Patent Application
20230000760
This application contains a Sequence Listing, which has been submitted in XML format via EFS-Web, and is hereby incorporated by reference in its entirety. Said XML copy, created on Aug. 30, 2022, is named 072396_0936 SLST26.xml and is 8,661 bytes in size.
The present disclosure relates to methods of treating ocular disorders using a composition including a human trabecular meshwork stem cell (TMSC) secretome, wherein the composition can reduce or treat the impairment of ocular cells.
Glaucoma can be a cause of blindness, as there is irreversible damage to the optic nerve, which connects the eye to the brain, due to increased intraocular pressure (IOP) in the eye. This damage can result in permanent vision loss.
Increased IOP in glaucoma is due to the dysregulation of the major outflow pathway in the eye comprised of the trabecular meshwork (TM), causing accumulation of aqueous humor (AH) inside the eye. This accumulation causes an increased IOP, which can lead to damage of the optic nerve head and death of retinal ganglion cells (RGCs). IOP is mainly regulated by the TM and the Schlemm's canal endothelium by providing resistance to the aqueous humor outflow. Primary open-angle glaucoma (POAG), which accounts for up to 90% of all forms of glaucoma, is characterized by an elevation of IOP without pain. A certain range of IOP and anterior chamber angle can appear normal and remain asymptomatic until patients realize that there is a significant permanent loss of eyesight. There is a reduction of TM cells with age at the rate of 0.58% per year. There is a reduction in TM cellularity in glaucoma, and this loss can be aggravated by pathological conditions involving TM thickness, hyperplasia, abnormal ECM deposition, leading to increased outflow resistance.
Hypoxia is implicated in the death of RGCs in glaucoma through increased VEGF production, causing retinal edema, elevated reactive oxygen species (ROS), and increased accumulation of glutamate extracellularly, which causes excessive Ca+ influx and RGC death by ionotropic and metabotropic receptor activation and enhanced production of inflammatory cytokines.
Existing treatment options for glaucoma include eye drops (beta-blockers, alpha agonists, carbonic anhydrase inhibitors, rho kinase inhibitors, and prostaglandin analogs), laser procedures, and surgery. However, all such treatments have various limitations and side effects. Therefore, there is a need in the art for improved techniques to treat ocular disorders without significant adverse side effects.
The disclosed subject matter relates to compositions and methods for treating an ocular disorder. It is based, at least in part, on the discovery that the administration of the disclosed composition can treat and/or prevent glaucoma by reducing impairment of retinal ganglion cells (RGCs). For example, the impairment of RGCs in glaucoma can include cell apoptosis, axon loss, vision loss, along with abnormalities of trabecular meshwork which can cause increased intraocular pressure, dysregulation of aqueous humor outflow, and/or combinations thereof.
The disclosed subject matter provides a composition comprising an effective amount of a human trabecular meshwork stem cell (TMSC) secretome, wherein the effective amount can be present in an amount to reduce impairment of retinal ganglion cells (RGC).
In certain embodiments, the TMSC secretome can include proteins involved in neuroprotection. The neuroprotection is selected from the group consisting of axon guidance, neurogenesis, negative regulation of neuron death, clearance of neuron apoptotic bodies, and combinations thereof. In non-limiting embodiments, the TMSC secretome can be a cell-free secretome. In certain embodiments, the TMSC secretome can be harvested from TMSCs by incubating the TMSCs with serum-free media. In non-limiting embodiments, the TMSC secretome can be a concentrated formulation from TMSC. The TMSC secretome can be concentrated by a factor of about 25 after harvesting.
In certain embodiments, the disclosed composition can be formulated in a form. The form can be selected from the group consisting of a solution, a suspension, a semi-solid, gel, a gel, an emulsion, semi-liquid, an ointment, a cream, foam gel, a controlled-release/sustain-release vehicle, and combinations thereof. In non-limiting embodiments, the disclosed composition can be formulated as an eye drop. In non-limiting embodiments, the disclosed composition can be formulated in the form of a solution for injection into the subject. The injection can be selected from the group consisting of a systemic injection, an intravenous injection, an intramuscular injection, and combinations thereof.
The disclosed subject matter also provides a method for treating an ocular disorder of a subject. In certain embodiments, the method can include administering an effective amount of a TMSC secretome to reduce impairment of RGCs to a target tissue of the subject.
In non-limiting embodiments, the method can further include harvesting the TMSC secretome by incubating the TMSCs with serum-free media. In certain embodiments, the method can further include concentrating the TMSC secretome by a factor of about 25.
In certain embodiments, the effective amount of the TMSC secretome can be perioculary administered to the target tissue. The target tissue can be an eye tissue of the subject. In non-limiting embodiments, the effective amount of the TMSC secretome is administered in appropriate amounts divided into multiple portions.
In certain embodiments, the TMSC secretome can include proteins involved in neuroprotection. The neuroprotection is selected from the group consisting of axon guidance, neurogenesis, negative regulation of neuron death, clearance of neuron apoptotic bodies, and combinations thereof.
In certain embodiments, the target tissue can be an eye of the subject. In non-limiting embodiments, the impairment can be selected from the group consisting of cell apoptosis, axon loss, vision loss, increased intraocular pressure, dysregulation of aqueous humor outflow, and combinations thereof. In non-limiting embodiments, the ocular disorder can be glaucoma.
In certain embodiments, the composition can be formulated in a form. The form can be selected from the group consisting of a solution, a suspension, a semi-solid gel, a gel, an emulsion, semi-liquid, an ointment, a cream, foam gel, a controlled-release/sustain-release vehicle, and combinations thereof. In non-limiting embodiments, the effective amount of the TMSC secretome can be administered to the target tissue via an injection. The injection can be selected from the group consisting of a systemic injection, an intravenous injection, an intramuscular injection, and combinations thereof.
In certain embodiments, the effective amount of the TMSC secretome can be present in an amount to decrease the intraocular pressure of the subject by increasing an expression level of a neuroprotective factor. The neuroprotective factor can be selected from the group consisting of an axon guidance factor, a neurogenesis factor, a negative regulation of neuron death factor, a clearance of neuron apoptotic bodies factor, and combinations thereof. In non-limiting embodiments, the axon guidance factor can be selected from the group consisting of Tubulin beta-2A (TUBB2A), ACTR3, ARPC4, Semaphorin 5A (SEMA5A), and combinations thereof. In non-limiting embodiments, the neurogenesis factor can be selected from the group consisting of NEO1, SPTBN1, GAS6, SDC2, and combinations thereof. In non-limiting embodiments, the negative regulation of neuron death factor can be selected from the group consisting of PARK7, HYOU1, NONO, PIN1, and combinations thereof. In non-limiting embodiments, the clearance of neuron apoptotic bodies factor can be selected from the group consisting of NQO1, HSP90AB1, G6PD, UBE2V2, and combinations thereof.
In certain embodiments, the effective amount of the TMSC secretome can be present in an amount to reduce gliosis by promoting the regeneration of the RGC. In non-limiting embodiments, the effective amount of the TMSC secretome can be present in an amount to increase autophagy in the RGC and regenerate the RGC.
The disclosed subject matter provides compositions for treating ocular disorders. The compositions include an effective amount of a human trabecular meshwork stem cell (TMSC) secretome to reduce or treat the impairment of ocular cells. The disclosed subject matter further provides methods for ocular disorders. In certain embodiments, the disclosed compositions can be administered to the target tissue for treating ocular cell apoptosis, axon loss, vision loss, increased intraocular pressure, dysregulation of aqueous humor outflow, and/or combinations thereof.
As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. Abbreviations used herein have their conventional meaning within the chemical and biological arts.
