members of the microRNA-200 family (miR-200a, miR-. 200b, miR-200c, miR-141 and miR-429) and miR-205 were markedly downregulated in epithelial cells ...
CHAPTER
Driss Zoukhri Helen P. Makarenkova
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Role of Stem Cells in Lacrimal and Meibomian Gland Development and Regeneration
INTRODUCTION The non-keratinized epithelia of the ocular surface are constantly challenged by environmental insults such as smoke, dust, or airborne pathogens. Protection of eye surface is mediated, in part by the secretions of two major glands, the lacrimal (LG) and meibomian (MG). In humans, the LG is the primary contributor to the aqueous layer of the tear film. The meibomian gland (MG) is responsible for the production of meibum, an oily secretion that retards evaporation of the preocular tear film. When LG and MG secretions decrease, or the composition of their secretions change, it affects the health of the ocular surface. The LG and MG are dynamic tissues that are capable of responding to physiological stimuli (i.e., environmental insults or severe injury) by mounting a well-orchestrated regenerative response that restores their cytoarchitecture. However, the mechanisms of LG and MG regeneration and the source of progenitor cells are still not very well defined. Here, we provide a timely review of LG and MG development, maintenance, and repair with the focus on emerging areas in stem cell research.
DISORDERS OF THE TEAR FILM DUE TO LACRIMAL AND MEIBOMIAN GLAND DISEASES Tears are the sole physical protective barrier for the ocular surface. The preocular tear film can be divided into three interacting layers: 1) an inner mucous layer that coats the cornea and conjunctiva; 2) an aqueous middle layer; and 3) an outer lipid layer which floats on the aqueous layer. Each layer of the tear film is secreted by a different set of glands.1-3 The mucous layer is secreted by the conjunctival goblet cells,4 the lipid layer is secreted by the MG that line the eyelid,1 and the aqueous layer is secreted by the main and accessory LGs.2 Production of tears in insufficient quantity or of inadequate quality results in constant irritation of the ocular surface leading to dry eye disease also referred to as keratoconjunctivitis sicca (KCS).5 Dry eye disease secondary to LG deficiency is known as aqueousdeficient type of dry eye.5 The second major class of dry eye is called evaporative dry eye and is mainly due to MG deficiency.5,6
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STEM CELLS IN OPHTHALMOLOGY Patients with KCS can experience intense pain due to eye irritation, gritty/scratchy feeling in the eyes, blurry vision and light sensitivity.5 LG dysfunction that occurs as a result of autoimmunity, such as Sjögren’s syndrome or rheumatoid arthritis, accounts for an estimated 1-4 million patients in the United States suffering from dry eye syndromes.7-9 Furthermore, KCS occurring in the absence of an underlying autoimmune disease is most prevalent in people over the age of 50 and postmenopausal women.10,11 Furthermore, dry eye due to MG dysfunction is thought to account for a large number of the dry eye patient population. Recent epidemiological studies reported that an estimated 25-30 million Americans suffer from dry eye syndromes.5,10-15 These numbers are expected to rise significantly in the next decades with the aging population.5,13 Aqueous, as well as evaporative dry eye, are usually linked with inflammation-induced damage to the lacrimal and meibomian glands with consequent immune-mediated destruction of the glandular secretory units. Therefore, the question arises as to how one could halt or better yet reverse this cellular loss and trigger tissue regeneration. Recent studies conducted using various animal models suggest that both glands are capable of repair following experimentally induced injury and that they contain stem/progenitor cells that are recruited during the repair phase.
