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How Does Amniotic Membrane Work?
Laboratory Science JAMES V. JESTER, PHD, SECTION EDITOR
How Does Amniotic Membrane Work? SCHEFFER C. G. TSENG, MD, PHD,1,2 EDGAR M. ESPANA, MD,1 TETSUYA KAWAKITA, MD,1 MARIO A. DI PASCUALE, MD,1 WEI LI, MD,1 HUA HE, PHD,1 TZONG-SHYNE LIU, MD,2 TAE-HEE CHO, MD,2 YING-YING GAO, MD,2 LUNG-KUN YEH, MD,3 AND CHIA-YANG LIU, PHD3
ABSTRACT Transplantation of amniotic membrane as a temporary or permanent graft promotes epithelial wound healing and exerts potent anti-inflammatory and anti-scarring effects on the ocular surface. These actions depend on the killing of allogeneic amniotic cells and preservation of the cytokine-containing matrix during the preparation of the amniotic membrane. This review describes how these actions inherently operate in utero and how amniotic membrane transplantation aims to recreate such a fetal environment to exert these actions by insulating the surgical site from the host environment. These actions also render the amniotic membrane a unique niche capable of expanding both epithelial and mesenchymal progenitor cells ex vivo, while maintaining their normal cell phenotypes. As a result, the amniotic membrane becomes an ideal substrate for engineering different types of ocular surface tissues for transplantation. Further studies investigating the exact molecular mechanism by which the amniotic membrane works will undoubtedly unravel additional applications in reconstruction and engineering of both ocular and nonocular tissues in the burgeoning field of regenerative medicine. Accepted for publication May 2004 From 1Ocular Surface Center and TissueTech, Inc., 2Ocular Surface Research & Education Foundation, and 3Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, Florida, USA Supported in part by research grants RO1 EY06819 and R43 EY014768 (to SCGT) from National Eye Institute of National Institutes of Health, a research grant from TissueTech, Inc., and an unrestricted grant from Ocular Surface Research & Education Foundation, Miami, FL. Dr. Tseng and his family are more than 5% shareholders of TissueTech, Inc., which owns US Patents Nos. 6,152,142 and 6,326,019 on the method of preparation and clinical uses of human amniotic membrane distributed by Bio-Tissue, Inc. A patent has been filed by Dr. Tseng and Dr. Espana related to keratocytes and licensed to TissueTech, Inc. Single copy reprint requests to: Scheffer C.G. Tseng, MD, PhD (address below). Corresponding author: Scheffer C. G. Tseng, MD, PhD, Ocular Surface Center, 7000 SW 97 Avenue, Suite 213, Miami, FL 33173, USA. Tel: (305) 274-1299. Fax: (305) 274-1297. E-mail: [email protected] Abbreviations are printed in boldface where they first appear with their definitions. ©2004 Ethis Communications, Inc. The Ocular Surface ISSN: 15420124. Tseng SCG, et al. How does amniotic membrane work? 2004; 2(3):177-187.
KEYWORDS amniotic membrane, anti-inflammatory action, anti-scarring action, cytokines, in utero environment, ocular surface reconstruction, stromal matrix, transforming growth factor-β
I. INTRODUCTION linical use of amniotic membrane (AM) as a dressing in general medicine dates back to the early 20th century, and its ophthalmic use as a dressing dates back to at least half a century ago (for more historical information, see a recent review1). The use of AM as a graft for ocular surface reconstruction was first reported by Kim and Tseng in 1995.2 The popularity of this surgical procedure has since increased (Figure 1) based on a method of graft preparation (Bio-Tissue, Inc, Miami, Florida, USA [Tseng SCG: Grafts made from amniotic membrane; methods for separating, preserving, and using such grafts in surgeries. TissueTech, Inc. 2000, #6,152,142; 2001, #6,326,019]) that kills allogeneic amniotic cells3 but maintains the integrity of AM cytokine-rich extracellular matrix; thus, the AM graft exerts potent anti-inflammatory, anti-scarring and anti-angiogenic actions and promotes epithelial healing in recipient eyes. In 2001, the US Food and Drug Administration (FDA) ruled that an AM graft thus prepared is homologous to the ocular surface, and hence approved it as a “tissue,” so AM transplantation for ocular surface reconstruction does not require premarket approval. In contrast, dehydrated and decellularized AM is not considered a “tissue” by FDA when used as a graft and requires premarket approval if advertised for wound repair. (See http://www.fda.gov/cber/tissue/trgfyrpts.htm FDA website, Tissue Action Plan, Tissue Reference Group Annual Reports.) In January 2004, AM transplantation as a graft for ocular surface reconstruction was approved as a standard surgical procedure by Medicare in the USA. Amniotic membrane has been used as either a permanent or temporary graft. As a permanent graft, AM is applied in one or multiple layers for corneal, conjunctival, or entire ocular surface reconstruction. It can be used alone or in conjunction with transplantation of conjunctival epithelial stem cells via conjunctival autograft or with trans-
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HOW DOES AM WORK? / Tseng, et al OUTLINE I. Introduction II. In utero function of the amniotic membrane A. Anti-inflammatory action B. Anti-scarring action III. Maintenance of keratocyte phenotype during ex vivo expansion on amniotic stromal matrix IV. Restoration of stromal niche important for ex vivo expansion of limbal epithelial progenitor cells V. Is the amniotic membrane an alternative progenitor cell niche?
plantation of limbal epithelial stem cells via conjunctival limbal autograft, keratolimbal allograft, or living-related conjunctival limbal allograft (see classification4). As a permanent graft, the surrounding host epithelial cells will migrate onto the amniotic basement membrane, while host mesenchymal cells will migrate into the amniotic stromal matrix. As a result, transplanted AM will be integrated into the recipient site. As a temporary graft or patch or dressing, the host epithelial cells will migrate underneath AM, and upon healing, the transplanted AM will invariably dissolve. Figure 2 lists the clinical indications for AM transplantation based on Donor-Recipient Information retrieved by Bio-Tissue, Inc. (Miami, FL, USA) during 1997 to 2002, (i.e., prior to Medicare approval). The main objective of this review is to describe the action mechanisms of AM transplantation for ocular sur-
Figure 1. Number of papers published regarding AM transplantation in ophthalmology. Data were obtained from Medline search of English-language literature.
face reconstruction. We retrieved from a PubMed search all articles based on the key words amniotic membrane, eye, and function, and we included only those that contain experimental (basic or clinical) data related to the possible action mechanisms. We excluded articles that describe only results of clinical studies. Published abstracts without accompanying papers were also excluded. We focus on antiinflammatory and anti-scarring effects in this review. Readers who are interested in clinical studies on the use of AM should consult the following reviews and references therein,1,5-7 as well as the review by Bouchard and John that is published in this issue of The Ocular Surface.8
Figure 2. Clinical indications for AM transplantation. Data was obtained from Donor-Recipient Information retrieved during 1997 to 2002 at Bio-Tissue, Inc. (Miami, FL). (N = 3,354 Data Source: Bio-Tissue, Inc.)