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, a reference to “a compound” includes mixtures of compounds.
As used herein, the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
As used herein, the term “administering” can mean any suitable route, e.g., via topical administration, intraocular administration, or periocular administration without limitation to other routes of administration.
The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of” the embodiments or elements presented herein, whether explicitly set forth or not.
The term “effective amount,” as used herein, refers to that amount of active agent sufficient to treat, prevent, or manage a disease. Further, a therapeutically effective amount with respect to the second targeting probe of the disclosure can mean the amount of active agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of the disease, which can include a decrease in the severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The term can encompass an amount that improves overall therapy, reduces or avoids unwanted effects, or enhances the therapeutic efficacy of or synergies with another therapeutic agent.
The term “secretome,” as used herein, refers to an array of secretory cytokines, growth factors, small RNAs, non-coding RNAs, ECM mediators, and proteins secreted by a cell. For example, a human trabecular meshwork stem cell (TMSC) secretome can mean a set of proteins expressed by the TMSCs and secreted into the extracellular space.
A “subject” may be a human or a non-human animal, for example, but not by limitation, a non-human primate, a dog, a cat, a horse, a rodent, a cow, a goat, a rabbit, a mouse, etc.
The term “dosage” is intended to encompass a formulation expressed in terms of total amounts for a given timeframe, for example, as μg/kg/hr, μg/kg/day, mg/kg/day, or mg/kg/hr. The dosage is the amount of an ingredient administered in accordance with a particular dosage regimen. A “dose” is an amount of an agent administered to a mammal in a unit volume or mass, e.g., an absolute unit dose expressed in mg of the agent. The dose depends on the concentration of the agent in the formulation, e.g., in moles per liter (M), mass per volume (m/v), or mass per mass (m/m). The two terms are closely related, as a particular dosage results from the regimen of administration of a dose or doses of the formulation. The particular meaning in any case will be apparent from the context.
As used herein, “ocular disorder,” “ophthalmic disease,” “ophthalmic disorder,” and the like, includes, but is not limited to, glaucoma, cataracts, leucoma, or retinal degeneration in a subject in need of such treatment comprising administering, to the subject, an effective amount of a compound as set forth above.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. Ranges disclosed herein, for example, “between about X and about Y” are, unless specified otherwise, inclusive of range limits about X and about Y as well as X and Y. With respect to sub-ranges, “nested sub-ranges” that extend from either endpoint of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 can include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.
The terms “treat,” “treating” or “treatment,” and other grammatical equivalents as used herein, include alleviating, abating, ameliorating, or preventing a disease, condition or symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition. The terms further include achieving a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient can still be afflicted with the underlying disorder.
For clarity of presentation, and not by way of limitation, the detailed description of the disclosed subject matter is divided into the following subsections:
8.1 Compositions; and
8.2 Methods of Treatment.
The disclosed subject matter provides a composition for treating ocular disorders. In certain non-limited embodiments, the composition can include an effective amount of a secretome to treat ocular disorders. In certain embodiments, the secretome can be harvested from a human trabecular stem cell (TMSC). For example, TMSCs can be cultured as clonal culture and be used for harvesting secretome at passages 1-10. In non-limiting embodiments, the TMSCs can be used for harvesting secretomes at passages 4-7. In certain embodiments, TMSCs can be cultured with at least about 1 ml, at least about 3 ml, at least about 5 ml, at least about 10 ml, at least about 25 ml, at least about 50 ml, at least about 75 ml, or at least about 100 ml of a basal media for harvesting secretome. In non-limiting embodiments, the volume of the basal media can be from about 1 ml to about 10 ml, from about 1 ml to about 25 ml, from about 1 ml to about 50 ml, from about 1 ml to about 100 ml, from about 10 ml to about 50 ml, from about 10 ml to about 100 ml, or from about 50 ml to about 100 ml. In certain embodiments, the number of TMSCs cultured for harvesting secretome can be at least about 1×105, at least about 1×106, at least about 1×107, at least about 1×108, at least about 1×109, or at least about 1×1010. In non-limiting embodiments, the range of TMSC population can be from about 1×105 to about 1×106, from about 1×106 to about 1×107, from about 1×107 to about 1×108, from about 1×108 to about 1×109, or from about 1×109 to about 1×1010. In some embodiments, for secretome harvesting, TMSC cells can be cultured to the log phase and incubated with basal media without serum and growth factors at about 60% to about 70% confluence for a predetermined period. The predetermined period can be at least about 1 hour, at least about 3 hours, at least about 5 hours, at least about 10 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, or at least about 72 hours.
In non-limiting embodiments, the harvested secretome can be further concentrated. The harvested secretome can be concentrated up to about 2×, about 5×, about 10×, about 20×, about 25×, about 50×, about 75×, or about 100×. For example, the harvested secretome can be concentrated using a centrifugal filter to 25× and stored at about −80° C. until use to avoid any growth factor/protein degradation.
In certain embodiments, the disclosed secretome can be filtered to remove any cell debris. The cell-free secretome can avoid any cytotoxicity, immune responses, and/or side effects related to cell debris. In non-limiting embodiments, the disclosed secretome can include stem cell secretome for stem cell-free therapy.
In certain embodiments, the effective amount of the TMSC secretome can be at least about 1 μl of the concentrated TMSC secretome, at least about 5 μl of the concentrated TMSC secretome, at least about 10 μl of the concentrated TMSC secretome, at least about 15 μl of the concentrated TMSC secretome, at least about 20 μl of the concentrated TMSC secretome, at least about 30 μl of the concentrated TMSC secretome, at least about 50 μl of the concentrated TMSC secretome, at least about 100 μl of the concentrated TMSC secretome, at least about 250 μl of the concentrated TMSC secretome, at least about 500 μl of the concentrated TMSC secretome, or at least about 1000 μl of the concentrated TMSC secretome.
In certain embodiments, the disclosed composition can be formulated for topical administration or for injection into the eye (e.g., periocular injection) or a component of a visual apparatus (e.g., the eye itself, nerves innervating the eye, and associated musculature). Accordingly, the disclosed composition can be formulated in the form of a solution, a suspension, a gel, an emulsion, an ointment, a cream, or a controlled-release/sustain-release vehicle. In non-limiting embodiments, the disclosed composition can be formulated as an eye drop or eye ointment. It is desirable that the pharmaceutical composition is sterile or sterilizable.
In certain embodiments, the TMSC secretome can include a set of proteins that relate to axon guidance, neurogenesis, neuron death, neuron apoptosis, or combinations thereof. For example, the TMSC secretome can include proteins related to the axon guidance pathways, such as TUBB2A (Tubulin beta-2A), ACTR3, ARPC4, and Semaphorin 5A (SEMA5A). The TMSC secretome can also include proteins involved in neurogenesis (e.g., NEO1, SPTBN1, GAS6, and/or SDC2), proteins involved in negative regulation of neuron death (e.g., PARK7, HYOU1, NONO, and/or PIN1), proteins involved in the clearance of neuron apoptotic process (e.g., NQO1, HSP90AB1, G6PD, and/or UBE2V2). In some embodiments, the TMSC secretome can include proteins involved in positive regulation of neuron differentiation (e.g., CAPRIN1, CFL1, CRABP2, and CTSZ), positive regulation of neurogenesis (e.g., CRKL, CSF1, DPYSL3, and/or EIF4G1), neuron projection maintenance and development (e.g., APP, INS, MAP1A, ACTR2, ARHGDIA, CAPRIN1, and/or CFL1) and positive regulation of neuron projection development (e.g., DPYSL3, FN1, HGF, and/or ITGA3).