STRUCTURE AND DEVELOPMENT OF THE LG The LG develops through branching morphogenesis. In branching morphogenesis, repetitive epithelial cleft and bud formation creates complex three-dimensional branching structures. The LG has both epithelial and mesenchymal components16,17 and its morphogenesis involves a series of reciprocal interactions between epithelium and mesenchyme that result in the formation of a three-dimensional tubular network.18 The epithelial component of the LG is of tubulo-acinar morphology and consists of three cell lineages: ductal, acinar and myoepithelial cells. Acinar cells of the LG secrete mucous, seromucous, and serous.19 LG mesenchyme is derived from periorbital cells of neural crest origin. Several growth factors, including fibroblast growth factors 7 and 10 (FGF7, FGF10) and bone morphogenetic protein 7 (BMP7) have been shown to be the main regulators of LG branching
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morphogenesis.16-18,20-22 Thus, over-expression of FGF7 or 10 in the embryonic cornea induces differentiation of corneal epithelial cells into functional lacrimal gland epithelium.20,21 These studies suggest that correct spatial and temporal expression of FGFs plays a critical role in normal lacrimal gland induction and development.23,24 Human LG consists of two lobes, which are both located superior to the orbit on the temporal side. One lobe is orbital, while the other lies in the palpebral region. There are multiple lacrimal duct openings in the dorsal eyelid.25 The development of the human LG has been studied extensively.26-28 LG morphogenesis in humans starts at O’Rahilly’s stages 19–20, by a thickening of the superior fornix epithelium near mesenchymal condensation. At the O’Rahilly’s stage 21, the first epithelial bud invaginates into the surrounding mesenchyme and later at stage 23, lumina appear within the glandular epithelial buds.27 The gland maturates during the weeks 9–16, acquiring morphology of adult LG.27 In mice, the two lobes of the LG are located differently. The mature LG also consists of a small intraorbital lobe and a much larger exorbital lobe situated adjacent to the external auditory apparatus. Both lobes are connected by a single duct that opens onto the rim of the lower eyelid at the temporal terminus.17 Using a transgene reporter construct based on an ectodermal enhancer from the Pax6 gene (P6 5.0-lacZ) we were able to characterize murine LG development in great detail16,17,20,29-31 (Figure 1). The first morphological indication of murine LG development is an ectodermal invagination from the conjunctival epithelium at the temporal edge of the eye (Figure 1A-D) while nasal invagination of the conjunctival epithelium (Figure 1E) gives rise to harderian gland. The lacrimal bud extends away from the eye but remains as a single bud until E15.5 (Figure 1F). The first branches in the developing gland appear between E15.5 and E16.5 (Figure 1G) and by E19.5, further branches are apparent in both the intraand exorbital lobes.17
LG REGENERATION AND EPITHELIAL MESENCHYMAL TRANSITION LG has a high regenerative potential and is able to repair itself even after substantial damage.32 LG regeneration is accompanied by a process called epithelial mesenchymal transition (EMT). During EMT, epithelial cells lose cell-cell attachment, polarity and epithelial-specific markers, undergo cytoskeletal
CHAPTER 13: Role of Stem Cells in Lacrimal and Meibomian Gland Development and Regeneration
Figure 1 - Lacrimal gland development. (A) Section of the eye in an E12.5 P6 5.0 lacZ reporter mouse. lacZ expression is indicated by the blue X-gal labeling in the lens epithelium (le) and the future conjunctival epithelium on the temporal side of the eye (red arrowhead). (B) Head region of E13.5 P6 5.0 lacZ reporter mouse showing X-gal-labeled corneal epithelium and lens. The lacrimal bud (red arrowhead) is observed projecting towards the auditory canal (ac). (C) As in B, but at higher magnification. The X-gal labeled lens epithelium (l) can be seen through the cornea, the epithelium (c) of which is also labeled. The lacrimal gland bud (red arrowhead) projects form the X-gal-labeled conjunctival epithelium. (D and E) Histological sections through the eye in an E13.5 P6 5.0 lacZ reporter mouse. (D) Blue X-gal labeling is apparent in the continuous layer of the embryonic corneal and conjunctival epithelium (ce) and the early LG bud (red arrowhead) is seen extending from the deepest aspect of the conjunctival epithelium (the fornix). (E) Reporter construct expression is absent from the fornix on the nasal side (black arrowhead). Early budding of the harderian gland primordium is apparent on the surface of the epithelium on the retinal side and is also negative for reporter expression (blue arrowhead). (F) The tip of the lacrimal bud has extended to a position dorsal to the stapedial artery (sa) and caudal to the bifurcation of its supraorbital (dashed lines – sas) and infraorbital (dashed lines – sai) branches by E15.5. (G) By E16.5 the first branching events are apparent. Adapted from Makarenkova et al., 2000.
remodeling, and gain a mesenchymal phenotype.33-36 Several reports suggest that epithelial cells of tissues undergoing EMT might have a certain level of plasticity and are able to dedifferentiate and then redifferentiate when regeneration is completed.37,38 However, it still remains unclear how much stem/progenitor cells or EMT contributes to the regeneration process.
