Trabecular meshwork cells are a valuable resource for cellular therapy of glaucoma

Abstract Trabecular meshwork (TM) contains a subset of adult stem cells or progenitors that can be differentiated into corneal endothelial cells, adipocytes and chondrocytes, but not osteocytes or keratocytes. Accordingly, these progenitors can be utilized as a cell‐based therapy to prevent blindness caused by glaucoma, corneal endothelial dysfunction and other diseases in general. In this review, we review in vitro expansion techniques for TM progenitors, discuss their phenotypic properties, and highlight their potential clinical applications in various ophthalmic diseases.

glaucoma can be associated with trauma, inflammation, tumour or other medical conditions. Elevated intraocular pressure (IOP) is one of leading risk factors of glaucoma. 9 Experimental animal models support the notion that increased IOP may lead to optic nerve damage, similar to that of glaucoma. 10 Current therapies to control IOP include pharmacologic agents for reduction in aqueous humour and surgical methods to increase out-flow. These therapies are relatively effective. However, they have significant side effects, for example, toxicity, complications and other medical conditions. Despite tremendous effort by research scientists in this field, an effective cure has not been discovered as a result of the lack of understanding of the etiology of glaucoma at the molecular and cellular levels.

| CHANG E S OF TR AB ECUL AR ME S HWORK CELL S IN G L AUCOMA
The main out-flow path of aqueous humour in eyes includes endothelial cell-lined channels in the anterior chamber consisting of the trabecular meshwork (TM), the collector channels, Schlemm's canal and the episcleral venous system. The TM is believed to be most resistant to aqueous out-flow. TM cells have two major functions: assisting maintenance of aqueous out-flow across the trabecular lamellae 12 and secretion of extracellular matrix, specific enzymes and phagocytosis of excreted debris from the aqueous humour. 11 Cellular dysfunction in TM is associated with aging, elevated out-flow resistance and IOP, 13,14 suggesting that TM cells may play a critical role in maintaining normal IOP and thus preventing glaucoma. Therefore, cell-based functional restoration of TM in eyes with glaucoma is a potential effective therapy not yet investigated because of lack of methods to sustain TM phenotype in vitro. Several studies have reported that the cells in TM retain properties of adult stem cells. 14 However, characterization of the presumptive TM stem cells (TMSCs) is incomplete. In addition, it is unclear whether TMSCs can be expanded without loss of their phenotype, and if so, whether such expanded TMSCs can be used for regenerative therapy of glaucoma is unclear. Interestingly in glaucoma patients, the expression of TGFβ2 is abnormally higher than that from normal people, suggesting TGFβ signalling may play an important role in the progression of glaucoma.

| PATHOLOGY OF TM CELL S IN G L AUCOMA
Pathological changes of TM cells in glaucoma are best described in Sihota et al. 15 In acute primary angle-closure glaucoma (PACG) eyes, widened spaces can be seen among the trabecular beams, with significantly increased accumulation of pigment granules under the microscopy. Phenotypically, trabecular endothelial cells are long, tapering, attenuated without regular cellular components. Elastic van Gieson staining shows a parallel distribution of the collagen and elastic components of the TM.
In chronic PACG eyes, significant alteration of the trabecular sheets with irregular trabecular spaces are observed. Endothelial cells can be observed with more immature collagen. In addition, melanin pigments are in the stroma compartment of fused trabecular beams. Under electron microscopy, the trabecular meshwork has many electron dense bodies with fibrillar-structured. Certain cells also contain ill-defined vesicles without intracytoplasmic organelles, characteristic of degenerative changes.

| TGFβ
The transforming growth factorβ (TGFβ) superfamily is a group of structurally related multifunctional regulatory proteins. The members of this superfamily include TGF-β1-3, BMPs and other structurally related signalling molecules. 14 These proteins share six conserved cysteine residues required to form covalently linked dimers, which can then interact with their respective receptors. 14 Intracellular TGFβ signalling begins with ligand binding and subsequent activation of the TGFβ type I receptor via phosphorylation, which leads to bridging of TGFβ type I and II receptors on the cell membrane.
This activates intracellular proteins called SMADs, which form an oligomeric complex with co-SMAD, also an intracellular protein, to mediate the transcription of target genes and cause downstream signalling of TGFβ. 16,17 The cellular effects of TGFβ molecules depend on the specific TGFβ isoform, its concentration and the target tissue. 18 In normal physiology, cell processes influenced by TGFβ are proliferation, recognition, differentiation and apoptosis. 17

| TGFβ in aqueous humour
Numerous growth factors and cytokines are found within the aqueous humour (AH). In 1990, Granstein et al concluded that levels of TGFβ in the AH were sufficient to serve a purpose in normal ocular physiology. 19 Significant levels of TGFβ in the AH of normal eyes have also been repeatedly demonstrated in animal studies. 19,20 It has been shown that TGF-β2 is produced locally by ocular tissues and there is no correlation between AH and serum TGF-β2 concentrations. 20