In certain embodiments, the disclosed TMSC secretome can include proteins related to neuroprotection and rescue RGCs from apoptosis. For example, the disclosed TMSC secretome can include a neuralized E3 ubiquitin-protein ligase 1 protein (NEURL1, formation of functional synapses), a neurofascin protein (neurite extension, axonal guidance, synaptogenesis, myelination, and neuron-glial cell interactions), and/or a neuroligin-1/3/4X protein (synapse function and synaptic signal transmission).
In certain embodiments, the disclosed composition can include an array of neuroprotective proteins, which can regulate different aspects of neurogenesis and provide neuroprotective effects on RGCs.
In certain embodiments, the disclosed composition can include an effective amount of the TMSC secretome, wherein the effective amount can be present in an amount to reduce impairment of RGCs, wherein the TMSC secretome can include proteins involved in neuroprotection, wherein the neuroprotection can be selected from the group consisting of axon guidance, neurogenesis, negative regulation of neuron death, neuron apoptosis, clearance of neuron apoptotic bodies, or combinations thereof, wherein the TMSC secretome can be a cell-free secretome, wherein the TMSC secretome can be harvested from TMSCs by incubating the TMSCs with serum-free media, wherein the TMSC secretome can be concentrated by a factor of about 25, wherein the impairment can be selected from the group consisting of cell apoptosis, axon loss, vision loss, increased intraocular pressure, dysregulation of aqueous humor outflow, and combinations thereof, wherein the ocular disorder can be glaucoma, wherein the composition can be formulated in a form, wherein the form can be selected from the group consisting of a solution, a suspension, a semi-solid gel, a gel, an emulsion, semi-liquid, an ointment, a cream, foam gel, a controlled-release/sustain-release vehicle, an eye drop, and combinations thereof, and wherein the composition is formulated in a form for injection into the subject, wherein the injection can be selected from the group consisting of a systemic injection, an intravenous injection, an intramuscular injection, and combinations thereof.
In certain embodiments, the disclosed subject matter provides a method of treating an ocular disorder comprising administering an effective amount of a TMSC secretome to reduce impairment of RGCs in a target tissue of the subject. In non-limiting embodiments, the target tissue can be an eye or a component of the visual apparatus (e.g., the eye itself, nerves innervating the eye, and associated musculature).
In some embodiments, the disclosed composition can be administered to the target tissue by any method known in the art, including, but not limited to, topical instillation, periocular injection, intravitreal injection, systemic administration, or the insertion of a reservoir that provides sustained release of the composition. In certain embodiments, the disclosed composition can be periocularly administered. For example, a composition including about 1 ul to about 1000 ul of the concentrated TMSC secretome can be perioculary injected into a target tissue. In certain embodiments, the volume of the concentrated TMSC secretome can be from about 1 μl to about 500 μl, from about 1 μl to about 400 μl, from about 1 μl to about 300 μl, from about 1 μl to about 200 μl, from about 1 μl to about 100 μl, from about 1 μl to about 50 μl, from about 1 μl to about 10 μl, or from about 1 μl to about 5 μl. In certain embodiments, the volume of the concentrated TMSC secretome can be from about 500 μl to about 1000 μl, from about 500 μl to about 900 μl, from about 500 μl to about 800 μl, from about 500 μl to about 700 μl, or from about 500 μl to about 600 μl. In certain embodiments, the volume of the concentrated TMSC secretome can be from about 100 μl to about 900 μl, from about 100 μl to about 800 μl, from about 100 μl to about 700 μl, from about 600 μl to about 500 μl, from about 100 μl to about 400 μl, from about 100 μl to about 300 μl, from about 100 μl to about 200 μl, or from about 100 μl to about 150 μl.
In one non-limiting embodiment, the disclosed composition can be administered to an affected eye topically, for example, as eye drops or as an ointment. For example, but not by way of limitation, the administration can be at least once a day, at least twice a day, at least once a week, at least twice a week, at least once a month, at least twice a month, at least six times a year, at least four times a year, at least twice a year or at least once a year, and/or up to twice a day, up to three times a day, up to once a week, up to twice a week, up to three times a month, up to six times a year, or up to four times a year. In a specific non-limiting embodiment, where the composition can be administered as eye drops, about 1 μl to about 1000 μl of the composition comprising the concentrated TMSC secretome can be administered to the affected eye at a time with the appropriate number of drops. The secretome proteins can cross the corneal barrier when they are administered in the form of eye drops.
In non-limiting embodiments, the disclosed subject matter can be used to treat the impairment of RGCs. In some embodiments, the impairment can include cell apoptosis, axon loss, vision loss, increased intraocular pressure, dysregulation of aqueous humor outflow, or combinations thereof. In particular non-limiting embodiments, the impairment can be cell apoptosis and increased intraocular pressure. The disclosed composition can also be used for any ocular disorders associated with the impairment of RGCs. Without being bound by any particular theory, it is believed that such impairment can affect and induce ocular disorders (e.g., glaucoma, cataracts, leucoma, and/or retinal degeneration).
Ocular disorders that can be treated according to the disclosed subject matter include, but are not limited to, glaucoma, cataracts, leucoma, and retinal degeneration. Retinal ganglion cell damage can be a contributor to a number of optic neuropathies (e.g., glaucoma), and it can be implicated in other diseases (e.g., multiple sclerosis). During development, RGCs can be produced in about a two-fold excess, but the insufficiency to form an efficient synaptic connection in the brain and lack of signal from neighboring retinal neurons can lead to the death of RGCs. During adulthood, pathological conditions can disrupt the synaptic connectivity or induce RGC death after optic nerve injury due to the deprivation of various factors (e.g., growth factor) or changes in electrical activity. Most optic neuropathies that involve damage to an optic nerve (e.g., traumatic optic neuropathies, ischaemic optic neuropathies, and glaucoma) can lead to RGC death by apoptosis. RGCs can be damaged in a variety of diseases that involve acute diseases (e.g., ischaemic optic neuropathy, optic neuritis, and glaucoma). For example, retinal ischemia, retinal artery, or vein occlusions can directly injure RGC cell bodies in the ganglion cell layer and can lead to vision loss. In certain embodiments, RGC viability (neuroprotection) and/or RGC function (neuroenhancement) can be enhanced by the disclosed subject matter. In non-limiting embodiments, the disclosed subject matter can be used to treat the impairment of retinal ganglion cells (RGCs). The impairment can be cell apoptosis, axon loss, vision loss, increased intraocular pressure, dysregulation of aqueous humor outflow, and/or combinations thereof. The disclosed composition can also be used for any ocular disorders associated with the impairment of RGCs. Without being bound by any particular theory, it is believed that such impairment can affect and induce ocular disorders (e.g., glaucoma, cataracts, leucoma, and/or retinal degeneration). The disclosed TMSC secretome can reduce the impairment of RGCs directly and/or indirectly.
In certain embodiments, administering the effective does of the disclosed composition can reduce the death of ocular cells and preserve cellular activities. In particular, the disclosed TMSC secretome can include proteins related to neuroprotection and rescue RGCs from apoptosis. In some embodiments, the disclosed TMSC secretome can protect RGCs from apoptosis through the neurotrophin and/or PI3-Akt signaling pathway. In certain embodiments, the disclosed TMSC secretome can prevent axon loss and vision loss. Since the RGC apoptosis leading to axon loss in the optic nerve can be the main reason for vision loss in glaucoma, the disclosed TMSC secretome can prevent/treat vision loss in genetic glaucoma by protecting RGCs from cell death. In non-limiting embodiments, the disclosed TMSC secretome can preserve RGCs' functions. Preservation of RGC functions can be important for treating primary open-angle glaucoma (POAG) and reducing IOP. In certain embodiments, administering the effective does of the disclosed composition can reduce IOP. For example, administering the disclosed composition can result in maintaining IOP at normal ranges without causing damages to the optic nerve.