tissue remodeling during embryogenesis39 (Figure 2). The role of EMT in tissue repair/regeneration is well described. Several studies reported the occurrence of EMT during tissue repair in several tissues including mammary glands, liver, kidney, and lung.40-46 It is generally well accepted that induction of EMT generates cells with mesenchymal stem-like properties.41,47,48
Epithelial-Mesenchymal Transition During LG Regeneration
Down-regulation of E-cadherin gene expression, an adherens junction protein, is critical for initiation of EMT and is associated with loss of cell polarity.33,34,36 Several transcription factors have been shown to regulate EMT.35,36,39 Thus, Snai1 seems to be a master regulator
Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) play major roles in
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STEM CELLS IN OPHTHALMOLOGY of EMT and acts partly by repressing expression of epithelial markers, such as E-cadherin and induction of mesenchymal markers, such as vimentin expression.46,49-51
and transcription factors controlling the expression of MMPs at different stages of LG development or disease progression.
It was recently shown that EMT is activated during repair of murine LG and generates cells with mesenchymal phenotype referred to as mesenchymal stem cells (MSC) that migrate to the site of injury and initiate LG repair.52 It was suggested that, during the later phases of LG repair, the MET process is initiated and MSC differentiate and form acinar and ductal epithelial cells.52 MSCs can be isolated from injured LGs and propagated in vitro. However, additional studies are needed to characterize the molecular mechanisms controlling both EMT and MET during LG repair. Such studies could unravel novel strategies to restore functional LG tissue and hence adequate production of the aqueous layer of the tear film.
We recently reported that during LG development mesenchymally expressed FGF10 and epithelially expressed homeobox transcription factor Barx2 cooperate in regulation of epithelially and mesenchymally expressed MMPs. Moreover, we, and others found that Barx2 binds to regulatory sequences of different MMPs and controls epithelial cell migration through the ECM.53 Our findings that BMPs regulate Barx2 expression54 and that BMPs, FGFs, and Barx2 regulate MMP expression and control Notch signaling55-57 and cell proliferation, migration and differentiation in different cellular contexts suggest that this signaling pathway may be involved in EMT and could be considered for pharmacological intervention.
Although EMT is essential for embryonic development and regeneration of many exocrine glands it can also be destructive for the epithelium and could possibly lead to dry eye conditions or tumors if the mechanism or timing of EMT is deregulated. It is also known that LG of Sjögren’s syndrome patients do not regenerate efficiently, suggesting that this disease prevents reverse transition of the MSC, i.e., MET. Thus, defining the molecular mechanisms controlling EMT and MET processes would be extremely useful for development of new strategies to treat some types of dry eye conditions.
Several recent studies suggest that specific MicroRNAs (miRNAs) are involved in regulation of the EMT/MET58 in different tissues. MiRNAs are small non-coding RNAs that target multiple messenger RNAs (mRNAs) and function as post-transcriptional regulators of gene expression regulating many biological processes including embryogenesis, regeneration, and EMT. Recently, the miRNA-dependent post-transcriptional regulation of development-associated genes was proposed to play a role in regulation of tissue regeneration and EMT initiation. Several up-to-date potential microRNA modulators of EMT have been identified. Thus, all five members of the microRNA-200 family (miR-200a, miR200b, miR-200c, miR-141 and miR-429) and miR-205 were markedly downregulated in epithelial cells that had undergone EMT.59 It was reported that dysregulation of miR-204 and/or miR200 mediates EMT and migration and invasion of endometrial and lung cancer.58,60 MiR-204 is an important endogenous negative regulator of Runx2 and can act as a regulator of EMT during secondary cataract formation.61 Moreover, TGF-β receptor 2 (TGF-β R2) and Snail2 (found to be important for induction of MET in LG) are direct targets of miR204.62 These findings suggest that downregulation of microRNAs may be an important step in EMT-mediated epithelial tissue regeneration.