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HOW DOES AM WORK? / Tseng, et al II. IN UTERO FUNCTION OF THE AMNIOTIC MEMBRANE
A number of clinical studies have shown that AM transplantation facilitates epithelial wound healing and reduces inflammation, scarring, and angiogenesis. These actions resemble the scarless fetal wound healing observed in utero.9,10 Therefore, a closer look at the role of the AM during fetal development may shed light on how this tissue works. Anatomically, AM, or amnion, is the innermost membrane enwrapping the fetus in the amniotic cavity, which contains the amniotic fluid. As a result of enlargement of the fetus toward the end of the first trimester, the membrane is fused with the chorion to form the fetal membrane. From this point onward, these two membranes are contiguous with, and become a part of, the placenta. The placenta supports the development and survival of the fetus by providing exchange of metabolic and gaseous products, nutrients, and electrolytes, and by producing maternal antibodies and developmentally important hormones. Developmentally, both the placenta and fetal membrane are derived from the outer cell mass, while the fetus is derived from the inner cell mass of the blastocyst. Both outer cell mass and inner cell mass are developed from the fertilized egg, indicating that AM and the fetus share the same cell origin. The fetal membrane physically separates the fetus from the maternal environment (i.e., uterus). In the fetal membrane, the amnion and the chorion are only loosely attached, whereas the chorion and the underlying decidua tightly adhere to each other. Before the rupture of membrane prior to delivery, maternal cells, including circulating inflammatory and immune cells, exit from decidual postcapillary venules and encounter chorion trophoblasts. Because inappropriate activation of inflammatory cells in the membrane environment contributes to the pathogenesis of preterm labor, the integrity of the fetal membrane guards against such a potential threat to pregnancy. Because the chorion is vascularized and contiguous with the decidua of the placenta, the true protective layer of the fetal membrane resides in the AM. One important function of the AM is to protect the fetus from cellular insults derived from the maternal environment. This concept is evidenced by the facts that intentional amniotomy induces labor or abortion and that premature rupture of the membrane leads to delivery. The AM achieves this important function of protecting the fetus by exerting critical anti-inflammatory and anti-scarring effects, as described below. A. Anti-inflammatory Action
Although the cytokines present in the AM matrix are important, the cellular sources of these distinct cytokines have not been elucidated. The AM’s anti-inflammatory action may be mediated in part by interleukin-10 (IL-10). (We detect a significant amount of IL-10 in AM extracts using ELISA [unpublished data]). IL-10 is known to suppress or counteract the actions of pro-inflammatory cytokines such as IL-611 and tumor necrosis factor-alpha (TNF-α).12 IL-10 also suppresses amniotic cell produc-
tion of IL-8,13 which is a pro-inflammatory chemokine attracting the migration of neutrophils. Furthermore, the AM produces inhibin and activin; both belonging to the TGF-β superfamily. Activin promotes the production of prostaglandin E2 (PGE2).14,15 A low dose of activin stimulates, but a high dose of activin inhibits, the production of IL-6, IL-8, and PGE2 by the AM.16 No such effect is noted in the chorion or decidua. Furthermore, production of TNF-α is significantly inhibited by activin in the chorion and deciduas.16 The AM contains various protease inhibitors, including α1 anti-trypsin and inter-α-trypsin inhibitor,17 the latter of which may exert an anti-inflammatory effect [see review18]. Future studies are needed to determine whether IL-10, activin, protease inhibitors, and/or a combination of them are responsible for the anti-inflammatory action of AM when it is transplanted to the ocular surface. The AM contains IL-1 receptor antagonist (IL-1RA) and helps transport it to the amniotic fluid.19 IL-1RA is a potent inhibitor of IL-1, and, thus, will suppress the inflammation mediated by IL-1. Data from our laboratory have shown that limbal epithelial cells cultured on the AM stromal matrix downregulate the expression and production of IL-1, but upregulate the expression and production of IL-1RA, resulting in a higher ratio of IL-1RA/IL-1.20 Such an effect withstands the challenge of lipopolysaccharide, a known stimulator of IL-1 production.20 Collectively, these findings support the concept that the AM may exert its anti-inflammatory action by suppressing the signaling pathway via IL-1, at least in epithelial cells. Additional studies are needed to verify such an action in inflammatory or immune cells. Even if the AM contains the aforementioned anti-inflammatory mediators, its anti-inflammatory action may require a close contact with its stromal matrix. We have reported that activated neutrophils are rendered apoptotic 24 hours after excimer laser-induced phototherapeutic keratectomy or photorefractive keratectomy in rabbit corneas when in contact with human AM stroma matrix as a temporary graft, but not with the amniotic basement membrane.21,22 As a result, inflammatory cells are excluded from the ablated stroma, resulting in less corneal haze. A similar finding was also noted in a murine model of HSV1-induced keratitis, where infiltration of lymphocytes and macrophages was markedly reduced 48 hours after the cornea was covered with human AM as a temporary graft.23 Shimmura et al24 noted that both mononuclear and polymorphonuclear inflammatory cells and lymphocytes are trapped by AM stromal matrix and induced to undergo apoptosis. Clearance of granulocytes by pushing them into rapid apoptosis is an effective anti-inflammatory strategy25; thus, we speculate that further investigation into the molecular mechanism by which AM stromal matrix exerts its anti-inflammatory action may identify new important therapeutic directions for modulating innate immunity. Recently, Ueta et al26 also reported that human AM can suppress alloreactive responses and downregulate production of Th1 and Th2 cytokines in mouse lymphocytes in vitro. This finding suggests that the AM may also suppress acquired immunity.
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HOW DOES AM WORK? / Tseng, et al B. Anti-scarring Action
It has been recognized in the field of pediatric surgery that incisions made through the fetal skin before the third trimester do not result in scar formation at birth. This phenomenon termed scarless fetal wound healing has been a subject of intensive study for the last three decades.9,10,27 One natural explanation is that fetal fibroblasts are intrinsically different from adult fibroblasts in their response to scar-promoting cytokines.28-30 Nevertheless, there is strong evidence in the literature that the AM plays a direct and positive role in exerting an anti-scarring effect during fetal wound healing. As stated above, the AM contains the anti-inflammatory cytokine, IL-10, which can inhibit the production of IL-6.11 Diminished IL-6 production is one hallmark of scarless fetal wound repair.31 Furthermore, the phenomenon of fetal scarless wound healing was abolished in IL-10-knockout mice.32 The AM may exert an anti-scarring effect by supporting nerve growth in the fetus, which is essential to endow the fetus with scarless fetal wound healing. It has been reported that denervation (i.e., cutting the sciatic nerve) of the hind limb on one side will abolish the phenomenon of scarless fetal wound healing on that side, as compared to the intact innervated hind limb on the other side.33 It is intriguing to note that the AM is not innervated (i.e., not supplied by nerves), and yet amniotic epithelial cells synthesize various neurotransmitters,34 neuropeptides, and neurotrophins.35 The production of neurotrophic factors, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), is particularly significant, as these factors control the growth and targeting of sensory and autonomic nerves to the peripheral tissues (see review36). The fact that the AM contains and produces these neurotrophins strongly suggests that its main purpose is to help the development of the nervous system in the fetus, thereby ensuring scarless wound healing. Experimental data from our laboratory have shown that cryopreserved AM contains abundant amounts of NGF and permits the expression of NGF receptors by the limbal epithelial cells when grown over the membrane in culture.37 This new finding supports the concept that the AM plays an active role in supporting the target tissue to express and respond to such a neurotrophin as NGF. This finding may explain, in part, why AM transplantation is effective in treating neurotrophic corneal ulcers, which are caused by corneal denervation.38-40 Additionally, we have now gathered strong evidence that AM stromal matrix exerts a direct anti-scarring action on ocular surface fibroblasts. When human AM was transplanted into the rabbit corneal stromal pocket, we noted that epithelial-induced differentiation of corneal stromal fibroblasts (keratocytes) into myofibroblasts is inhibited.41 To confirm that such an in vivo anti-scarring action is not caused indirectly by suppressing inflammation via inflammatory cells or epithelial cells as described above, we investigated the expression of transforming growth factorbeta (TGF-β) genes and their receptors by ocular surface fibroblasts cultured directly on AM stromal matrix. Three different TGF-βs, i.e., TGF-β1, TGF-β2 and 180
TGF-β3, each encoded by separate genes, are expressed by mammalian cells. TGF-β turns out to be the most potent cytokine promoting myofibroblast differentiation by upregulating expression of α-SMA,42-45 integrin α5β1, and EDA domain-containing fibronectin (Fn)46 in a number of cell types, including fibroblasts (see reviews47-48). TGF-β also upregulates the expression of such matrix components as collagens and proteoglycans, downregulates proteinase and matrix metalloproteinases (MMP), and upregulates their inhibitors. Collectively, these actions result in increased cell-matrix interactions and adhesiveness, as well as deposition and formation of scar tissue.47-49 TGF-βs exert their actions via binding with TGF-β receptors (TGF-βRs) on the cell membrane. In human cells, there are three TGFβRs, namely, TGF-βR type I (TGF-βRI), type II (TGF-βRII), and type III (TGF-βRIII). TGF-βs, serving as ligands, bind with a serine, threonine kinase receptor complex made of TGF-βRI and TGF-βRII50; such a binding is facilitated by TGF-βRIII, which is not a serine, threonine kinase receptor. Binding with TGF-βRII activates TGF-βRI, which is responsible for direct phosphorylation of a family of effector proteins known as Smads, which modulate transcription of a number of target genes, including those described above, participating in scar formation (see reviews50,51). Our laboratory data strongly support the concept that AM stromal matrix exerts a direct, potent, anti-scarring action on ocular surface fibroblasts by suppressing the TGF-β signaling at the transcriptional level. In short, we discovered that transcript expression of TGF-β2 and TGF-β3, but not TGF-β1, as well as transcript expression of TGF-βRI, TGFβRII and TGF-βRIII, are markedly downregulated in fibroblasts derived from normal human cornea,52 limbus52 and conjunctiva53 and in abnormal fibroblasts derived from patients with pterygium,53 cultured in either a serum-containing or serum-free medium (Figure 3A and B). Such a pattern of transcript and protein inhibition can withstand the challenge by exogenous TGF-β1 in a medium with or without 1% FBS (Figure 3B). Furthermore, such transcript suppression is followed by marked suppression of both transcripts and proteins of downstream TGF-β target genes encoding α-SMA (Figure 3C and D), EDA-containing Fn, and integrin α5β1.52,53 Collectively, these findings support the concept that AM stromal matrix may exert a direct anti-scarring action on fibroblasts by suppressing TGF-β signaling at the transcriptional level, leading to downregulation of several downstream scar-forming genes responsible for scar formation. III. MAINTENANCE OF KERATOCYTE PHENOTYPE DURING EX VIVO EXPANSION ON AMNIOTIC STROMAL MATRIX
Suppression of TGF-β signaling by AM stromal matrix is not only pathologically important in preventing myofibroblast differentiation and scar formation, but it is also physiologically relevant in maintaining the normal phenotype of corneal keratocytes. Keratocytes embedded in the corneal stroma are a unique population of cranial neural crest-derived mesenchymal cells that play an important role in maintaining
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Figure 3. Northern hybridization. Transcription of TGF-β2, TGF-β3, TGF-βRI, TGF-βRII and TGF-βRIII, but not TGF-β1 is suppressed by AM in human corneal fibroblasts (HCF) and human limbal fibroblasts (HLF [upper left]), and in human conjunctival fibroblasts (HJF) and pterygium body fibroblasts (PBF [upper right],) as compared to collagen gel (lower left) or plastic (P) in both serum-containing (FBS or D-FBS) medium and serum-free (ITS or D-ITS) medium. In the serum-free medium, addition of 10 ng/ml TGF-β1 upregulates TGF-β1 mRNA but not others. Transcription of α-SMA is correspondingly downregulated by AM (lower left). Western blot (lower right) confirms that protein expression of α-SMA is upregulated on plastic cultures by 10 ng/ml TGF-β for 7 days in DMEM with 1% FBS, but remains suppressed on AM. (Modified with permission from J Cell Physiology,52 Curr Eye Res,53 and Invest Ophthalmol Vis Sci.60)
corneal transparency. Under the normal in vivo condition, keratocytes are characteristically mitotically quiescent. They exhibit a dendritic morphology with extensive intercellular contacts, and synthesize the collagens I, V, VI, and XII54 and keratan sulfate-containing proteoglycans, such as lumican, keratocan, and mimecan.55,56 They express several keratan sulfate-rich proteoglycans, including keratocyte-specific keratocan57,58 and CD34.59,60 To study keratocyte biology and the role of keratocytes in maintaining corneal stromal transparency, it is important to isolate and maintain these cells in culture. Interestingly,
keratocytes isolated from bovine56 and rabbit45 corneal stroma and cultured on plastic in a serum-containing medium rapidly lose their dendritic characteristics and acquire a fibroblastic morphology.58 At the same time, they turn into myofibroblasts with the expression of integrin α5β161 and α-SMA,63 especially when seeded at a low density. One way to preserve the ability of keratocytes to secrete lumican, keratocan, and mimecan is to culture them in a serum-free culturing condition.58,64 However, this serum-free culturing method precludes ex vivo expansion and subculturing (see review48).
