER to SCEC cultured alone without statistical significance. These results are shown in FIG. 11. VE-Cad expression in the SCEC with TMC was similar to that of SCEC cultured alone, which was significantly reduced in the SCEC with TMSC. These results are shown in FIG. 12.

[0123] Human TMSC were able to reduce the permeability of co-cultured SCEC which will reduce IOP, while TMC had little effect on the SCEC permeability. It reveals that TMSC might secrete important factors that can regulate SCEC permeability while TMC might need to be in contact with SCEC to regulate SCEC function.Example 6

[0124] Trabecular meshwork stem cells (TMSC) can reconstruct the trabecular meshwork (TM) and reduce intraocular pressure (IOP) in mouse glaucoma models. In this study, human Schlemm’s canal endothelial cells (SCEC) were co-cultured with TMSC or TM cells (TMC) using transwells in 6- well plates. This study aimed to explore if TMSC can further affect the SCEC’s structure and function for glaucoma treatment.

[0125] SCEC express Fibulin-2, Prox- 1 , JAM-3, ZO- 1 , VE-Cad, Na+ / K+ATPase identified by immunostaining and / or qPCR. These results are shown in FIG. 13 and FIG. 14. SCEC co- cultured with TMSC started to show reduced transendothelial electrical resistance (TEER) from day 7 up to day 12. ** p < 0.01, *** p < 0.001. Two-way Anova followed by Turkey posttest. These results are shown in FIG. 11. SCEC co-cultured with TMSC significantly increase the expression of Na+ / K+ATPase detected by qPCR, loosen cell-cell junction, and widen pores by SEM, similar to Y27632. These results are shown in FIGS. 15A-15B. Gene expression profiles were different between human TMSC & SCEC, and 519 DEGs were obtained by microarray. These results are shown in FIGS. 16A-16D.

[0126] Human TMSC were able to reduce the cultured SCEC’s permeability, even without contact with each other. This uncovers one of the mechanisms that TMSC treatment can reduce IOP.Example 7

[0127] Glaucoma is a leading cause of irreversible blindness, with elevated intraocular pressure (IOP) as the most important risk factor. The trabecular meshwork (TM) and Schlemm’s canal are the main components of the proximal aqueous humor outflow pathway, and their dysfunction is a major contributor to IOP elevation. This study aims to identify and validate key biomarkers in TM and Schlemm’s canal endothelial (SCE) cells.

[0128] Methods: Microarray was performed on identified human TM and SCE cells to analyze their transcriptome profiling. Differentially expressed genes (DEGs) were identified and cross-referenced with published single-cell RNA sequencing (scRNA-Seq) datasets to ensure cell-specific relevance. Further validation was performed using qPCR. One-way ANOVA was used for statistical analysis and p<0.05 was considered significant. A diagram of this method is shown in FIG. 17.

[0129] Results: Cultured human TM cells and SCE cells between passages 3 and 5 underwent immunofluorescent staining and qPCR to confirm the distinct identities of TM and SCE cells. These results are shown in FIGS. 18A-18P. Microarray revealed 341 DEGs, with TM cells enriched in metabolic and signaling pathways, and SCE cells enriched in adhesion, immune, and morphogenesis-related processes. These results are shown in FIGS. 19A-19F. Cross-referencing with scRNA-Seq data refined the list of candidate biomarkers and qPCR confirmed the significant differences in gene expression between TM and SCE cells. CTTNBP2 and MGARP were highly expressed in TM cells. JAM2, PODXL, and IFI27 were significantly upregulated in SCE cells. These results are shown in FIGS. 20A-20H.

[0130] The validated biomarkers offer insights into glaucoma pathophysiology and lay the groundwork for targeted therapies.

[0131] The following patents, applications and publications as listed below and throughout this document are hereby incorporated by reference in their entirety herein.