Role of Mesenchymal Cells in Controlling the LG EMT/MET Epithelial structure of the LG is maintained by cellcell and cell extracellular matrix (ECM) interactions. Mesenchymal cells contribute to formation and maintenance of the ECM by production of different matrix components or matrix remodeling enzymes such as MMPs. Mesenchymal cells are also a source to multiple signaling molecules that can bind the ECM and control and guide epithelial growth. For example, several FGFs, and specifically FGF10, regulate all stages of LG morphogenesis. These findings suggest that signals leading to EMT within the LG could be produced by mesenchymal cells. The necessity to control the proteolytic activity of MMPs for treatment of diseases such as cancer or Sjögren’s syndrome has been acknowledged, however little progress has been made in this direction. The reason for this could be due to our limited knowledge of signaling pathways
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LG REGENERATION AND STEM/ PROGENITOR CELLS
CHAPTER 13: Role of Stem Cells in Lacrimal and Meibomian Gland Development and Regeneration Evidence for the Presence of LG Stem Cells The LG is composed of multiple cell types including epithelial, mesenchymal, neuronal, and endothelial cell lineages that are essential for normal physiological function and maintenance of the gland. At the same time, secretory function of the LG completely depends on development and differentiation of LG epithelial components. Lacrimal and salivary glands are ectodermal appendages that develop through similar cellular mechanisms that involve an intimate crosstalk between the epithelium and mesenchyme. However, mutations that perturb primary epithelial bud formation leads to the complete absence of LG epithelial component and therefore secretory cells, while the mesenchymal component of the gland is still present.17,24,63,64 These findings suggest that the epithelial component of the LG is essential for development of a functional LG. In addition, this suggests that epithelial and mesenchymal components of the LG might contain separate lineage specific stem/progenitor cells. Both LG and salivary glands are organs with a high risk of being damaged due to their constant secretory activity, and it becomes extremely important that they retain their ability to regenerate during the whole life span. Based on their similar epithelial origin, LG and submanidibular glands (SMG) have many common characteristics, and as suggested in recent publications, may also have a similar putative stem cell population that gives rise to multipotent stem/progenitor cells in each epithelial organ.65 Several studies indicate that multipotent progenitor cells are present in the lacrimal gland and are involved in their regeneration.66-68 However, the nature of these cells is still not well defined, with more recent studies indicating that myoepithelial cells (MECs) and/or MCSs may have the capacity to act as stem cells and mediate regeneration.66-68 In addition to MECs and ductal epithelial cells, progenitor populations have been proposed but not identified.69,70 Stem cells are generally considered to be slowly dividing cells and are the precursors of progenitor cells, which are more proliferative, lineage-committed, with less capacity to self-renew, and may be lineage-specific. The common method for identifying slow cycling cells consists of using the 5-bromo-2’-deoxyuridine (BrdU) pulse-chase technique. BrdU is a thymidine analog that incorporates into the DNA of dividing cells (during the S phase of the cell cycle), rendering them detectable by immunohistochemical means. Subsequent cell
divisions in the absence of label (chase period) dilute the incorporated BrdU and only cells with the lowest replication profile could be detected.71-73 In a recent study, we showed that LG contains the BrdU-label retaining slow cycling cells that are mobilized to proliferate during LG repair.66 These findings support the idea that slow cycling stem cells with regeneration potential are present within the adult LG. LG Epithelial Progenitor Cells Acinar cell: LG acinar cells are highly polarized cells that secrete proteins, electrolytes and water. Although LG acinar cells are fully differentiated highly specialized secretory cells, they have a certain level of plasticity. Thus, highly purified preparations of murine lacrimal acinar cells cultured in Matrigel rafts resulted in the formation of functional acini that mimic function of acinar cells in uninjured LG.74-76 Another group was able to induce the efficient proliferation of primary rabbit lacrimal gland acinar cells by treating cells with epidermal growth factor (EGF) and dihydrotestosterone (DHT).75 A recent study of human LGs also suggests the presence of stem cells within the acinar cells.77 Although it is possible that LG acinar cells have their own stem/ progenitor cells, this hypothesis needs to be supported by lineage tracing experiments. Thus, isolated LG acinar cells may still contain centroacinar cells. In fact centroacinar cells (found within majority of endocrine glands) are an extension of the intercalated duct cells closely associated with the gland acini and were shown to possess stem cell properties.78-81 Myoepithelial cells: (MECs) are also found in multiple glandular organs such as LG, salivary, harderian, sweat and mammary glands. They surround glandular secretory epithelium, express SMA, and can contract helping secretory function of these exocrine glands.82-87 In the LG and other exocrine glands, MECs synthesize the basement membrane and form a functional network around the acinar and ductal cells separating them from the basement membrane and the mesenchymal stromal cells.88-90 MECs are enriched in expression of muscarinic and purinergic receptors.91,92 The superficial to the basement membrane location of MECs in sweat and mammary glands suggests that these cells are epithelial.93,94 Lack of vimentin expression (marker of mesenchymal cells) in MECs of salivary glands and both major and accessory LGs further supports the idea that MECs are cells of ectodermal origin.95 It has been suggested that MECs retain some proliferative potential
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STEM CELLS IN OPHTHALMOLOGY
Figure 2 - Schematic representation of EMT and MET initiation. In healthy LGs, the acinar epithelial cells have an apico-basal polarity created by the tight junction and interactions with the basement membrane. The cells express markers of the epithelial lineage such as E-cadherin, a protein of the adherens junctions. Following injury (or inflammation), the epithelial cells undergoing EMT (steps 1 and 2) lose cell-cell and cell-extracellular matrix (ECM) contact, lose epithelial markers and express mesenchymal ones (such as vimentin), acquire front-back polarity (a morphological characteristic of migratory cells, wherein the front -leading edge- and the back show morphological and functional asymmetry) and enhanced ability to migrate. During tissue repair or resolution of inflammation (step 3), MET is initiated and the cells re-acquire their epithelial phenotype. The molecular mechanisms controlling MET are still poorly understood.