| TGFβ in different types of glaucoma
To compare the various types of glaucoma, Inatani et al 23 showed that the level of biologically active TGF-β2, but not total TGF-β2, is higher in the AH of eyes with primary open angle glaucoma (POAG) than that with primary angle closure glaucoma, pseudoexfoliative glaucoma (XFG) and uveitis-associated secondary glaucoma. Other studies have measured multiple isoforms of TGFβ, showing that only TGF-β2 is significantly elevated in POAG compared with nonglaucomatous eyes, but that TGF-β1 and TGF-β3 are elevated in other forms of glaucoma. 24

| TGFβ and IOP
Ex vivo and in vitro studies indicate that TGFβ is involved in the pathogenesis of ocular hypertension. In an animal anterior eye segment perfusion culture, it was demonstrated that TGF-β2 infusion for 14 days significantly increased IOP. 25 Furthermore, TGF-β2 antagonized the IOP-lowering effect of interleukin (IL)-1 in another perfusion model. 26 Shepard et al 27 observed an increased IOP and reduced AH out-flow in rodents following adenoviral gene transfer of active human TGF-β2, as well as the elevation of TM expression of TGF-β2, 28 which led to anatomical changes in the anterior segment that resulted in increased IOP. These studies indicate that TGFβ is able to cause elevated IOP and is not merely an effect of elevated IOP. The TM gene expression profile also undergoes changes with IOP elevation. 29 Vittitow and Borras 29 revealed that in eyes with elevated IOP, genes are up-regulated and one-third of them are involved in signal transduction pathways. Notably, expression of matrix GLA protein (MGP) was down-regulated by TGF-β1. It was suggested that MGP contributes to TM tissue softening and plays a key role in maintaining pressure-induced ocular homeostasis. 29 This down-regulation of MGP is another way TGFβ may impact the extracellular cellular matrix (ECM) environment and promote conditions that elevate the IOP. Therefore, it can be assumed that patients with significantly elevated levels of TGFβ in the AH are at an increased risk to develop elevated IOP, putting them at risk of optic nerve damage and vision loss.

| TGFβ, the number and phenotype of TM cells
Several growth factor receptor mRNAs, including those for TGFβ isoforms, have been detected in cultured human TM cells and ex vivo human TM tissue from healthy and glaucomatous eyes. 30 These receptors are densely dispersed in the TM. Cultured porcine TM cells express TGFβ receptors with an estimated density of 4000 per cell. 31 These studies suggest that the human eye, in particular the TM, is sensitive to TGFβ, further implying that the elevated levels of TGFβ seen in the AH of patients suffering from glaucoma have its biological actions in the TM. TGF-β2 decreases the TM cell population by inhibiting cell proliferation, 30 inducing dose-dependent TM cell apoptosis 32 and phagocytosis of TM cells. 33 TGF-β2 has also been shown to induce a secretory phenotype in cultured human TM cells by promoting collagen synthesis. 34 This synthesis of collagen increases the ECM and may lead to TM obstruction and decreased out-flow facility.
TGF-β1 induces a myofibroblast-like phenotype in human TM cells, as reflected by a dose-dependent increase in the expression and production of α-smooth muscle actin (αSMA) in vitro. 35 αSMA-positive human TM cells are spindle shaped and contain stress fibres, signifying an increase in contractility and decrease in out-flow facility. 35 Altered actin cytoskeletal fibres have also been shown to play a crucial role in pathogenesis of POAG and steroidinduced glaucoma. 36 Contrary to the previous findings, Robertson et al 28 found that adenoviral transfer of active TGF-β1 led to decreased αSMA. It is important to note, however, that this was TGF-β1 isomer, and that the anatomic changes seen were more consistent with primary angle closure glaucoma as opposed to POAG. 28 This may be one of the differences between the effects of TGF-β1 and TGF-β2 on the eye, and a reason that elevated TGF-β2 is seen in POAG and TGF-β1 seen more commonly in other forms of glaucoma. with controls, which likely correlates with increased IOPs in vivo. 37 TGF-β1 in vitro has been shown to increase human TM cell expression of connective tissue growth factor 38 and elastin which could potentially contribute to out-flow resistance. 39 The TGF-β2 isomer induces expression of connective tissue growth factor, thrombospondin-1, fibronectin, collagen types IV and VI and plasminogen activator inhibitor-1 (PAI-1) from cultured human TM cells and bovine TM cells. 40 TGF-β2 also inhibits hyaluronic acid expression, most notably in the juxtacanalicular tissue. 40 In an ex vivo model of human eye anterior segment, TGF-β2-induced IOP elevation was associated with increased secretion of fibronectin and PAI-1, which was blocked by TGFβ type I receptor inhibitors in vitro TM culture. 41 PAI-1 is an inhibitor of plasminogen required for the activation of matrix metalloproteinases (MMPs). Therefore, increased PAI-1 leads to decreased activation of MMPs and may contribute to the increase in ECM of TM in glaucomatous eyes. 42 It is also well documented that in glaucoma, MMPs are altered, as are their inhibitory enzymes, TIMPs. 43 Therefore, the dysregulation of MMPs by increased levels of TGF-β2 may contribute to the decreased degradation of ECM and abnormal organization commonly seen in the TM of patients with glaucoma.