Claims

CLAIMSWhat is claimed is:

1. A method of increasing wound healing, proliferation, and / or permeability of Schlemm’s canal endothelial cells (SCEC) in a subject in need thereof, the method comprising administering a therapeutically effective amount of trabecular meshwork cell (TMC) secretome and / or trabecular meshwork stem cell (TMSC) secretome to a Schlemm’s canal of the subject.

2. The method of claim 1 , wherein SCEC wound closure reaches about 80% or more following administration of the TMC secretome and / or TMSC secretome.

3. The method of any one of claims 1-2, wherein the method increases Ki-67 expression in the SCEC by about 1.5-fold or more following administration of the TMC secretome and / or TMSC secretome.

4. The method of any one of claims 1-3, wherein the method increases permeability of the SCEC by about 20% or more following administration of the TMC secretome and / or TMSC secretome.

5. A method of increasing ciliogenesis and / or intercellular pore formation in Schlemm’s canal endothelial cells (SCEC) in a subject in need thereof, the method comprising administering a therapeutically effective amount of trabecular meshwork cell (TMC) secretome and / or trabecular meshwork stem cell (TMSC) secretome to a Schlemm’s canal of the subject.

6. The method of claim 5, wherein the method increases the length of cilia on the SCEC by about 5% or more.

7. The method of any one of claims 5-6, wherein the method increases the prevalence of cilia on the SCEC by about 0.25% or more.

8. The method of any one of claims 5-7, wherein the method yields a density of intercellular pores between the SCEC of about 0.2 pores per cell or more.

9. A method of decreasing cellular stiffness and / or storage modulus of Schlemm’s canal endothelial cells (SCEC) in a subject in need thereof, the method comprising administering a therapeutically effective amount of trabecular meshwork cell (TMC) secretome and / or trabecular meshwork stem cell (TMSC) secretome to a Schlemm’s canal of the subject.

10. The method of claim 9, wherein the method decreases cellular stiffness of the SCEC by from about 40% to about 100% following administration of the TMC secretome and / or TMSC secretome.

11. The method of any one of claims 9-10, wherein the method decreases storage modulus of the SCEC by from about 40% to about 100% following administration of the TMC secretome and / or TMSC secretome.

12. The method of any one of claims 1-11, wherein the method comprises: a) determining if the subject has increased intraocular pressure (IOP), a thin retinal nerve fiber layer (RNFL), and / or an enlarged optic nerve cup to disc ratio; and b) administering a therapeutically effective amount of TMC secretome and / or TMSC secretome to the Schlemm’s canal of the subject.

13. The method of any one of claims 1-12, wherein the method results in reduced aqueous humor (AH) outflow resistance and / or reduced intraocular pressure (IOP).

14. The method of any one of claims 1-13, wherein the SCEC are glaucomatous and / or the subject has glaucoma.

15. The method of any one of claims 1-14, wherein the subject has a family history of glaucoma.

16. The method of any one of claims 1-15, wherein the subject has high interocular pressure (IOP).

17. The method of any one of claims 1-16, wherein the method comprises administering whole trabecular meshwork cells (TMCs) and / or whole trabecular meshwork stem cells (TMSCs) to the Schlemm’s canal of the subject; and wherein the TMCs and / or TMSCs secrete the therapeutically effective amount of the TMC secretome and / or TMSC secretome in vivo.

18. The method of any one of claims 1-16, wherein the TMC secretome and / or TMSC secretome is administered as a composition comprising from about 10% to about 100% total secretome.

19. The method of any one of claims 1-18, wherein the TMC secretome and / or TMSC secretome is administered intraocularly or via eye drops.

20. The method of any one of claims 1-19, wherein the TMC secretome and / or TMSC secretome is substantially free of cells and / or cellular debris.

21. A composition comprising trabecular mesh cell (TMC) secretome and trabecular mesh stem cell (TMSC) secretome.

22. The composition of claim 21, wherein the TMC secretome and the TMSC secretome are present in a ratio of from about 0.1:1 to about 10:1.

23. The composition of any one of claims 21-22, wherein the composition comprise from about 10% to about 100% total secretome.

Images