in adult uninjured LG and salivary glands, and that they have a high level of plasticity and participate in glands gland regeneration.68,96 Thus, MECs of parotid glands show a strong increase (up to 23%) in their proliferative rate 5-days following gland injury, while proliferation of other epithelial cell types (ductal and acinar) increases much later on days 7-10 after injury.96 This quick response of MECs to gland injury suggests that a MEC sub-population may contain faster proliferating committed progenitor cells.
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Ductal cells: Similar to the duct system in the salivary glands, the LG ducts can also be divided using the same anatomical/structural divisions: intercalated, intralobular, interlobular, intralobar, interlobar, and main excretory ducts.97 Of note is the absence of striated ducts in the LG, which are commonly found in the salivary glands. LG ducts are composed of a single layer of cuboidal, eosinophilic cells that are arranged in a smooth pattern. The primary function of ductal cells is to modify the electrolyte composition of the primary LG
CHAPTER 13: Role of Stem Cells in Lacrimal and Meibomian Gland Development and Regeneration fluid (originating from the acinar cells). Several studies on regenerating salivary glands reported increased proliferation of ductal cells suggesting their involvement in tissue repair.98,99 Our own studies showed increased expression of Ki67 (a cell proliferation marker) in ductal cells of regenerating LGs.67 Furthermore, we also reported initiation of EMT within ductal cells of injured LGs suggesting the involvement of ductal cells in LG repair.52 In addition, several studies in salivary glands, as well as pancreatic and mammary glands, propose that ducts might be a niche for stem/progenitor cells capable of differentiating into other epithelial cells.100-103 Regulation of LG Development and Regeneration Our previous studies showed that LG mesenchyme plays an instructive role for epithelial cells and produces multiple growth factors that regulate LG morphogenesis and LG stem/progenitor cell specification.17,20 We, and others showed that several signaling pathways, such as FGF, Wnt and TGF-beta signaling are important for LG morphogenesis.16,17,22,104,105 For example, the FGF pathway has been shown to be particularly important for multiple aspects of embryonic development including LG epithelial bud outgrowth.16,17,106 The biological activities of FGFs and other morphogens are regulated by the ECM that establishes and maintains their gradients during development.29,64,107,108 ECM and associated growth factors are also important components of the stem cell niches.109,110 In human genetic syndromes caused by mutations of the FGF/FGFR, signaling pathway was shown to be associated with lacrimo-auriculo-dentodigital syndrome (LADD: OMIM 149730) and aplasia of lacrimal and salivary glands (ALSG:OMIM 602115). These mutations result in haplo-insufficiency of FGF10 or its receptor FGFR2b and induce severe defects in the survival and function of LG epithelial progenitor cells. Transcription factors are important regulators of tissue morphogenesis and stem cell function.111-113 We previously showed that paired-box transcription factor Pax6 is necessary for LG development and is a competence factor for LG bud outgrowth.17,20 We and others also found that the homeobox transcription factor Barx2, forkhead/winged helix transcription factor Foxc1, and Runt-related transcription factors (RUNX) are expressed in epithelial LG ducts and are necessary for normal LG development.53,114,115 Thus, autosomal dominant mutations in human FOXC1 cause eye disorders such as Axenfeld-Rieger Syndrome and glaucoma iris
hypoplasia, resulting from malformation of the anterior segment of the eye.115 More recently, we have determined that Barx2 regulates LG cell proliferation, migration and plasticity and its expression was upregulated during LG regeneration (HPM unpublished observations). LG Stem/Progenitor Cell Markers Several recent studies indicate that, similar to other exocrine glands (pancreas, salivary, and mammary),96,116-118 the LG has a high regenerative potential and that multipotent progenitor cell population(s) may be involved in their regeneration.66-68 The question that remains to be answered is whether or not LG epithelial cells have a common or specific progenitor cells. Shatos and co-authors showed that within uninjured LG nestin (an intermediate filament