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Figure 4. Morphology and expression of keratocan by RT-PCR. Primary cultures of collagenase-isolated human corneal fibroblasts cultured on plastic (P) are more dendritic in a serum-free medium (0% FBS), but become fibroblastic when 10% FBS is added. In contrast, cells cultured on AM stromal matrix retain dendritic morphology and intensive cell-cell contact even in the presence of 10% FBS. Expression of keratocan transcript was dramatically increased from plastic to AM cultures even when FBS was added (peaking at 5% FBS). K: RNA extracted directly from human cornea in vivo. (Modified from Espana EM et al65 with permission of Invest Ophthalmol Vis Sci.)
Our recent laboratory data showed that AM stromal matrix allows ex vivo human corneal keratocytes (HCF) to effectively expand for up to five passages in a serum-containing medium, while maintaining their characteristic dendritic morphology and continuous expression of keratocan (Figure 4).65 A similar result was reproduced in mouse keratocytes for up to passage eight (not shown). As shown in Figure 3D, we also noted that expression of CD34, another new membrane marker for keratocytes,60 was similarly maintained. To further delineate the experimental matrix conditions governing keratocyte maintenance (i.e., expression of keratocan and CD34) or myofibroblast differentiation (i.e., expression of αSMA), we subcultured HCF between AM and plastic from primary cultures. Figure 5 shows that continuous passages on AM (e.g., AA or AAA) were essential for maintaining CD34 expression, while subpassage ending up on plastic rendered α-SMA expression whether or not the cells were first exposed to AM. Interestingly, subculturing cells from plastic to AM suppressed α-SMA expression, but did not regain expression of CD34 (Figure 5). Therefore, we have established culturing conditions to modulate keratocyte phenotype from 182
keratocytes, myofibroblasts, and the “transitional state” of fibroblasts. Future studies are underway to confirm that the latter state is indeed a transitional state between keratocytes and myofibroblasts by their expression of α5β1 integrins and EDA-containing Fn.
Figure 5. Western blot . Primary culture of HCF was established on either AM (A) or plastic (P) and subpassaged to either A or P (and PP, PPP, etc.). Equal amounts of cell extracts by RIPA buffer were loaded as verified by β-actin and analyzed by antibodies to CD34 and α-SMA, respectively. CD34 was expressed only by continuous subculturing on AM (e.g., AA or AAA). In contrast, α-SMA was markedly promoted when cells were continuously cultured on P (e.g., PP or PPP) or ended up on P (e.g., AP or PAP). [Modified from Espana et al60 with permission of Invest Ophthalmol Vis Sci.)
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E
Figure 6. Ex vivo expansion of human limbal progenitor cells. Human limbal explants prepared from corneoscleral buttons were seeded on either intact or epithelially-denuded AM and cultured in SHEM (A). Epithelial outgrowth started from the limbal region (A, arrows) and migrated onto the amniotic membrane after 5 days (B). The outgrowth on intact AM sometimes adhered over the amniotic epithelial cells (C, arrows) and consisted of a monolayer of small and compact cuboidal epithelial cells (D). The epithelial outgrowth was faster on denuded AM than on intact AM (E). (Modified from Meller et al67 with permission of Br J Ophthalmol.)
IV. RESTORATION OF STROMAL NICHE IMPORTANT FOR EX VIVO EXPANSION OF LIMBAL EPITHELIAL PROGENITOR CELLS
Besides maintaining the normal keratocyte phenotype, the AM has also been used as a substrate in several protocols to expand limbal epithelial progenitor cells directly from limbal explants,66-69 cell suspensions from the limbal epithelium,70 or indirectly from prior expansion on 3T3 fibroblast feeder layers.71 As shown in Figure 6, a compact monolayer of small epithelial cells with a scanty cytoplasm migrates from the limbal explant to the basement membrane surface of the AM. Similar to the findings of Koizumi et al,66 we noted that epithelial outgrowth reaches a diameter of 2030 mm in 18-25 days on intact AM (retaining the amniotic epithelial cells), whereas it takes 11-18 days for epithelially denuded AM. Such an epithelial outgrowth is rarely generated from human peripheral or central cornea.67 On intact AM, the resultant epithelial phenotype of the ex vivo expanded limbal epithelial cell layer has been characterized to be a limbal epithelium manifesting features of limbal basal epithelial progenitor cells, such as: 1) poor differentiation, i.e., lack of expression of a cornea-specific keratin K3,72 2) slower proliferation rate, resembling the in vivo slow-cycling property,73 and 3) lack of expression of connexin 43 (Cx43),74 a gap junction protein controlling intercellular communication upon xenotransplantation to the subcutaneous plane of nude mice to induce stratification and differentiation (Figure 7).67,68 In contrast, though growing faster (Figure 6), the resultant epithelial phenotype is corneal epithelium on denuded AM. Due to the lack of definitive marker(s) for limbal epithelial stem cells, future studies are needed to
confirm the necessity of including devitalized amniotic epithelial cells during ex vivo expansion on AM. In a rabbit model of unilateral limbal stem cell deficiency, we have recently confirmed the long-term (more than 1 year) efficacy of transplanting ex vivo expanded limbal epithelial progenitor cells on rabbit AM based on a small limbal biopsy taken from the other eye.75-77 Collectively, the findings described above strongly support the concept that ex vivo expansion of limbal epithelial progenitor cells can be achieved by AM and explain why this new surgical procedure is useful for treatment of human patients with unilateral71,78 and bilateral79,80 total limbal stem cell deficiency. Recently, AM has also been used to expand progenitor cells from the oral mucosa as a substitute for treating corneas with bilateral limbal stem cell deficiency.81 V. IS THE AMNIOTIC MEMBRANE AN ALTERNATIVE PROGENITOR CELL NICHE?
The findings that the AM can be used as a substrate to support ex vivo expansion of limbal epithelial progenitor cells and keratocytes strongly support the concept that the AM functions as an alternative niche.82 Therefore, future studies evaluating the molecular signaling mediated by the AM should yield important information regarding how AM works. In this regard, we have identified NGF signaling as one critical pathway exerted by the AM to help expand limbal epithelial stem cells in culture.37,68 We noted that NGF protein level is readily measured by ELISA as 35.6 ± 9.1 and 41 ± 12.5 pg/mg protein in the homogenate of the intact and epithelially denuded AM, respectively, indicating that NGF is
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nic pathway mediated by MAP kinase.92,93 We speculate that both PI3KAkt-FKHRL1 and Ras-Raf-MAPK pathways may functionally be linked with suppression of TGF-β signaling in order to explain how AM may exert antiscarring action and, at the same time, support ex vivo expansion of limbal epithelial stem cells. This hypothesis is in part supported by a recent finding showing that cell survival and proliferation maintained by the Akt signaling pathway is linked to phosphorylation of FKHRL1, which is then dissociated from its DNA binding site of the promoter, leading to downregulation of Figure 7. Characterization of ex vivo expanded human limbal epithelium after TGF-β2.94 Our recent bio-informatic xenotransplantation to nude mice. The monolayer shown in Figure 6 was labeled with BrdU analysis and unpublished data disclosed for 7 days and chased for 9 days after transplantation. H & E staining showed a stratified that putative Forkhead DNA binding epithelium (A). Immunostaining confirmed basal negativity to to Cx43 (B) and K3 keratin (C), both features of the limbal epithelial phenotype in vivo. The majority of BrdU-labeled sites are present and functional not only cells remained in the basal cell layer (D). Arrows indicate the basement membrane. (Modiin the promoter of TGF-β2 as refied from Meller et al67 with permission of Br J Ophthalmol.) ported,94 but also in those of TGF-β3, TGF-βRI, TGF-βRII and TGF-βRIII, but predominantly found in the stromal matrix. uniquely absent in that of TGF-β1. This pattern is identical to The biological effects of NGF are mediated by a low the aforementioned pattern of inhibition of TGF-β and TGFaffinity receptor (p75NTR), and a high-affinity, tyrosine βR transcripts by AM (Figure 3). We are currently testing the kinase transducing receptor (TrkA).83-85 It is believed that hypothesis that AM downregulates transcription of TGF-β signaling through TrkA promotes cell survival,86,87 while and TGF-βR genes by activating the Akt-FKHRL1 pathway signaling through p75NTR promotes apoptosis and difin cultured human corneal fibroblasts. It remains to be deterferentiation.88 Immunostaining of the normal human mined whether NGF-TrkA signaling linked to TGF-β signalcorneoscleral button showed that a strongly positive TrkA ing is also utilized by AM to exert its anti-inflammatory acstaining is localized at the basal epithelial cell layer of nortion to PMNs or immune cells as mentioned above. mal corneal and limbal epithelia, with the highest intenTherefore, not only the AM provides the means to genersity noted in the stem cell-containing limbus (Figure 8). In contrast, positive staining of p75NTR is localized in the full-thickness of the corneal epithelium, but limited to the superficial layers of the limbus. This finding suggests that the limbal epithelial stem cells (located at the limbal basal epithelial layer) preferentially utilize TrkA, but not p75NTR, to sustain its unique anti-apoptotic survival. To prove that NGF signaling indeed plays an important role in maintaining expansion of limbal epithelial stem cells, we compared the epithelial outgrowth using limbal explant cultures on intact AM.67-69 As shown in Figure 9, the control culture showed vivid outgrowth in all six wells after two weeks of culturing on AM. Nevertheless, the experimental culture treated with K252a, a small MW specific inhibitor for TrkAmediated phosphorylation,89 resulted in markedly-inhibited epithelial outgrowth in all six wells. These results confirm that the NGF signaling exerted by AM is involved in promotFigure 8. Expression of TrkA and p75NTR by normal human corneolimbal epithelia. Immunostaining showed strong TrkA staining expansion of limbal epithelial stem cells. ing confined to basal and some suprabasal epithelial layers of the Signaling triggered by NGF-TrkA leads to one intracellulimbus and the peripheral and central cornea. In contrast, p75NTR 90 lar signaling pathway operated by PI3K-Akt-FKHRL1, staining was seen only in superficial epithelial layers of the limbus and the full-thickness layers of the peripheral and the central corwhich has been shown to be a dominant pathway governing nea. (Modified from Touhami et al37 with permission of Invest cell survival in both neuronal and non-neuronal cells (see Ophthalmol Vis Sci.) review91). NGF-TrkA signaling also leads to another mitoge184
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Figure 9. Outgrowth rate of HLE expanded on intact AM treated with or without K252a. In the course of 3 weeks, addition of K252a significantly suppressed explant outgrowth on AM. (Modified from Touhami et al37 with permission of Invest Ophthalmol Vis Sci.)