| TGFβ and TM contraction
Contraction and relaxation of the TM cells is believed to regulate out-flow of the AH, and thus to control IOP. It is believed that TGF-β1 may affect the contraction of TM cells, providing another mechanism by which TGFβ affects the IOP. In an in vitro culture of bovine TM cells in collagen gel, application of TGF-β1 caused a dosedependent contraction of the collagen gel. 44 Formation of actin stress fibres in TM cells triggered by TGF-β1 is mediated by protein kinase C and Rho GTPase. 44 Stress fibres are highly bundled actin filaments, which generate contractile force in the cell by connecting the cytoskeleton to the ECM. In vitro, fibronectin enhances this TM cell-mediated collagen contraction by promoting the expression of integrin α5, thus facilitating the connection of TM cells to the surrounding matrix and the formation of stress fibres. 45

| Interaction of TGFβ with TM cells
TGFβ is involved in the pathogenesis of glaucoma. 41

| PROG ENITOR CELL S IN TR AB ECUL AR ME S HWORK
Somatic stem cells are critical for maintenance and repair of various tissues. 50 Adult progenitor/stem cells are present in adult bone marrow, brain, heart, skeletal muscle, limbus 52 and trabecular meshwork. 53 These cells are crucial for tissue renewal and can be expanded in vitro for tissue regeneration in vivo. 51 Present evidence reveals that there is a population of progenitors in Schwalbe's Ring, the transitional area between the periphery of corneal endothelium (CE) and the anterior non-filtering portion of the TM. 54 This hypothesis primarily comes from the observation of an increase in TM cell division localized to the anterior non-filtering portion of the TM after argon laser trabecuoplasty (ALT). 55 A study has supported the notion, showing positive BrdU labelling, a marker for cell division, in the TM and posterior limbus. 56 In order to isolate TM cell, collagenase digestion is often used, which does not disrupt cell-cell junctions and thus retains the cell phenotype (Table 1).  56,58 A report suggested that progenitors from human TM expanded in vitro showed their ability to differentiate into TM cells in vivo in mice. In this study, DiO-labelled TMSCs injected into the anterior chamber of normal mice were localized primarily in TM, remaining in the tissue at least 4 months. In 1 week, TMSCs expressed TM marker CHI3L1. Almost no apoptosis could be seen in injected TM tissue and IOP was not increased in the experiment. 60 In another study, Nadri et al have isolated a group of mesenchymal stem cells from human TM, which can be differentiated into photoreceptor-like cells on amniotic membrane. 59 However, it is unclear how the trabecular meshwork progenitors can be maintained and expanded in vitro without change in their phenotype. In addition, it is also unclear whether TM progenitors can differentiate into corneal endothelium (CE), fat and cartilage, as we know they may have or may not have a similar origin and a close anatomical position. Thus, the potential to repair or replace the human corneal TM cells, to generate endothelial or other tissues by TM progenitors is also an important area that needs to be explored.

| 2-D MATRI G EL A S THE CULTURE SUBS TR ATE FOR TM CELL S
According to Table 1

| TM progenitors
One interesting report 62

| CURRENT S TATUS OF CELL THER APY IN ANIMAL OR CLINI C AL S TUD IE S US ING TM CELL S
Only until recently, two significant mouse models have been established for studies of TM cell therapies. 83

| CON CLUS ION
Trabecular meshwork cells contain adult stem cells which can be expanded in vitro without differentiation and differentiated into corneal endothelial cells, chondrocytes and adipocytes, but not keratocytes or osteocytes. Thus, these stem cells can be potentially deployed for cell-based therapy for glaucoma, corneal endothelial dysfunction and other ophthalmic diseases in general. Therefore, it is pivotal for us to continue the studies of defining TM stem cell properties and transitioning these results to clinical applications.

ACK N OWLED G EM ENTS
This work was supported by Grant 2015A030313774, Natural Science Fund of Guangdong Province, China.

CO N FLI C T O F I NTE R E S T
No conflict of interest is declared.