protein progenitor cell marker) expression was colocalized with expression of SMA and Pax6, suggesting that these MEC populations could be of epithelial origin.68 Cultured MECs expressed mesenchymal markers, such as α-actinin, vimentin and adenylyl cyclase II.92 Similar to LG, increased proliferation of MECs was described during atrophy and regeneration of rat parotid glands.96 Further studies are warranted to determine whether or not MECs could be considered as LG stem cells or differentiated cells with increased cell plasticity (i.e., that they are able to dedifferentiate and re-differentiate again). In a recent study, we have shown that Runt family transcription factors Runx1-3 are expressed in the LG epithelial component and inhibition of their expression by siRNAs blocks LG morphogenesis.114 Specifically, a high level of Runx1 expression was found within the LG ductal component and centroacinar cells. Moreover, we found that Runx1, Runx3, and cytokeratin-5 expression (putative stem cell marker) increased significantly in regenerating LGs suggesting their involvement in regulation of regeneration. Runx1 is involved in stem cell specification, proliferation and differentiation, and influences tissue homeostasis of many different organs.119-121 Runx1 was recently shown to be involved in differentiation of the epithelial secretory cell lineage in colon.122 Involvement of Runx1 in LG morphogenesis and regeneration, combined with the reported contribution of Runx1 in controlling multiple stem cell lineages, suggests that high level of Runx1 expression might mark LG epithelial progenitor cells.
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STEM CELLS IN OPHTHALMOLOGY In addition, we reported that nestin positive cells are present in the murine lacrimal gland and that the nestin protein level increased during LG regeneration.32 We extended these findings by showing that proliferating cells within the injured murine LG were nestinpositive.66,123 These cells expressed mesenchymal, as well as progenitor cell markers such as vimentin, SMA, nestin, ABCG2 and Sca-1, and high level of proliferation as measured by Ki67 expression.66,123 In addition, a subpopulation of these cells also expressed the epithelial cell marker, Pax6, suggesting the involvement of epithelial progenitor cells in LG repair.
STRUCTURE AND DEVELOPMENT OF THE MEIBOMIAN GLANDS Because the MGs are greatly enlarged, modified sebaceous glands without a hair follicle, their structure and development is very similar to sebaceous gland morphogenesis in the skin.94 Similar to the LG, the MG is a tubuloacinar gland whose primary function is to secrete the lipid layer of the tear film.1 The MGs are embedded in the lower tarsal plates of the lid margins.75 There are approximately 30-40 glands in humans that are up to 10 mm long in the upper lid and fewer (2030) and shorter (up to 6 mm) in the lower lid.75,124 The MGs consist of a single lobule (acinus) or a collection of lobules that coalesce into a system of ducts.75,124 The duct of each gland opens directly onto the inner margin of the eyelids. Lipids are synthesized within the acinus and are then secreted into small ducts, which lead to a single, straight duct lined by modified keratinized squamous epithelium.124 MGs produce lipids that are the main component of the superficial lipid layer of the tear film. This lipid layer is thought to prevent or retard the evaporation of the aqueous layer of the tear film and to stabilize the tear film by lowering surface tension.1,75 A single meibomian gland is composed of clusters of secretory acini that are arranged circularly around a long central duct.75 The MG has holocrine secretion mode, the central duct opens at the eyelid margin, where the oily secretion is delivered onto the tear meniscus.75 MG acini can be subdivided according to their stage of differentiation and location into undifferentiated cells, maturing cells and necrotic cells.75,124 The undifferentiated cells are germinal basal cells in contact with the basement membrane and do not synthesize or secrete lipids.75,124 The maturing cells are larger and have an elaborate smooth endoplasmic reticulum and Golgi apparatus for the synthesis of lipids, which are