ate a therapeutic agent for today’s therapy, but this tissue may play an invaluable role in the struggle for the definition of the natural niche, and thereby its study should facilitate future medical drug therapy, with or without AM. Further studies determining the exact molecular mechanism underlying AM effects will undoubtedly unravel additional applications in reconstruction and engineering of both ocular and nonocular tissues in the burgeoning field of regenerative medicine. REFERENCES 1. Dua HS, Gomes JA, King AJ, Maharajan VS. The amniotic membrane in ophthalmology. Surv Ophthalmol 2004;49:51-77 2. Kim JC, Tseng SC. Transplantation of preserved human amniotic membrane for surface reconstruction in severely damaged rabbit corneas. Cornea 1995;14:473-84 3. Kruse FE, Joussen AM, Rohrschneider K, et al. Cryopreserved human amniotic membrane for ocular surface reconstruction. Graefe’s Arch Clin Exp Ophthalmol 2000;238:68-75 4. Holland EJ, Schwartz GS. The evolution of epithelial transplantation for severe ocular surface disease and a proposed classification system. Cornea 1996;15:549-56 5. Kruse FE, Rohrschneider K, Volcker HE. [Techniques for reconstruction of the corneal surface by transplantation of preseved human amniotic membrane]. Ophthalmologe 1999;96:673-8 6. Sippel KC, Ma JJ, Foster CS. Amniotic membrane surgery. Curr Opin Ophthalmol 2001;12:269-81 7. Tseng SCG, Tsubota K. Amniotic membrane transplantation for ocular surface reconstruction, in Holland EJ, Mannis MJ (eds). Ocular surface disease. NY, Berlin, Heidelberg: Springer; 2002, pp 226-31 8. Bouchard CS, John T: Amniotic membrane transplantation in the management of severe ocular surface disease: Indications and outcomes. Ocular Surface 2004;2:201-11 9. Mast BA, Diegelmann RF, Krummel TM, Cohen IK. Scarless wound healing in mammalian fetus. Surg Gyencol Obstet
1992;174:441-51 10. Adzick NS, Lorenz HP. Cells, matrix, growth factors, and the surgeon. The biology of scarless fetal wound repair. Ann Surg 1994;220:10-8 11. Foutunato SJ, Menon R, Swan KF, Lombardi SJ. Interleukin10 inhibition of interleukin-6 in human amniochorionic membrane: transcriptional regulation. Am J Obstet Gynecol 1996;175:1057-65 12. Fortunato SJ, Menon R, Lombardi SJ. Interleukin-10 and transforming growth factor-beta inhibit amniochorion tumor necrosis factor-alpha production by contrasting mechanisms of action: therapeutic implication in prematurity. Am J Obstet Gynecol 1997;177:803-9 13. Fortunato SJ, Menon R, Lombardi SJ. The effect of transforming growth factor and interleukin-10 on interleukin-8 release by human amniochorion may regulate histologic chorioamnionitis. Am J Obstet Gynecol 1998;179:794-9 14. Petraglia F, Anceschi MM, Calza L, et al. Inhibin and activin in human fetal membranes: evidence for a local effect on prostaglandin release. J Clin Endocrinol Metab 1993;77:542-8 15. Riley SC, Leask R, Balfour C, et al. Production of inhibin forms by the fetal membranes, decidua, placenta and fetus at parturition. Hum Reprod 2000;15:578-83 16. Keelan JA, Zhou RL, Mitchell MD. Activin A exerts both proand anti-inflammatory effects on human term gestational tissues. Placenta 2000;21:38-43 17. Na BK, Hwang JH, Kim JC, et al. Analysis of human amniotic membrane components as proteinase inhibitors for development of therapeutic agent of recalcitrant keratitis. Trophoblast Res 1999;13:459-66 18. Salier JP, Rouet P, Raguenez G, Daveau M. The inter-a-inhibitor family: from structure to regulation. Biochem J 1996;315:1-9 19. Romero R, Gomez R, Galasso M, et al. The natural interleukin1 receptor antagonist in the fetal, maternal, and amniotic fluid compartments: the effect of gestational age, fetal gender, and intrauterine infection. Am J Obstet Gynecol 1994;171:912-21 20. Solomon A, Rosenblatt M, Monroy D, et al. Suppression of interleukin 1alpha and interleukin 1beta in human limbal epithelial cells cultured on the amniotic membrane stromal matrix. Br J Ophthalmol 2001;85:444-9 21. Park WC, Tseng SC. Modulation of acute inflammation and keratocyte death by suturing, blood and amniotic membrane in PRK. Invest Ophthalmol Vis Sci 2000;41:2906-14 22. Wang MX, Gray TB, Parks WC, et al. Reduction in corneal haze and apoptosis by amniotic membrane matrix in excimer laser photoablation in rabbits. J Cataract Refract Surg 2001;27:310-9 23. Heiligenhaus A, Meller D, Meller D, et al. Improvement of HSV-1 necrotizing keratitis with amniotic membrane transplantation. Invest Ophthalmol Vis Sci 2001;42:1969-74 24. Shimmura S, Shimazaki J, Ohashi Y, Tsubota K. Antiinflammatory effects of amniotic membrane transplantation in ocular surface disorders. Cornea 2001;20:408-13 25. Savill J, Haslett C. Granulocyte clearance by apoptosis in the resolution of inflammation. Semin Cell Biol 1995;6:385-93 26. Ueta M, Kweon MN, Sano Y, et al. Immunosuppressive properties of human amniotic membrane for mixed lymphocyte
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HOW DOES AM WORK? / Tseng, et al reaction. Clin Exp Immunol 2002;129:464-70 27. Dostal G, Gamelli RL. Fetal wound healing. Surg Gynecol Obstet 1993;176:299-306 28. Gosiewska A, Yi CF, Brown LJ, et al. Differential expression and regulation of extracellular matrix-associated genes in fetal and neonatal fibroblasts. Wound Repair Regen 2001;9:213-22 29. Chin GS, Kim WJ, Lee TY, et al. Differential expression of receptor tyrosine kinases and Shc in fetal and adult rat fibroblasts: toward defining scarless versus scarring fibroblast phenotypes. Plast Reconstr Surg 2000;105:972-9 30. Chin GS, Lee S, Hsu M, et al. Discoidin domain receptors and their ligand, collagen, are temporally regulated in fetal rat fibroblasts in vitro. Plast Reconstr Surg 2001;107:769-76 31. Liechty KW, Adzick NS, Crombleholme TM. Diminished interleukin 6 (IL-6) production during scarless human fetal wound repair. Cytokine 2000;12:671-6 32. Liechty KW, Kim HB, Adzick NS, Crombleholme TM. Fetal wound repair results in scar formation in interleukin-10-deficient mice in a syngeneic murine model of scarless fetal wound repair. J Pediatr Surg 2000;35:866-72; discussion 872-3 33. Stelnicki EJ, Doolabh V, Lee S, et al. Nerve dependency in scarless fetal wound healing. Plast Reconstr Surg 2000;105:140-7 34. Sakuragawa N, Elwan MA, Uchida A, et al. Non-neuronal neurotransmitters and neurotrophic factors in amniotic epithelial cells: expression and function in humans and monkey. Jpn J Pharmacol 2001;85:20-3 35. Uchida S, Inanaga Y, Kobayashi M, et al. Neurotrophic function of conditioned medium from human amniotic epithelial cells. J Neurosci Res 2000;62:585-90 36. Lewin GR, Barde YA. Physiology of the neurotrophins. Annu Rev Neurosci 1996;19:289-317 37. Touhami A, Grueterich M, Tseng SC. The role of NGF signaling in human limbal epithelium expanded by amniotic membrane culture. Invest Ophthalmol Vis Sci 2002;43:987-94 38. Lee SH, Tseng SC. Amniotic membrane transplantation for persistent epithelial defects with ulceration. Am J Ophthalmol 1997;123:303-12 39. Chen HJ, Pires RT, Tseng SC. Amniotic membrane transplantation for severe neurotrophic corneal ulcers. Br J Ophthalmol 2000;84:826-33 40. Solomon A, Touhami A, Sandoval H, Tseng SC. Neurotrophic keratopathy: basic concepts and therapeutic strategies. Comprehensive Ophthalmol Update 2000;3:165-74 41. Choi TH, Tseng SC. In vivo and in vitro demonstration of epithelial cell-induced myofibroblast differentiation of keratocytes and an inhibitory effect by amniotic membrane. Cornea 2001;20:197-204 42. Ronnov-Jessen L, Petersen OW. Induction of alpha-smooth muscle actin by transforming growth factor-beta 1 in quiescent human breast gland fibroblasts. Implications for myofibroblast generation in breast neoplasia. Lab Invest 1993;68:696-707 43. Verbeek MM, Otte-Holler I, Wesseling P, et al. Induction of alpha-smooth muscle actin expression in cultured human brain pericytes by transforming growth factor-beta 1. Am J Pathol 1994;144:372-82 44. Hales AM, Schulz MW, Chamberlain CG, McAvoy JW. TGF-