In certain embodiments, the disclosed subject matter can treat ocular disorders by regenerating TM cells. In particular, the disclosed composition can preserve gene expression related to extracellular matrix (ECM) remodeling. For example, administering the disclosed composition can preserve the expression level of the CHI3L1 gene, which is a TM cell marker and involved in ECM remodeling. In non-limiting embodiments, the disclosed subject matter can also reduce mutations in genes, which can cause ocular disorders. For example, myocilin and ANGPTL7 are glaucoma associated genes, and mutations in these can be associated with glaucoma. The increased expression of Myoc and ANGPTL7 of glaucoma patients can be reduced by TMSC secretome, preventing steroid-induced glaucoma. In some embodiments, the disclosed composition can reduce the excess formation of fibrous tissues after a wound in ocular cells. For example, the disclosed subject matter can reduce fibrotic gene expression including a secreted protein acidic and cysteine-rich (SPARC) gene, fibronectin, and/or a connective tissue growth factor (CTGF) gene.
In certain embodiments, the disclosed subject matter can be used to treat ocular disorders by activating neuroprotective proteins, enzymes, or signaling pathways. For example, the expression level of TMEM177, COX2, PGE2, or combinations thereof can increase after administering the disclosed secretome of TMSC into the subject. TMEM177 can stabilize or increase the biogenesis of COX2. Increased TMEM177 after the secretome of the TMSC treatment can increase COX2 stabilization and biogenesis, leading to increased PGE2 expression that can reduce IOP. In non-limiting embodiments, the increased PGE2 can reverse glaucomatous changes and promote the self-renewal and proliferation of stem cells (e.g., ABCB5+ and OCT4+ endogenous stem cells). In non-limiting embodiments, the TMSC can be transplanted to increase the expression level of TMEM177, COX2, PGE2, or combinations thereof.
In certain embodiments, the disclosed subject matter can be used to treat gliosis. Gliosis can be caused by neurodegeneration of RGC (e.g., alteration of structure, function, and gene expression profile of glial cells). Administering the disclosed composition, including the effective amount of TMSC secretome, can reduce gliosis and further enhance regeneration in RGC. The proteins in the secretome can induce axon guidance pathway activation, neurogenesis, negative regulation of neuron death, and clearance of neuron apoptotic process leading to the enhanced RGC protection, survival, and function. In non-limiting embodiments, the TMSC secretome can increase autophagy to promote RGC survival. For example, the expression level of Beclin1 and Atg5, which controls autophagy, can be restored or increased.
In certain embodiments, the method of treating an ocular disorder can further include harvesting the TMSC secretome by incubating the TMSCs with serum-free media. For example, TMSC cells can be incubated with basal media without serum and growth factors at about 60-70% confluence for predetermined periods. The predetermined period can be at least about 1 hour, at least about 3 hours, at least about 5 hours, at least about 10 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, or at least about 72 hours.
In certain embodiments, the method of treating an ocular disorder can further include concentrating the TMSC secretome. For example, the harvested secretome can be filtered to remove any cell debris and further concentrated. The harvested secretome can be concentrated up to about 2×, about 5×, about 10×, about 20×, about 25×, about 50×, about 75×, or about 100×.
In certain embodiments, the effective amount of the TMSC secretome can be from about 0.1 mg/ml to about 1 mg/ml, from about 0.1 mg/ml to about 0.9 mg/ml, from about 0.1 mg/ml to about 0.8 mg/ml, from about 0.1 mg/ml to about 0.7 mg/ml, from about 0.1 mg/ml to about 0.6 mg/ml, from about 0.1 mg/ml to about 0.5 mg/ml, from about 0.1 mg/ml to about 0.4 mg/ml, from about 0.1 mg/ml to about 0.3 mg/ml, from about 0.1 mg/ml to about 0.2 mg/ml, from about 0.2 mg/ml to about 1 mg/ml, from about 0.2 mg/ml to about 0.9 mg/ml, from about 0.2 mg/ml to about 0.8 mg/ml, from about 0.2 mg/ml to about 0.7 mg/ml, from about 0.2 mg/ml to about 0.6 mg/ml, from about 0.2 mg/ml to about 0.5 mg/ml, from about 0.2 mg/ml to about 0.4 mg/ml, or from about 0.2 mg/ml to about 0.3 mg/ml.
In certain embodiments, the effective volume of the TMSC secretome can be from about 0.1 μl to about 1000 μl, from about 0.1 μl to about 900 μl, from about 0.1 μl to about 800 μl, from about 0.1 μl to about 700 μl, from about 0.1 μl to about 600 μl, from about 0.1 μl to about 500 μl, from about 0.1 μl to about 400 μl, from about 0.1 μl to about 300 μl, from about 0.1 μl to about 200 μl, from about 0.1 μl to about 100 μl, from about 0.1 μl to about 50 μl, from about 0.1 μl to about 10 μl, from about 0.5 μl to about 1000 μl, from about 0.5 μl to about 900 μl, from about 0.5 μl to about 800 μl, from about 0.5 μl to about 700 μl, from about 0.5 μl to about 600 μl, from about 0.5 μl to about 500 μl, from about 0. μl to about 400 μl, from about 0.5 μl to about 300 μl, from about 0.5 μl to about 200 μl, from about 0.5 μl to about 100 μl, from about 0.5 μl to about 50 μl, from about 0.5 μl to about 10 μl, from about 1 μl to about 1000 μl, from about 1 μl to about 900 μl, from about 1 μl to about 800 μl, from about 1 μl to about 700 μl, from about 1 μl to about 600 μl, from about 1 μl to about 500 μl, from about 1 μl to about 400 μl, from about 1 μl to about 300 μl, from about 1 μl to about 200 μl, from about 1 μl to about 100 μl, from about 1 μl to about 50 μl, or from about 1 μl to about 10 μl.
In certain embodiments, a treatment regimen using the disclosed composition can be combined with a regimen of treatment using other pharmaceutical agents.
In certain embodiments, the disclosed method can include harvesting the TMSC secretome by incubating the TMSCs with serum-free media, concentrating the TMSC secretome by a factor of about 25, and administering an effective amount of the TMSC secretome to reduce impairment of the RGC to a target tissue of the subject, wherein the effective amount of the TMSC secretome can be perioculary administered to the target tissue, wherein the effective amount of the TMSC secretome is administered in appropriate amounts divided into multiple portions, wherein the TMSC secretome can include proteins involved in neuroprotection, wherein the neuroprotection can be selected from the group consisting of axon guidance, neurogenesis, negative regulation of neuron death, neuron apoptosis, clearance of neuron apoptotic bodies, and combinations thereof, wherein the target tissue is an eye of the subject, wherein the impairment can be selected from the group consisting of cell apoptosis, axon loss, vision loss, increased intraocular pressure, dysregulation of aqueous humor outflow, and combinations thereof, wherein the ocular disorder can be glaucoma, wherein the composition can be formulated in a form, wherein the form can be selected from the group consisting of a solution, a suspension, a semi-solid gel, a gel, an emulsion, semi-liquid, an ointment, a cream, foam gel, a controlled-release/sustain-release vehicle, and combinations thereof, wherein the effective amount of the TMSC secretome can be administered to the target tissue via an injection, wherein the injection can be selected from the group consisting of a systemic injection, an intravenous injection, an intramuscular injection, and combinations thereof, wherein the effective amount of the TMSC secretome can be present in an amount to decrease intraocular pressure of the subject by increasing an expression level of a neuroprotective factor, wherein the neuroprotective factor can be selected from the group consisting of an axon guidance factor, a neurogenesis factor, a negative regulation of neuron death factor, a clearance of neuron apoptotic bodies factor, and combinations thereof, wherein the axon guidance factor can be selected from the group consisting of Tubulin beta-2A (TUBB2A), ACTR3, ARPC4, Semaphorin 5A (SEMA5A), and combinations thereof, wherein the neurogenesis factor can be selected from the group consisting of NEO1, SPTBN1, GAS6, SDC2, and combinations thereof, wherein the negative regulation of neuron death factor can be selected from the group consisting of PARK7, HYOU1, NONO, PIN1, and combinations thereof, wherein the clearance of neuron apoptotic bodies factor can be selected from the group consisting of NQO1, HSP90AB1, G6PD, UBE2V2, and combinations thereof, wherein the effective amount of the TMSC secretome can be present in an amount to reduce gliosis by promoting regeneration of RGC, wherein the effective amount of the TMSC secretome can be present in an amount to increase autophagy in the RGC and regenerate the RGC.