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then stored in secretory granules.75,124 The necrotic cells are located in the innermost zone and are larger and full of secretory granules that are ready for release. Lipid secretion occurs through a holocrine manner: the secretory granules fuse to form large aggregates and lipid droplets and cell debris are released following lysis of the necrotic cells.75,124 Although development of sebaceous glands is well documented, little is known about MG development especially in humans.125,126 Mouse MG development starts at E18.5 with the formation of an epithelial placode within the fused eyelid margin epithelium and associated with placodes mesenchymal condensations.94 The epithelial invagination into the eyelid mesenchyme appears at P0 and at P3.5 is observed as a solid cord without lumen (Figure 3A). By P8 (Figure 3B) the developing MGs show distinct differentiation into ductal and acinar regions. By P15, the MGs appear to obtain an appearance of mature glands94 (Figure 3C-E). Mature MGs form a regular pattern in adult mouse eyelids (Figure 3F). Lipid synthesis was first detected at P3 and at P15 and it was present in the acini of the mature MG.94
MG REGENERATION AND MG STEM/PROGENITOR CELLS The information on stem cells, stem cell types and cell dynamics in the MG is very limited. Some of the currently available information on MG stem cells can be found in the review by Knop et al.127 Due to similarities with sebaceous glands, most of the information about MG stem cells is derived from studies of cell dynamics in the sebaceous glands. The holocrine type of secretion with continuous loss of acinar cells requires a mechanism that allows a continuous cell turnover and differentiation within the acinus. Several studies show that in mouse MG the rapidly dividing meibocytes are located in the basal acinar epithelium; however, these cells are most likely progenitor cells rather than stem cells, which are defined as a slow-cycling cell population. Olami and co-authors showed that meibocytes have a generation time of 4.1 days between each division.128 Interestingly, similar to other glands slow-cycling, label retaining, putative MG stem cells have been found concentrated in the ductal epithelium of the MG.129 Similarity of the MG and sebocyte suggests that MGs stem/progenitor cells may express markers found in stem/progenitor cells of sebocytes. Thus, it has been reported that sebocytes stem/progenitor cells express Blimp1 Krt5 and Krt14, while activated proliferating cells express myc, Krt5 and
CHAPTER 13: Role of Stem Cells in Lacrimal and Meibomian Gland Development and Regeneration
Figure 3 - Meibomian gland (MG) development and structure. (A) Confocal images using actin (phalloidin, red) and nuclear DNA staining of eyelids at P3.5 (postnatal day 8). At P3.5, prominent epidermal tubular-like MG invaginations can be observed (white arrows). (B) Propidium iodide nuclei staining (red) and CD31/PECAM-1 (green) staining at P8, show distinct development of acinar regions of MGs (white arrows) and blood vessels. (C and D) Hematoxylin and Eosin staining of P15 eyelids show the presence of MGs (blue dashed line) in the eyelid. (E) β-Galactosidase staining shows Runx1 expression in MGs of P15 mouse eyelids with well developed acinar region and the duct opening (red arrow, nuclei stained with fast red). Note the high level of Runx1 expression in the ductal regions and the undifferentiated meibocytes (black arrow). (F) Adult mouse eyelid showing regular patterning of fully developed MGs. Labels: hf - hair follicles, ms – muscle of eyelid.
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STEM CELLS IN OPHTHALMOLOGY Krt14.130,131 Whether MG stem/progenitor cells express these markers has yet to be determined.
CONCLUDING REMARKS Inflammatory diseases of the lacrimal and meibomian glands that lead to tissue destruction account for the bulk of the dry eye conditions that can lead to ocular surface damage. Emerging studies from several laboratories show that both glands are capable of repair and that stem/progenitor cells play a crucial role in the repair process. Despite the modest progress, identification of lineage (epithelial and mesenchymal) specific markers for stem/progenitor cells present in the LG and MG is still lacking. Identification of these markers would therefore be an essential step toward defining the stem/ progenitor cell populations and their roles in LG and MG regeneration. Furthermore, these cells would be a great asset in the emerging field of regenerative medicine and tissue engineering, as treatment modalities to aqueousdeficient, as well as evaporative dry eye diseases. References 1.
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