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beta 1 induces lens cells to accumulate alpha-smooth muscle actin, a marker for subcapsular cataracts. Curr Eye Res 1994;13:885-90 45. Jester JV, Barry-Lane PA, Cavanagh HD, Petroll WM. Induction of alpha-smooth muscle actin expression and myofibroblast transformation in cultured corneal keratocytes. Cornea 1996;15:505-16 46. Serini G, Bochaton-Piallat M-L, Ropraz P, et al. The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by transforming growth factor-beta 1. J Cell Biol 1998;142:873-81 47. Grande JP. Role of transforming growth factor-beta in tissue injury and repair. Proc Soc Exp Biol Med 1997;214:27-40 48. Jester JV, Petroll WM, Cavanagh HD. Corneal stromal wound healing in refractive surgery: the role of myofibroblasts. Prog Retin Eye Res 1999;18:311-56 49. Lawrence DA. Transforming growth factor-beta: a general review. Eur Cytokine Netw 1996;7:363-74 50. Massague J, Chen YG. Controlling TGF-beta signaling. Genes and Development. 2000;14:627-44 51. Derynck R, Feng XH. TGF-beta receptor signaling. Biochim Biophys Acta 1997;1333:F105-F150 52. Tseng SC, Li DQ, Ma X. Suppression of transforming growth factor isoforms, TGF-beta receptor II, and myofibroblast differentiation in cultured human corneal and limbal fibroblasts by amniotic membrane matrix. J Cell Physiol 1999;179:325-35 53. Lee SB, Li DQ, Tan DT, et al. Suppression of TGF-beta signaling in both normal conjunctival fibroblasts and pterygial body fibroblasts by amniotic membrane. Curr Eye Res 2000;20:325-34 54. Birk DE, Lande MA, Fernandez-Madrid FR. Collagen and glycosaminoglycan synthesis in aging human keratocyte cultures. Exp Eye Res 1981;32:331-9 55. Funderburgh JL, Funderburgh ML, Mann MM, et al. Synthesis of corneal keratan sulfate proteoglycans by bovine keratocytes in vitro. J Biol Chem 1996;271:31431-6 56. Beales MP, Funderburgh JL, Jester JV, Hassell JR. Proteoglycan synthesis by bovine keratocytes and corneal fibroblasts: Maintenance of the keratocyte phenotype in culture. Invest Ophthalmol Vis Sci 1999;40:1658-63 57. Liu CY, Siraishi A, Kao CW, et al. The cloning of mouse keratocan cDNA and genomic DNA and the characterization of its expression during eye development. J Biol Chem 1998;273:22584-8 58. Berryhill BL, Kader R, Kane B, et al. Partial restoration of the keratocyte phenotype to bovine keratocytes made fibroblastic by serum. Invest Ophthalmol Vis Sci 2002;43:3416-21 59. Toti P, Tosi GM, Traversi C, et al. CD-34 stromal expression pattern in normal and altered human corneas. Ophthalmology 2002;109:1167-71 60. Espana EM, Kawakita T, Liu C-Y, Tseng SCG. CD-34 expression by cultured human keratocytes is downregulated during myofibroblast differentiation induced by TGF-b1. Invest Ophthalmol Vis Sci 2004, in press, 2004 61. Masur SK, Cheung JK, Antohi S. Identification of integrins in cultured corneal fibroblasts and in isolated keratocytes. Invest Ophthalmol Vis Sci 1993;34:2690-8 62. Desmouliere A, Geinoz A, Gabbiani F, Gabbiani G. Transforming
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HOW DOES AM WORK? / Tseng, et al growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J Cell Biol 1993;122:103-11 63. Masur SK, Dewal HS, Dinh TT, et al. Myofibroblasts differentiate from fibroblasts when plated at low density. Proc Natl Acad Sci U S A 1996;93:4219-23 64. Berryhill BL, Beales MP, Hassell JR. Production of prostaglandin D synthase as a keratan sulfate proteoglycan by cultured bovine keratocytes. Invest Ophthalmol Vis Sci 2001;42:1201-7 65. Espana EM, He H, Kawakita T, et al. Human keratocytes cultured on amniotic membrane stroma preserve morphology and express keratocan. Invest Ophthalmol Vis Sci 2003;44:5136-41 66. Koizumi N, Fullwood NJ, Bairaktaris G, et al. Cultivation of corneal epithelial cells on intact and denuded human amniotic membrane. Invest Ophthalmol Vis Sci 2000;41:2506-13 67. Meller D, Pires RT, Tseng SC. Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures. Br J Ophthalmol 2002;86:463-71 68. Grueterich M, Tseng SC. Human limbal progenitor cells expanded on intact amniotic membrane ex vivo. Arch Ophthalmol 2002;120:783-90 69. Grueterich M, Espana E, Tseng SC. Connexin 43 expression and proliferation of human limbal epithelium on intact and denuded amniotic membrane. Invest Ophthalmol Vis Sci 2002;43:63-71 70. Koizumi N, Cooper LJ, Fullwood NJ, et al. An evaluation of cultivated corneal limbal epithelial cells, using cell-suspension culture. Invest Ophthalmol Vis Sci 2002;43:2114-21 71. Schwab IR, Reyes M, Isseroff RR. Successful transplantation of bioengineered tissue replacements in patients with ocular surface disease. Cornea 2000;19:421-6 72. Schermer A, Galvin S, Sun TT. Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol 1986;103:49-62 73. Cotsarelis G, Cheng SZ, Dong G, et al. Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: Implications on epithelial stem cells. Cell 1989;57:201-9 74. Matic M, Petrov IN, Chen S, et al:. Stem cells of the corneal epithelium lack connexins and metabolite transfer capacity. Differentiation 1997;61:251-60 75. Ti SE, Anderson D, Touhami A, et al. Factors affecting outcome following transplantation of ex vivo expanded limbal epithelium on amniotic membrane for total limbal deficiency in rabbits. Invest Ophthalmol Vis Sci 2002;43:2584-92 76. Ti SE, Grueterich M, Espana EM, et al. Correlation of longterm phenotypic and clinical outcomes following limbal epithelial transplantation cultivated on amniotic membrane in rabbits. Br J Ophthalmol 2003;88:422-7 77. Espana EM, Ti SE, Grueterich M, et al. Corneal stromal changes following reconstruction by ex vivo expanded limbal epithelial cells in rabbits with total limbal stem cell deficiency. Br J Ophthalmol 2003;87:1509-14
78. Tsai RJ, Li LM, Chen JK. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med 2000;343:86-93 79. Koizumi N, Inatomi T, Suzuki T, et al. Cultivated corneal epithelial transplantation for ocular surface reconstruction in acute phase of Stevens-Johnson syndrome. Arch Ophthalmol 2001;119:298-300 80. Koizumi N, Inatomi T, Suzuki T, et al. Cultivated corneal epithelial stem cell transplantation in ocular surface disorders. Ophthalmology 2001;108:1569-74 81. Nakamura T, Endo K-I, Cooper LJ, et al. The successful culture and autologous transplantation of rabbit oral mucosal epithelial cells on amniotic membrane. Invest Ophthalmol Vis Sci 2003;44:106-16 82. Grueterich M, Espana EM, Tseng SC. Ex vivo expansion of limbal epithelial stem cells: amniotic membrane serving as a stem cell niche. Surv Ophthalmol 2003;48:631-46 83. Kaplan DR, Hempstead BL, Martin-Zanca D, et al. The trk proto-oncogene product: a signal transducing receptor for nerve growth factor. Science 1991;252:554-8 84. Smeyne RJ, Klein R, Schnapp A, et al. Severe sensory and sympathetic neuropathies in mice carrying a disrupted Trk/NGF receptor gene. Nature 1994;368:246-9 85. Barbacid M. Structural and functional properties of the TRK family of neurotrophin receptors. Ann N Y Acad Sci 1995;766:442-58 86. Dechant G, Barde YA. Signalling through the neurotrophin receptor p75NTR. Curr Opin Neurobiol 1997;7:413-8 87. Carter BD, Lewin GR. Neurotrophins live or let die: does p75NTR decide? Neuron 1997;18:187-90 88. Carter BD, Kaltschmidt C, Kaltschmidt B, et al. Selective activation of NF-kappa B by nerve growth factor through the neurotrophin receptor p75. Science 1996;272:542-5 89. Koizumi S, Contreras ML, Matsuda Y, et al. K-252a: a specific inhibitor of the action of nerve growth factor on PC 12 cells. J Neurosci 1988;8:715-21 90. Zheng WH, Kar S, Quirion R. FKHRL1 and its homologs are new targets of nerve growth factor Trk receptor signaling. J Neurochem 2002;80:1049-61 91. Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts. Genes Dev 1999;13:2905-27 92 Amino S, Itakura M, Ohnishi H, et al. Nerve growth factor enhances neurotransmitter release from PC12 cells by increasing Ca(2+)-responsible secretory vesicles through the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase. J Biochem (Tokyo). 2002;131:887-94 93. Barnabe-Heider F, Miller FD. Endogenously produced neurotrophins regulate survival and differentiation of cortical progenitors via distinct signaling pathways. J Neurosci 2003;23:5149-60 94. Samatar AA, Wang L, Mirza A, et al. Transforming growth factor-beta 2 is a transcriptional target of Akt/protein kinase B via forkhead transcription factor. J Biol Chem 2002;277:2811826. Epub 2002 May 14