The following examples are merely illustrative of the presently disclosed subject matter, and they should not be considered as limiting the scope of the disclosed subject matter in any way.
The primary objective of this research study was to evaluate the effect of human trabecular meshwork stem cell (TMSC) secretome in steroid-induced and genetic mouse models of glaucoma. Four-months old wildtype (WT) C57BL/6J mice were purchased from Jackson Laboratory as wildtype control (WT). Adult C57BL/6J mice were periocularly injected with 20 μl of dexamethasone acetate (Dex-Ac) (DE122, Spectrum Chemicals) at 10 mg/ml once a week. In the genetic POAG model, transgenic mice with myocilin Y437H mutation (Tg-MyocY437H) were obtained from North Texas Eye Research Institute and bred with C57BL/6J WT mice and genotyped to confirm the myocilin mutation. Littermates were used as controls. Primary human TMSC, TM cells, and corneal fibroblasts were derived from deidentified corneas unsuitable for corneal transplantation obtained from the Center for Organ Recovery and Education (CORE, Pittsburgh, Pa.) or from the corneal rims after corneal transplantation. Proper informed consent for using the donated corneas for research purposes was obtained from all the donors by the CORE. Induced pluripotent stem cells (iPSC) cell lines were used and maintained in the laboratory. Blinded methods were used for study outcomes and designs. In particular, the histological evaluations of cell immunofluorescence for different antibodies and TM and retinal ganglion cell (RGC) counts were performed by two independent researchers blindly. Axon count in the optic nerve sections was performed by an expert in a blinded fashion. qPCR experiments were performed by another researcher in a blinded manner. The sample size was estimated based on the variability of different assays and potential outliers. The sample size for Dex-Ac and Tg-MyocY437H mice cohorts was estimated by power analysis before the initiation of the experiments. The number of samples (n) used for each experiment is specified in the captions for each figure.
TMSCs were cultured in Opti-MEM (Invitrogen, Carlsbad, Calif.), with supplements including 5% fetal bovine serum (ThermoFisher, Pittsburgh, Pa.), 0.08% chondroitin sulfate, 100 μg/ml bovine pituitary extract (Life Technologies, Carlsbad, Calif.), 20 μg/ml ascorbic acid, 10 ng/ml epidermal growth factor (Sigma-Aldrich, St. Louis, Mo.), and 200 μg/ml calcium chloride (Sigma-Aldrich), 50 mg/ml gentamicin, 100 mg/ml streptomycin, and 100 IU/ml penicillin (ThermoFisher). For stem cell characterization, TM cells were cultivated in DMEM: HAM's F12 (1:1) medium with 10% FBS and confirmed by responsiveness to 100 nM Dex. TMSC were used between passages 4-7. Secretome treatment was given 1) in cell culture together with Dex (referred as “parallel TMSC-Scr”) for five days (for preventive effect); 2) TM cells were treated with Dex for five days, and then secretome was supplemented in cell culture in the presence of Dex for another five days (referred as “post TMSC-Scr”) (for reverse effect). Human corneal fibroblasts were collagenase digested and cultured in DMEM/F12 with 10% FBS and used between passage 4-7.
Table 1 provides a listing of antibodies used in the disclosed subject matter. The acronyms provided in Table 1 are as follows: FITC is fluorescein isothiocyanate, PE is phycoerythrin, APC is allophycocyanin, HC is a heavy chain, LC is a light chain. IF is immunofluorescence. WB is western blotting. FC is flow cytometry.
RGC Induction from Human iPSCs
iPSCs reprogrammed from human dermal fibroblasts using four Yamanaka factors OCT4, KLF4, SOX2, and cMyc were cultured in mTeSR Plus media (StemCell Technologies) on Matrigel (Corning)-coated plates. These cells were induced to differentiate into RGC in Neurobasal: DMEM/F12 (1:1) medium containing Glutamax, N2 and B27 supplement (Invitrogen), and 25 μM forskolin (Stemcell Technologies) for 40 days. Differentiated RGC were characterized by immunofluorescent staining with RGC markers RBPMS and Thy1.1. After 40-day induction, cells were treated with 500 μM CoCl2 (Cobalt Chloride, Sigma-Aldrich) for 48 h to induce apoptosis or in the presence of a secretome to detect the protection effect.
Differentiated RGC treated with different conditions were dissociated using Accutase and stained with Annexin V and 7-AAD (BD Biosciences) as per manufacturer's instructions. 2×104 cells were acquired immediately on BD FACS Aria (BD Biosciences). Cells with no staining, containing Annexin V or 7-AAD alone, were taken as controls. RGCs were handled gently during the entire procedure to prevent any mechanical damage to the cells. For stem cell characterization, TMSC were blocked in 1% bovine serum albumin (BSA) for one hour and incubated with fluorochrome-conjugated antibodies for 30-min on ice in the dark. 5′104 cells were acquired per tube on BD FACS Aria (BD Biosciences). Compensations were adjusted using proper controls, and isotype controls were used to rule out spectral bleeding. Cell apoptosis was assessed by Annexin V/7-AAD staining. Data were analyzed using the FlowJo V10 software (FlowJo, Ashland, Oreg.).
Post secretome harvesting (˜90% confluence), TMSC and corneal fibroblasts were stained for 15 minutes in the dark with the viability dyes Hoechst 33342 (1:2000) and Calcein AM (1:1000) (Invitrogen). Live cells were captured at excitation filters of the wavelength of 361 nm and 565 nm, respectively, employing TE 200-E (Nikon Eclipse).
To assess the cell viability post secretome harvesting, 5×103 TM cells were cultured per well in 96-well plates in optimum culture conditions as described above. These cells were then incubated with secretome from three different TMSCs for 48 hours. MTT reagent (Millipore, Burlington, Mass.) was used to assess the formation of formazan crystals at the endpoint. The optical density was measured at ELISA reader (Tecan) using 570 nm wavelength and considering 600 nm as a reference to eliminate any background noise. Cells grown in optimum culture media without secretome were taken as control.
Whole mouse eyes were enucleated into freshly prepared 1% PFA in PBS and fixed for at least 48 h at 4° C. For plastic sectioning, eyes were embedded in glycol methacrylate A resin (JB-4, Polysciences) according to the manufacturer's instructions. Specifically, the eyes were placed in 70% ethanol and then dehydrated in increasing concentrations of ethanol to 100%. Fully dehydrated samples were infiltrated with resin monomer and its catalyst overnight. The next day, an accelerant was added, and the samples were embedded. Then 3-μm sections were made on a rotary microtome (RM2235, Leica). Slides were immediately stained for 1 minute in Mayer's hematoxylin solution (Electron Microscopy Sciences). Sections were photographed in DIC color mode using 40× oil objective (Olympus). For RGC counting, the retina was imaged at nasal and temporal sides, and three images were captured per eye. RGC counting was done in 3-4 eyes per group in both Dex-Ac and Tg-MyocY437H models using automated cell counter mode in ImageJ (NIH). Total counted RGC in a defined length of the retina were normalized to calculate cells/pm for each group.