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How Does Amniotic Membrane Work? SCHEFFER C. G. TSENG, MD, PHD,1,2 EDGAR M. ESPANA, MD,1 TETSUYA KAWAKITA, MD,1 MARIO A. DI PASCUALE, MD,1 WEI LI, MD,1 HUA HE, PHD,1 TZONG-SHYNE LIU, MD,2 TAE-HEE CHO, MD,2 YING-YING GAO, MD,2 LUNG-KUN YEH, MD,3 AND CHIA-YANG LIU, PHD3
ABSTRACT Transplantation of amniotic membrane as a temporary or permanent graft promotes epithelial wound healing and exerts potent anti-inflammatory and anti-scarring effects on the ocular surface. These actions depend on the killing of allogeneic amniotic cells and preservation of the cytokine-containing matrix during the preparation of the amniotic membrane. This review describes how these actions inherently operate in utero and how amniotic membrane transplantation aims to recreate such a fetal environment to exert these actions by insulating the surgical site from the host environment. These actions also render the amniotic membrane a unique niche capable of expanding both epithelial and mesenchymal progenitor cells ex vivo, while maintaining their normal cell phenotypes. As a result, the amniotic membrane becomes an ideal substrate for engineering different types of ocular surface tissues for transplantation. Further studies investigating the exact molecular mechanism by which the amniotic membrane works will undoubtedly unravel additional applications in reconstruction and engineering of both ocular and nonocular tissues in the burgeoning field of regenerative medicine. Accepted for publication May 2004 From 1Ocular Surface Center and TissueTech, Inc., 2Ocular Surface Research & Education Foundation, and 3Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, Florida, USA Supported in part by research grants RO1 EY06819 and R43 EY014768 (to SCGT) from National Eye Institute of National Institutes of Health, a research grant from TissueTech, Inc., and an unrestricted grant from Ocular Surface Research & Education Foundation, Miami, FL. Dr. Tseng and his family are more than 5% shareholders of TissueTech, Inc., which owns US Patents Nos. 6,152,142 and 6,326,019 on the method of preparation and clinical uses of human amniotic membrane distributed by Bio-Tissue, Inc. A patent has been filed by Dr. Tseng and Dr. Espana related to keratocytes and licensed to TissueTech, Inc. Single copy reprint requests to: Scheffer C.G. Tseng, MD, PhD (address below). Corresponding author: Scheffer C. G. Tseng, MD, PhD, Ocular Surface Center, 7000 SW 97 Avenue, Suite 213, Miami, FL 33173, USA. Tel: (305) 274-1299. Fax: (305) 274-1297. E-mail: [email protected] Abbreviations are printed in boldface where they first appear with their definitions. ©2004 Ethis Communications, Inc. The Ocular Surface ISSN: 15420124. Tseng SCG, et al. How does amniotic membrane work? 2004; 2(3):177-187.
KEYWORDS amniotic membrane, anti-inflammatory action, anti-scarring action, cytokines, in utero environment, ocular surface reconstruction, stromal matrix, transforming growth factor-β
I. INTRODUCTION linical use of amniotic membrane (AM) as a dressing in general medicine dates back to the early 20th century, and its ophthalmic use as a dressing dates back to at least half a century ago (for more historical information, see a recent review1). The use of AM as a graft for ocular surface reconstruction was first reported by Kim and Tseng in 1995.2 The popularity of this surgical procedure has since increased (Figure 1) based on a method of graft preparation (Bio-Tissue, Inc, Miami, Florida, USA [Tseng SCG: Grafts made from amniotic membrane; methods for separating, preserving, and using such grafts in surgeries. TissueTech, Inc. 2000, #6,152,142; 2001, #6,326,019]) that kills allogeneic amniotic cells3 but maintains the integrity of AM cytokine-rich extracellular matrix; thus, the AM graft exerts potent anti-inflammatory, anti-scarring and anti-angiogenic actions and promotes epithelial healing in recipient eyes. In 2001, the US Food and Drug Administration (FDA) ruled that an AM graft thus prepared is homologous to the ocular surface, and hence approved it as a “tissue,” so AM transplantation for ocular surface reconstruction does not require premarket approval. In contrast, dehydrated and decellularized AM is not considered a “tissue” by FDA when used as a graft and requires premarket approval if advertised for wound repair. (See http://www.fda.gov/cber/tissue/trgfyrpts.htm FDA website, Tissue Action Plan, Tissue Reference Group Annual Reports.) In January 2004, AM transplantation as a graft for ocular surface reconstruction was approved as a standard surgical procedure by Medicare in the USA. Amniotic membrane has been used as either a permanent or temporary graft. As a permanent graft, AM is applied in one or multiple layers for corneal, conjunctival, or entire ocular surface reconstruction. It can be used alone or in conjunction with transplantation of conjunctival epithelial stem cells via conjunctival autograft or with trans-
C
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HOW DOES AM WORK? / Tseng, et al OUTLINE I. Introduction II. In utero function of the amniotic membrane A. Anti-inflammatory action B. Anti-scarring action III. Maintenance of keratocyte phenotype during ex vivo expansion on amniotic stromal matrix IV. Restoration of stromal niche important for ex vivo expansion of limbal epithelial progenitor cells V. Is the amniotic membrane an alternative progenitor cell niche?
plantation of limbal epithelial stem cells via conjunctival limbal autograft, keratolimbal allograft, or living-related conjunctival limbal allograft (see classification4). As a permanent graft, the surrounding host epithelial cells will migrate onto the amniotic basement membrane, while host mesenchymal cells will migrate into the amniotic stromal matrix. As a result, transplanted AM will be integrated into the recipient site. As a temporary graft or patch or dressing, the host epithelial cells will migrate underneath AM, and upon healing, the transplanted AM will invariably dissolve. Figure 2 lists the clinical indications for AM transplantation based on Donor-Recipient Information retrieved by Bio-Tissue, Inc. (Miami, FL, USA) during 1997 to 2002, (i.e., prior to Medicare approval). The main objective of this review is to describe the action mechanisms of AM transplantation for ocular sur-
Figure 1. Number of papers published regarding AM transplantation in ophthalmology. Data were obtained from Medline search of English-language literature.
face reconstruction. We retrieved from a PubMed search all articles based on the key words amniotic membrane, eye, and function, and we included only those that contain experimental (basic or clinical) data related to the possible action mechanisms. We excluded articles that describe only results of clinical studies. Published abstracts without accompanying papers were also excluded. We focus on antiinflammatory and anti-scarring effects in this review. Readers who are interested in clinical studies on the use of AM should consult the following reviews and references therein,1,5-7 as well as the review by Bouchard and John that is published in this issue of The Ocular Surface.8
Figure 2. Clinical indications for AM transplantation. Data was obtained from Donor-Recipient Information retrieved during 1997 to 2002 at Bio-Tissue, Inc. (Miami, FL). (N = 3,354 Data Source: Bio-Tissue, Inc.)
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HOW DOES AM WORK? / Tseng, et al II. IN UTERO FUNCTION OF THE AMNIOTIC MEMBRANE
A number of clinical studies have shown that AM transplantation facilitates epithelial wound healing and reduces inflammation, scarring, and angiogenesis. These actions resemble the scarless fetal wound healing observed in utero.9,10 Therefore, a closer look at the role of the AM during fetal development may shed light on how this tissue works. Anatomically, AM, or amnion, is the innermost membrane enwrapping the fetus in the amniotic cavity, which contains the amniotic fluid. As a result of enlargement of the fetus toward the end of the first trimester, the membrane is fused with the chorion to form the fetal membrane. From this point onward, these two membranes are contiguous with, and become a part of, the placenta. The placenta supports the development and survival of the fetus by providing exchange of metabolic and gaseous products, nutrients, and electrolytes, and by producing maternal antibodies and developmentally important hormones. Developmentally, both the placenta and fetal membrane are derived from the outer cell mass, while the fetus is derived from the inner cell mass of the blastocyst. Both outer cell mass and inner cell mass are developed from the fertilized egg, indicating that AM and the fetus share the same cell origin. The fetal membrane physically separates the fetus from the maternal environment (i.e., uterus). In the fetal membrane, the amnion and the chorion are only loosely attached, whereas the chorion and the underlying decidua tightly adhere to each other. Before the rupture of membrane prior to delivery, maternal cells, including circulating inflammatory and immune cells, exit from decidual postcapillary venules and encounter chorion trophoblasts. Because inappropriate activation of inflammatory cells in the membrane environment contributes to the pathogenesis of preterm labor, the integrity of the fetal membrane guards against such a potential threat to pregnancy. Because the chorion is vascularized and contiguous with the decidua of the placenta, the true protective layer of the fetal membrane resides in the AM. One important function of the AM is to protect the fetus from cellular insults derived from the maternal environment. This concept is evidenced by the facts that intentional amniotomy induces labor or abortion and that premature rupture of the membrane leads to delivery. The AM achieves this important function of protecting the fetus by exerting critical anti-inflammatory and anti-scarring effects, as described below. A. Anti-inflammatory Action
Although the cytokines present in the AM matrix are important, the cellular sources of these distinct cytokines have not been elucidated. The AM’s anti-inflammatory action may be mediated in part by interleukin-10 (IL-10). (We detect a significant amount of IL-10 in AM extracts using ELISA [unpublished data]). IL-10 is known to suppress or counteract the actions of pro-inflammatory cytokines such as IL-611 and tumor necrosis factor-alpha (TNF-α).12 IL-10 also suppresses amniotic cell produc-
tion of IL-8,13 which is a pro-inflammatory chemokine attracting the migration of neutrophils. Furthermore, the AM produces inhibin and activin; both belonging to the TGF-β superfamily. Activin promotes the production of prostaglandin E2 (PGE2).14,15 A low dose of activin stimulates, but a high dose of activin inhibits, the production of IL-6, IL-8, and PGE2 by the AM.16 No such effect is noted in the chorion or decidua. Furthermore, production of TNF-α is significantly inhibited by activin in the chorion and deciduas.16 The AM contains various protease inhibitors, including α1 anti-trypsin and inter-α-trypsin inhibitor,17 the latter of which may exert an anti-inflammatory effect [see review18]. Future studies are needed to determine whether IL-10, activin, protease inhibitors, and/or a combination of them are responsible for the anti-inflammatory action of AM when it is transplanted to the ocular surface. The AM contains IL-1 receptor antagonist (IL-1RA) and helps transport it to the amniotic fluid.19 IL-1RA is a potent inhibitor of IL-1, and, thus, will suppress the inflammation mediated by IL-1. Data from our laboratory have shown that limbal epithelial cells cultured on the AM stromal matrix downregulate the expression and production of IL-1, but upregulate the expression and production of IL-1RA, resulting in a higher ratio of IL-1RA/IL-1.20 Such an effect withstands the challenge of lipopolysaccharide, a known stimulator of IL-1 production.20 Collectively, these findings support the concept that the AM may exert its anti-inflammatory action by suppressing the signaling pathway via IL-1, at least in epithelial cells. Additional studies are needed to verify such an action in inflammatory or immune cells. Even if the AM contains the aforementioned anti-inflammatory mediators, its anti-inflammatory action may require a close contact with its stromal matrix. We have reported that activated neutrophils are rendered apoptotic 24 hours after excimer laser-induced phototherapeutic keratectomy or photorefractive keratectomy in rabbit corneas when in contact with human AM stroma matrix as a temporary graft, but not with the amniotic basement membrane.21,22 As a result, inflammatory cells are excluded from the ablated stroma, resulting in less corneal haze. A similar finding was also noted in a murine model of HSV1-induced keratitis, where infiltration of lymphocytes and macrophages was markedly reduced 48 hours after the cornea was covered with human AM as a temporary graft.23 Shimmura et al24 noted that both mononuclear and polymorphonuclear inflammatory cells and lymphocytes are trapped by AM stromal matrix and induced to undergo apoptosis. Clearance of granulocytes by pushing them into rapid apoptosis is an effective anti-inflammatory strategy25; thus, we speculate that further investigation into the molecular mechanism by which AM stromal matrix exerts its anti-inflammatory action may identify new important therapeutic directions for modulating innate immunity. Recently, Ueta et al26 also reported that human AM can suppress alloreactive responses and downregulate production of Th1 and Th2 cytokines in mouse lymphocytes in vitro. This finding suggests that the AM may also suppress acquired immunity.
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HOW DOES AM WORK? / Tseng, et al B. Anti-scarring Action
It has been recognized in the field of pediatric surgery that incisions made through the fetal skin before the third trimester do not result in scar formation at birth. This phenomenon termed scarless fetal wound healing has been a subject of intensive study for the last three decades.9,10,27 One natural explanation is that fetal fibroblasts are intrinsically different from adult fibroblasts in their response to scar-promoting cytokines.28-30 Nevertheless, there is strong evidence in the literature that the AM plays a direct and positive role in exerting an anti-scarring effect during fetal wound healing. As stated above, the AM contains the anti-inflammatory cytokine, IL-10, which can inhibit the production of IL-6.11 Diminished IL-6 production is one hallmark of scarless fetal wound repair.31 Furthermore, the phenomenon of fetal scarless wound healing was abolished in IL-10-knockout mice.32 The AM may exert an anti-scarring effect by supporting nerve growth in the fetus, which is essential to endow the fetus with scarless fetal wound healing. It has been reported that denervation (i.e., cutting the sciatic nerve) of the hind limb on one side will abolish the phenomenon of scarless fetal wound healing on that side, as compared to the intact innervated hind limb on the other side.33 It is intriguing to note that the AM is not innervated (i.e., not supplied by nerves), and yet amniotic epithelial cells synthesize various neurotransmitters,34 neuropeptides, and neurotrophins.35 The production of neurotrophic factors, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), is particularly significant, as these factors control the growth and targeting of sensory and autonomic nerves to the peripheral tissues (see review36). The fact that the AM contains and produces these neurotrophins strongly suggests that its main purpose is to help the development of the nervous system in the fetus, thereby ensuring scarless wound healing. Experimental data from our laboratory have shown that cryopreserved AM contains abundant amounts of NGF and permits the expression of NGF receptors by the limbal epithelial cells when grown over the membrane in culture.37 This new finding supports the concept that the AM plays an active role in supporting the target tissue to express and respond to such a neurotrophin as NGF. This finding may explain, in part, why AM transplantation is effective in treating neurotrophic corneal ulcers, which are caused by corneal denervation.38-40 Additionally, we have now gathered strong evidence that AM stromal matrix exerts a direct anti-scarring action on ocular surface fibroblasts. When human AM was transplanted into the rabbit corneal stromal pocket, we noted that epithelial-induced differentiation of corneal stromal fibroblasts (keratocytes) into myofibroblasts is inhibited.41 To confirm that such an in vivo anti-scarring action is not caused indirectly by suppressing inflammation via inflammatory cells or epithelial cells as described above, we investigated the expression of transforming growth factorbeta (TGF-β) genes and their receptors by ocular surface fibroblasts cultured directly on AM stromal matrix. Three different TGF-βs, i.e., TGF-β1, TGF-β2 and 180
TGF-β3, each encoded by separate genes, are expressed by mammalian cells. TGF-β turns out to be the most potent cytokine promoting myofibroblast differentiation by upregulating expression of α-SMA,42-45 integrin α5β1, and EDA domain-containing fibronectin (Fn)46 in a number of cell types, including fibroblasts (see reviews47-48). TGF-β also upregulates the expression of such matrix components as collagens and proteoglycans, downregulates proteinase and matrix metalloproteinases (MMP), and upregulates their inhibitors. Collectively, these actions result in increased cell-matrix interactions and adhesiveness, as well as deposition and formation of scar tissue.47-49 TGF-βs exert their actions via binding with TGF-β receptors (TGF-βRs) on the cell membrane. In human cells, there are three TGFβRs, namely, TGF-βR type I (TGF-βRI), type II (TGF-βRII), and type III (TGF-βRIII). TGF-βs, serving as ligands, bind with a serine, threonine kinase receptor complex made of TGF-βRI and TGF-βRII50; such a binding is facilitated by TGF-βRIII, which is not a serine, threonine kinase receptor. Binding with TGF-βRII activates TGF-βRI, which is responsible for direct phosphorylation of a family of effector proteins known as Smads, which modulate transcription of a number of target genes, including those described above, participating in scar formation (see reviews50,51). Our laboratory data strongly support the concept that AM stromal matrix exerts a direct, potent, anti-scarring action on ocular surface fibroblasts by suppressing the TGF-β signaling at the transcriptional level. In short, we discovered that transcript expression of TGF-β2 and TGF-β3, but not TGF-β1, as well as transcript expression of TGF-βRI, TGFβRII and TGF-βRIII, are markedly downregulated in fibroblasts derived from normal human cornea,52 limbus52 and conjunctiva53 and in abnormal fibroblasts derived from patients with pterygium,53 cultured in either a serum-containing or serum-free medium (Figure 3A and B). Such a pattern of transcript and protein inhibition can withstand the challenge by exogenous TGF-β1 in a medium with or without 1% FBS (Figure 3B). Furthermore, such transcript suppression is followed by marked suppression of both transcripts and proteins of downstream TGF-β target genes encoding α-SMA (Figure 3C and D), EDA-containing Fn, and integrin α5β1.52,53 Collectively, these findings support the concept that AM stromal matrix may exert a direct anti-scarring action on fibroblasts by suppressing TGF-β signaling at the transcriptional level, leading to downregulation of several downstream scar-forming genes responsible for scar formation. III. MAINTENANCE OF KERATOCYTE PHENOTYPE DURING EX VIVO EXPANSION ON AMNIOTIC STROMAL MATRIX
Suppression of TGF-β signaling by AM stromal matrix is not only pathologically important in preventing myofibroblast differentiation and scar formation, but it is also physiologically relevant in maintaining the normal phenotype of corneal keratocytes. Keratocytes embedded in the corneal stroma are a unique population of cranial neural crest-derived mesenchymal cells that play an important role in maintaining
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Figure 3. Northern hybridization. Transcription of TGF-β2, TGF-β3, TGF-βRI, TGF-βRII and TGF-βRIII, but not TGF-β1 is suppressed by AM in human corneal fibroblasts (HCF) and human limbal fibroblasts (HLF [upper left]), and in human conjunctival fibroblasts (HJF) and pterygium body fibroblasts (PBF [upper right],) as compared to collagen gel (lower left) or plastic (P) in both serum-containing (FBS or D-FBS) medium and serum-free (ITS or D-ITS) medium. In the serum-free medium, addition of 10 ng/ml TGF-β1 upregulates TGF-β1 mRNA but not others. Transcription of α-SMA is correspondingly downregulated by AM (lower left). Western blot (lower right) confirms that protein expression of α-SMA is upregulated on plastic cultures by 10 ng/ml TGF-β for 7 days in DMEM with 1% FBS, but remains suppressed on AM. (Modified with permission from J Cell Physiology,52 Curr Eye Res,53 and Invest Ophthalmol Vis Sci.60)
corneal transparency. Under the normal in vivo condition, keratocytes are characteristically mitotically quiescent. They exhibit a dendritic morphology with extensive intercellular contacts, and synthesize the collagens I, V, VI, and XII54 and keratan sulfate-containing proteoglycans, such as lumican, keratocan, and mimecan.55,56 They express several keratan sulfate-rich proteoglycans, including keratocyte-specific keratocan57,58 and CD34.59,60 To study keratocyte biology and the role of keratocytes in maintaining corneal stromal transparency, it is important to isolate and maintain these cells in culture. Interestingly,
keratocytes isolated from bovine56 and rabbit45 corneal stroma and cultured on plastic in a serum-containing medium rapidly lose their dendritic characteristics and acquire a fibroblastic morphology.58 At the same time, they turn into myofibroblasts with the expression of integrin α5β161 and α-SMA,63 especially when seeded at a low density. One way to preserve the ability of keratocytes to secrete lumican, keratocan, and mimecan is to culture them in a serum-free culturing condition.58,64 However, this serum-free culturing method precludes ex vivo expansion and subculturing (see review48).