For cryosections, fixed whole mouse eyes were embedded sagittally in OCT compound (Tissue Plus, FisherScientific) and flash-frozen in liquid nitrogen chilled isopentane. 10-μm sections through the center of the eye were made on a motorized Cryostat (CM3050 S, Leica).
This staining was used to assess autophagy. RGC were incubated with 0.1 μg/ml of acridine orange (Molecular probes) for 15 minutes. Cells were washed and maintained in PBS and photographed immediately using confocal microscopy at an excitation maximum of 502 nm and an emission maximum of 525 nm.
Cells were cultured on glass coverslips and fixed with 4% paraformaldehyde (PFA) (Electron Microscopy Sciences). Fixed cells were permeabilized with 0.1% Triton X-100 and blocked with 1% BSA. Samples were stained with appropriate primary antibodies at 4° C. overnight. After washing with PBS, cells were then stained with corresponding secondary antibodies conjugated with fluorochromes FITC, PR, and APC. Nuclei were stained with DAPI. Samples were examined under a confocal laser scanning microscope (Olympus).
Quantitative Real-Time PCR (qPCR).
Cells were lysed with RLT buffer, and total RNAs were isolated using an RNA purification kit (RNeasy Mini Kit, Qiagen, Hilden, Germany). cDNAs were transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif.). Primers were designed using Primer3 and blasted to confirm the specificity. The primer sequences were as follows: the sequences are: MYOC (forward: AAGCCCACCTACCCCTACAC (SEQ ID NO: 1); reverse: TCCAGTGGCCTAGGCAGTAT (SEQ ID NO: 2)), ANGPTL7 (forward: GCACCAAGGACAAGGACAAT (SEQ ID NO: 3); reverse: GATGCCATCCAGGTGCTTAT (SEQ ID NO: 4)). RNA content was normalized by 18S rRNA (Forward: CCCTGTAATTGGAATGAGTCCAC (SEQ ID NO: 5), Reverse: GCTGGAATTACCGCGGCT (SEQ ID NO: 6)). Relative mRNA abundance was calculated as the Ct for amplification of a gene-specific cDNA minus the average Ct for 18S expressed as a power of 2 (2ΔΔCt). Three individual gene-specific values, thus calculated, were averaged to obtain mean±SD.
Secretomes were harvested from both TMSC and corneal fibroblasts. 1×106 cells were cultured per T75 flask in the log phase and incubated with basal media without serum and growth factors at 60-70% confluence for 48 h. Then, the secretome was centrifuged at 3000 rpm for 5 minutes to remove any cell debris, filtered, and concentrated using 3 kDa centricon devices (Amicon) to 25× and immediately stored at −80° C. until use to avoid any growth factor/protein degradation.
For cell culture, 1× secretome was mixed with basal DMEM/F12 (1:1) in dexamethasone (Dex, 100nM/ml, Sigma Aldrich) treated TM cell or mixed with neurobasal medium (1:1) in CoCl2 treated RGC cultures. For animal experiments, 20 μl of 25× concentrated secretome was periocularly injected into each mouse eye. In the Dex-Ac model, both TMSC and fibroblast secretomes were injected at week 3, continued once a week until week-6, and animals were sacrificed at week-8. In the Tg-MyocY437H model, the secretome derived from human TMSC (TMSC-Scr) was injected at week 0 when mice were 4 months old, continued once a week until week-7, and animals were sacrificed at week-10. IOP was measured once a week.
Two TMSC strains from two different donors at passage 4-7 were used for secretome isolation and mouse injection. Fibro-Scr was used as a control in the Dex-Ac induced model. For the Dex-Ac model, mice were divided in four groups: Dex-Ac treated mice (n=16), vehicle-injected with vehicle solution used for solubilizing Dex-Ac (n=16), TMSC-Scr group (n=16), and Fibro secretome group (n=16). For the genetic model, mice were divided into five groups: littermate group (WT, n=16), Tg-MyocY437H mice (Tg, n=16), Tg-MyocY437H mice with a periocular injection of the basal medium (Tg-Sham, n=16), and Tg mice with periocular injection of TMSC-Scr (Tg-TMSC, n=16 each). For TMSC injections, Tg-MyocY437H mice were divided into four groups as described above (n=3-4 per group) and injected with 5×104 DiO-green labeled TMSC intracamerally. In brief, mice were anesthetized with ketamine-xylazine. 20 μl of 25× concentrated secretome or basal medium was injected periocularly using a 33-gauge needle connected to a Hamilton syringe. An I-care tonometer was used to measure mouse IOP (TonoLab; Colonial Medical Supply, Windham, N.H.). All IOP measurements were performed between 1 PM and 3 PM. IOP measurement before injection served as a baseline, and measurements were conducted once a week for 8-10 weeks.
PERG was performed on the Celeris (Diagnosys LLC, Lowell, Mass.) to evaluate the RGC function. Mice (n=9-15 for each group) were dark-adapted overnight and anesthetized with intraperitoneal injections of the mixture of Ketamine and Xylazine. Mouse pupil was dilated with 0.5% Tropicamide and 2.5% Phenylephrine eye drops. A circular electrode centered on the cornea was placed in a plane perpendicular to the visual axis after applying GenTeal lubricant to avoid corneal dryness and prevent cataract formation. Pattern stimuli consisted of horizontal bars of variable spatial frequencies and contrasts that alternate at different temporal frequencies. The parameters for PERG amplitude were spatial frequency 0.155 cycles/degree, temporal frequency 2.1 reversals/sec, contrast 100%, and substantial averaging (600-1800 sweeps). The Amplitude of P1 was used to analyze the function of RGCs.
Optic nerves were removed from enucleated eyes and immediately fixed in Karnovsky's fixative containing 2% paraformaldehyde and 2.5% glutaraldehyde. Optic nerves were cut into thick sections of 350 nm using a cryotome and stained with toluidine blue. Sectioned nerves were photographed using a phase-contrast microscope (Oplympus) at 20× and 60× using oil objectives. Three images were acquired for each optic nerve, and each group included 3-4 optic nerves. Axons in the optic nerve were counted using automated analysis in Metamorph. A 400×400 pixel box was made for uniform counting that can be moved to an area that is in good focus, then segmented image with threshold segmentation. IMA was used to measure small and large axons and log out summary measurements to an excel spreadsheet. For gliosis measurement, we employed semiautomatic quantification of total glial/scar areas and degenerative axonal profiles in plastic cross-sections using threshold contrast-based segmentation in addition to shape factor and elliptical form factor. For accurate RGC axon count, the following shape measurements were taken into consideration: Perimeter—distance around the edge of the object, measuring from the midpoints of each pixel that defines its border; Shape factor—4piA/P2-A value from 0-1 representing how closely the object represents a circle. A value near 0 indicates a flattened object, whereas a value of 1.0 indicates a perfect circle; Elliptical form factor—length/breadth-the ratio of an object's breadth to its length. Shape factor cut-off for gliosis quantification was set between 0.0-0.4, and elliptical for factor cut-off was set between 1.2-10000.
Mouse limbus tissue and aqueous humor were lysed using RIPA buffer (Santa Cruz Biotechnology). Limbus tissue was sonicated to fine pieces. For secretory Myoc, aqueous humor was extracted from the eyes. Protein concentration was measured using BCA Protein Assay Kit (Pierce Biotechnology). Protein samples were loaded for each group in each well and run on 8-16% sodium dodecyl sulfate-polyacrylamide gel (ThermoFisher) for electrophoresis and then transferred to the PVDF membrane. After blocking with blocking buffer, membranes were incubated with primary antibodies overnight at 4° C. Corresponding secondary antibodies were incubated (IRDye 680LT and IRDye 800CW, LI-COR Biosciences) after three washes of 0.1% Tween 20 in Tris-buffered saline. Detection and capture of the fluorescent signal were performed using an infrared imager (Odyssey; LI-COR Biosciences). Image J was used for the quantification and analysis of protein expression with b-actin as an internal control.
Secreted prostaglandin E2 (PGE2) was measured in aqueous humor samples using a PGE2 ELISA kit (Enzo life Sciences, ADI-901-001), and the procedure was performed according to the manufacturer's instructions. Aqueous humor was used at a dilution of 2:100 for the assay. Optical density was measured at 405 nm using 570 nm and 600 nm as normalizing wavelengths using an ELISA reader. Optical density measurements were extrapolated to measure PGE2 concentration (pg/ml) reference to standards.
TCA-precipitated proteins were urea-denatured, reduced, alkylated, and digested with endoproteinase Lys-C (Roche) followed by modified trypsin (Promega). Peptide mixtures were loaded onto 250 μm fused silica microcapillary columns packed with strong cation exchange resin (Luna, Phenomenex) and 5-μm C18 reverse-phase (Aqua, Phenomenex), and then connected to a 100 μm fused silica microcapillary column packed with 5-μm C18 reverse-phase (Aqua, Phenomenex). Loaded microcapillary columns were placed in-line with a Quaternary Agilent 1100 series HPLC pump and a LTQ orbitrap Elite mass spectrometer equipped with a nano-LC electrospray ionization source (ThermoScientific). Fully automated 10-step MudPIT runs were carried out on the electrosprayed peptides. Tandem mass (MS/MS) spectra were interpreted using ProluCID v. 1.3.3 against a database consisting of 79096 non-redundant human proteins (NCBI, 2016-06-10 release), 193 usual contaminants. To estimate false discovery rates (FDRs), the amino acid sequence of each non-redundant protein entry was randomized to generate a virtual library. This resulted in a total library of 158578 non-redundant sequences against which the spectra were matched. All cysteines were considered as fully carboxamidomethylated (+57.0215 Da statically added), while methionine oxidation was searched as a differential modification (+15.9949 Da). DTASelect v 1.9 and swallow v. 0.0.1, an in-house developed software, were used to filter ProLuCID search results at given FDRs at the spectrum, peptide, and protein levels. Here all controlled FDRs were less than 5%. All 4 data sets were contrasted against their merged data set, respectively, using Contrast v 1.9 and in house developed sandmartin v 0.0.1. Our in-house developed software, NSAF7 v 0.0.1, was used to generate spectral count-based label-free quantitation results.
DAVID software, a free online tool (version DAVID 6.8), and GOstats (version 2.48.0) were used for the functional enrichment analysis of genes whose corresponding proteins had positively distributed normalized spectral abundance factor (dNSAF) values in both replicates of a specific cell type. Biological process (BP), cellular component (CC), and molecular function (MF) were three different categories according to which the GO terms classification was performed. The top 10 most significantly enriched GO categories for a given cell type were compared between fibroblasts and TMSC cells.
The hierarchical clustering analysis and heatmap plotting was performed using the R package heatmap (version 1.0.12). Comparative expression of the dNSAF protein values between TMSC and fibroblast cells was performed using heatmaps. For each row of the heatmap, the name of the gene that encodes the protein was used as the heatmap row name. Similar elements were classified in groups in a binary tree using hierarchical clustering.
Results were expressed as mean±standard error mean (SEM) or mean±standard deviation (SD). The statistical differences were analyzed by two-way ANOVA or one-way ANOVA, followed by Tukey posttest using PRISM. P-value less than 0.05 (p<0.05) was considered statistically significant.
TMSC was cultured from human donor eyes, and each TMSC strain was characterized from different donors by flow cytometry showing positive expression of stem cell markers CD90, CD73, CD105, CD166, SSEA4, OCT4, ABCG2, STRO-1, NOTCH-1, CD271, and negative expression of CD34 and CD45 (
To induce an ocular hypertension model, we periocularly injected 200 μg/20 μl of Dex-Ac into adult C57BL/6J mice once a week for 6 weeks and sacrificed the animals at week-8. The mouse IOP started to elevate from week-1 after Dex-Ac injection and remained elevated up to week-8 after Dex-Ac injection had been stopped for two weeks (
COX2 (cyclooxygenase) is a major protein that helps the biogenesis of prostaglandin E2 (PGE2) from prostaglandin H2 (PGH2) in response to physiological demand. The presence of PGE2 in TM cells plays an important role in the maintenance of IOP in a normal range. Human TM cells secrete PGE2, which is abrogated by glucocorticoid treatment. A dramatic reduction of COX2 expression was detected in Dex-treated TM cells in vitro, which was restored after TMSC-Scr treatment (
To further confirm the TMSC-Scr therapeutic effects, we also investigated a genetic mouse model of glaucoma, the Tg-MyocY437H mice(18), which start to have elevated IOP at 3-4-month of age. 20 μl of 25× concentrated TMSC-Scr was periocularly injected, or 20 μl plain medium was periocularly injected as sham control into the Tg-MyocY437H mice at 4-month of age (week-0) once a week until week-7, and the mice were sacrificed at week-10 (
Preserving and restoring the RGC and their function is the ultimate goal of glaucoma treatment. Pattern electroretinography (PERG) is an optimal approach to evaluate RGC function. PERG shows that TMSC-Scr treatment preserved the RGC function of Tg-MyocY437H mice as indicated by increased amplitude of P1 wave (7.74±1.75 μV), similar to that of WT (7.76±1.14 μV), while the sham treatment could not recover P1 amplitude (4.05±1.95 μV), similar to that of untreated Tg-MyocY437H (5.86±2.24 μV) (
To explore the potential of RGC regeneration in a more controlled system, human iPSCs were differentiated to RGC, which expressed RGC markers RNA-binding protein with multiple splicing (RBPMS) and Thy1.1 with extensive elongated axons (
The label-free proteomic identification and quantification of secretome proteins from two strains of human TMSC from different donors were performed and compared with the secretomes from two fibroblast strains. Total proteins expressed in secretomes of both TMSC strains and fibroblast strains were identified, and significant gene ontology (GO) terms uncovered in the proteomes of both TMSC and fibroblast secretomes were identified. Total GO terms discovered in the secretomes of TMSC and fibroblasts were also identified. 841 and 1196 total number of proteins were identified from the TMSC1 and TMSC2 secretomes, and 415 and 291 proteins in the secretomes of fibroblast1 and fibroblast2. Out of these, 549 proteins were commonly secreted by both TMSC strains, while 212 proteins were in both fibroblast strains with positive distributed normalized spectral abundance factor (dNSAF) average values. 165 proteins were expressed in both TMSC and fibroblast secretomes, and both cell types showed different expressions for these proteins (
STRING analysis of TMSC-Scr showed interaction patterns between proteins involved in response to hypoxia, wound healing, cell-matrix adhesion, and detoxification (
These results show that the TMSC-Scr is rich in the factors important for neuronal development and protection as well as cell survival, which can explain its protective effect on RGC and TM cells. A detailed summary of the therapeutic effect induced by TMSC-Scr on various aspects of glaucoma involving different pathways is shown in
The therapeutic potential of the secretome from human TMSC was explored, and potential mechanisms were discovered. TMSC-Scr was able to prevent as well as reverse Dex-induced TM cell changes in culture. By minimal invasive periocular injection, TMSC-Scr reduced the IOP, increased the TM cellularity, remodeled the ECM of the TM, activated the endogenous stem cells, promoted cell proliferation, and prevented and reversed RGC loss as well as preserved the RGC function in both steroid-induced and genetic myocilin mutant mouse models. The rejuvenating and therapeutic effects of TMSC-Scr on glaucoma are associated with the activation of COX2-PGE2 signaling as well as their neuroprotective proteins. Our novel discovery indicates the feasibility of stem cell-free therapies for glaucoma in preserving both the TM function and RGC function. Steroid-induced ocular hypertension is a common side-effect among patients using steroid therapy. Myocilin mutations have been reported to be the most common form of genetic glaucoma. The results show that Dex treatment leads to increased fibrotic ECM proteins, such as fibronectin as well as myocilin in the TM, resulting in protein misfolding, ER stress, and IOP elevation. A transgenic mouse model with myocilin Y437H mutation is characterized by increased IOP, persistent TM ER stress, and RGC loss and axonal degeneration, which resembles POAG in patients. All these characteristics of the Tg-MyocY437H mice were identified through the disclosed experiments. These two mouse models represent typical steroid-induced and genetic glaucoma. TM cell loss is associated with elevated IOP in glaucoma, and reducing IOP is the only effective treatment so far. TMSC-Scr treatment led to reduced IOP in both mouse models of glaucoma, which started as early as the following week of secretome periocular injection and effectively maintained at normal range two to three weeks after secretome withdrawal when experiments terminated.
CHI3L1 and AQP1 are involved in the TM cell function of remodeling ECM, maintaining TM homeostasis, and modulate aqueous outflow. After Dex-Ac treatment, TM cells lose these proteins and become stiffer. Increased CHI3L1 and AQP1 and reduced glaucoma-associated genes MYOCILIN and ANGPTL7 after TMSC-Scr treatment in Dex-treated TM cells reflect the rejuvenating ability of TMSC-Scr on TM cells. Postoperative fibrosis is a major complication in glaucoma, and fibronectin is the main fibrotic protein. TMSC-Scr treatment in Dex-treated cells shows TMSC-Scr mediated fibrosis reduction. Additionally, secretome treatment increased the number of TM cells reflecting its ability to regenerate TM tissue in vivo. Collagen forms a major connective tissue protein in ECM, and its degradation by matrix metalloproteinases (MMPs) is crucial for remodeling and repairing of tissue. TMSC-Scr treatment dramatically downregulated the fibrotic ECM components fibronectin and collagen IV, which can help TM cell survival. The TMSC-Scr proteins involved in ECM organization and collagen catabolic process can lead to enhanced ECM turnover and lower IOP. ER stress of the TM cells is involved in both glaucoma models. To counteract protein misfolding, cells activate the UPR pathway. ER chaperones GRP78 and CHOP are activated by UPR, resulting in the restoration of ER homeostasis by proteasome-mediated induction of ER-associated degradation. Here, the presence of UPR in TMSC-Scr can further induce the cellular responsiveness of TM cells to reduce ER stress.
Prostaglandin analogs are commonly used for glaucoma treatment to reduce IOP. COX2, a rate-limiting enzyme, is known to convert arachidonic acid to PGH2, which is isomerized to PGE2 by PGE2 synthase. COX2 modulates PGE2 synthesis in response to growth factors, inflammatory cytokines, and other physiological demands. Under normal conditions, COX2 is restricted mostly in the kidney. COX2 expression can be increased substantially in other tissues in response to proinflammatory cytokines or sheer stress. COX2 expression is completely lost in the non-pigmented secretory epithelium of the ciliary body and aqueous humor of end-stage POAG human eyes. Glucocorticoids are well known for inhibiting COX2 activity. Human TM cells can secrete PGE2, which was inhibited significantly after a moderate Dex treatment. Mitochondrial TMEM177 has also been reported to associate with COX2 to stabilize/increase the biogenesis of COX2. Hence, increased TMEM177 after TMSC-Scr treatment can be responsible for increased COX2 stabilization and biogenesis, which further indicates its therapeutic potential for glaucoma. Further evaluation of increased TMEM177/COX2 expression in Tg-MyocY437H mice after TMSC transplantation confirmed that both stem cell-based and cell-free therapy involves upregulation of COX2 to impart a therapeutic benefit.
Mobilization and maintenance of endogenous stem cells are very crucial for inducing tissue regeneration. Endogenous stem cell regeneration involves a complex interplay of cues in terms of growth factors, modulation in stem cell niche, and chemokines inducing differentiation, proliferation, and migration of these cells. Glaucoma and ER stress can decrease endogenous stem cells and increase apoptosis. PGE2 has been reported to increase the self-renewal and proliferation of stem cells. Increased PGE2 secretion in the aqueous humor of both mouse models after TMSC-Scr treatment can be crucial for maintaining the ABCB5+ and OCT4+ endogenous stem cells and reversing glaucomatous changes. The stem cell proliferation and renewal are further enhanced by TMSC-Scr by the presence of crucial proteins involved in promoting cell proliferation and stemness in progenitor cells. Hypoxia can lead to RGC death by inducing a number of degenerative changes. The increased secretion of response to hypoxia proteins in TMSC-Scr can be responsible for RGC survival/rescue in hypoxic conditions. The optic nerve is a tract of the central nervous system and is comprised of axons of RGC with various glial cells like oligodendrocytes, astrocytes, microglia as support cells. Neurodegeneration of RGC results in alteration of structure, function, and gene expression profile of glial cells, termed as gliosis. Gliosis is associated with neurodegeneration in chronic and age-related models of glaucoma. Reduced gliosis after TMSC-Scr treatment can further enhance regeneration in RGC. The proteins uncovered in TMSC-Scr involving proteins related to axon guidance pathway, neurogenesis, negative regulation of neuron death, and clearance of neuron apoptotic process can directly enhance RGC protection, survival, and function by activation of some intrinsic developmental pathways, which can result in RGC regeneration, which will be interesting for future investigations. Furthermore, therapeutic effects of TMSC-Scr given through the periocular route emphasize that secretome proteins can cross the corneal barrier and can be a good approach for the development of glaucoma eye drops. Autophagy has been reported to promote RGC survival following optic nerve axotomy. TMSC-Scr was able to restore the levels of Beclin1 and Atg5 indicating that TMSC-Scr induced increased autophagy is mediated by these proteins.
Stem cell secretome therapy, with low risk, minimal invasive administration, and effectiveness, is an attractive treatment strategy for glaucoma, which can be soon for clinical trials after confirmation in animals more relevant to humans like non-human primates. The key secretome proteins that have been identified in the current experiments can potentially lead to the designing of small molecule-based therapeutics for glaucoma in the future.
All patents, patent applications, publications, product descriptions, and protocols cited in this specification are hereby incorporated by reference in their entireties. In case of a conflict in terminology, the present disclosure controls.
While it will become apparent that the subject matter herein described is well calculated to achieve the benefits and advantages set forth above, the presently disclosed subject matter is not to be limited in scope by the specific embodiments described herein. It will be appreciated that the disclosed subject matter is susceptible to modification, variation, and change without departing from the spirit thereof. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.
This application is a continuation of International Application No. PCT/US2021/021416, filed on Mar. 8, 2021, which claims priority to U.S. Provisional Application Ser. No. 62/986,409, filed Mar. 6, 2020, the contents of which are hereby incorporated by reference in their entireties.
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.
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| 62986409 | Mar 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2021/021416 | Mar 2021 | US |
| Child | 17902050 | US |