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Figure 4. Morphology and expression of keratocan by RT-PCR. Primary cultures of collagenase-isolated human corneal fibroblasts cultured on plastic (P) are more dendritic in a serum-free medium (0% FBS), but become fibroblastic when 10% FBS is added. In contrast, cells cultured on AM stromal matrix retain dendritic morphology and intensive cell-cell contact even in the presence of 10% FBS. Expression of keratocan transcript was dramatically increased from plastic to AM cultures even when FBS was added (peaking at 5% FBS). K: RNA extracted directly from human cornea in vivo. (Modified from Espana EM et al65 with permission of Invest Ophthalmol Vis Sci.)
Our recent laboratory data showed that AM stromal matrix allows ex vivo human corneal keratocytes (HCF) to effectively expand for up to five passages in a serum-containing medium, while maintaining their characteristic dendritic morphology and continuous expression of keratocan (Figure 4).65 A similar result was reproduced in mouse keratocytes for up to passage eight (not shown). As shown in Figure 3D, we also noted that expression of CD34, another new membrane marker for keratocytes,60 was similarly maintained. To further delineate the experimental matrix conditions governing keratocyte maintenance (i.e., expression of keratocan and CD34) or myofibroblast differentiation (i.e., expression of αSMA), we subcultured HCF between AM and plastic from primary cultures. Figure 5 shows that continuous passages on AM (e.g., AA or AAA) were essential for maintaining CD34 expression, while subpassage ending up on plastic rendered α-SMA expression whether or not the cells were first exposed to AM. Interestingly, subculturing cells from plastic to AM suppressed α-SMA expression, but did not regain expression of CD34 (Figure 5). Therefore, we have established culturing conditions to modulate keratocyte phenotype from 182
keratocytes, myofibroblasts, and the “transitional state” of fibroblasts. Future studies are underway to confirm that the latter state is indeed a transitional state between keratocytes and myofibroblasts by their expression of α5β1 integrins and EDA-containing Fn.
Figure 5. Western blot . Primary culture of HCF was established on either AM (A) or plastic (P) and subpassaged to either A or P (and PP, PPP, etc.). Equal amounts of cell extracts by RIPA buffer were loaded as verified by β-actin and analyzed by antibodies to CD34 and α-SMA, respectively. CD34 was expressed only by continuous subculturing on AM (e.g., AA or AAA). In contrast, α-SMA was markedly promoted when cells were continuously cultured on P (e.g., PP or PPP) or ended up on P (e.g., AP or PAP). [Modified from Espana et al60 with permission of Invest Ophthalmol Vis Sci.)
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E
Figure 6. Ex vivo expansion of human limbal progenitor cells. Human limbal explants prepared from corneoscleral buttons were seeded on either intact or epithelially-denuded AM and cultured in SHEM (A). Epithelial outgrowth started from the limbal region (A, arrows) and migrated onto the amniotic membrane after 5 days (B). The outgrowth on intact AM sometimes adhered over the amniotic epithelial cells (C, arrows) and consisted of a monolayer of small and compact cuboidal epithelial cells (D). The epithelial outgrowth was faster on denuded AM than on intact AM (E). (Modified from Meller et al67 with permission of Br J Ophthalmol.)
IV. RESTORATION OF STROMAL NICHE IMPORTANT FOR EX VIVO EXPANSION OF LIMBAL EPITHELIAL PROGENITOR CELLS
Besides maintaining the normal keratocyte phenotype, the AM has also been used as a substrate in several protocols to expand limbal epithelial progenitor cells directly from limbal explants,66-69 cell suspensions from the limbal epithelium,70 or indirectly from prior expansion on 3T3 fibroblast feeder layers.71 As shown in Figure 6, a compact monolayer of small epithelial cells with a scanty cytoplasm migrates from the limbal explant to the basement membrane surface of the AM. Similar to the findings of Koizumi et al,66 we noted that epithelial outgrowth reaches a diameter of 2030 mm in 18-25 days on intact AM (retaining the amniotic epithelial cells), whereas it takes 11-18 days for epithelially denuded AM. Such an epithelial outgrowth is rarely generated from human peripheral or central cornea.67 On intact AM, the resultant epithelial phenotype of the ex vivo expanded limbal epithelial cell layer has been characterized to be a limbal epithelium manifesting features of limbal basal epithelial progenitor cells, such as: 1) poor differentiation, i.e., lack of expression of a cornea-specific keratin K3,72 2) slower proliferation rate, resembling the in vivo slow-cycling property,73 and 3) lack of expression of connexin 43 (Cx43),74 a gap junction protein controlling intercellular communication upon xenotransplantation to the subcutaneous plane of nude mice to induce stratification and differentiation (Figure 7).67,68 In contrast, though growing faster (Figure 6), the resultant epithelial phenotype is corneal epithelium on denuded AM. Due to the lack of definitive marker(s) for limbal epithelial stem cells, future studies are needed to
confirm the necessity of including devitalized amniotic epithelial cells during ex vivo expansion on AM. In a rabbit model of unilateral limbal stem cell deficiency, we have recently confirmed the long-term (more than 1 year) efficacy of transplanting ex vivo expanded limbal epithelial progenitor cells on rabbit AM based on a small limbal biopsy taken from the other eye.75-77 Collectively, the findings described above strongly support the concept that ex vivo expansion of limbal epithelial progenitor cells can be achieved by AM and explain why this new surgical procedure is useful for treatment of human patients with unilateral71,78 and bilateral79,80 total limbal stem cell deficiency. Recently, AM has also been used to expand progenitor cells from the oral mucosa as a substitute for treating corneas with bilateral limbal stem cell deficiency.81 V. IS THE AMNIOTIC MEMBRANE AN ALTERNATIVE PROGENITOR CELL NICHE?
The findings that the AM can be used as a substrate to support ex vivo expansion of limbal epithelial progenitor cells and keratocytes strongly support the concept that the AM functions as an alternative niche.82 Therefore, future studies evaluating the molecular signaling mediated by the AM should yield important information regarding how AM works. In this regard, we have identified NGF signaling as one critical pathway exerted by the AM to help expand limbal epithelial stem cells in culture.37,68 We noted that NGF protein level is readily measured by ELISA as 35.6 ± 9.1 and 41 ± 12.5 pg/mg protein in the homogenate of the intact and epithelially denuded AM, respectively, indicating that NGF is
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nic pathway mediated by MAP kinase.92,93 We speculate that both PI3KAkt-FKHRL1 and Ras-Raf-MAPK pathways may functionally be linked with suppression of TGF-β signaling in order to explain how AM may exert antiscarring action and, at the same time, support ex vivo expansion of limbal epithelial stem cells. This hypothesis is in part supported by a recent finding showing that cell survival and proliferation maintained by the Akt signaling pathway is linked to phosphorylation of FKHRL1, which is then dissociated from its DNA binding site of the promoter, leading to downregulation of Figure 7. Characterization of ex vivo expanded human limbal epithelium after TGF-β2.94 Our recent bio-informatic xenotransplantation to nude mice. The monolayer shown in Figure 6 was labeled with BrdU analysis and unpublished data disclosed for 7 days and chased for 9 days after transplantation. H & E staining showed a stratified that putative Forkhead DNA binding epithelium (A). Immunostaining confirmed basal negativity to to Cx43 (B) and K3 keratin (C), both features of the limbal epithelial phenotype in vivo. The majority of BrdU-labeled sites are present and functional not only cells remained in the basal cell layer (D). Arrows indicate the basement membrane. (Modiin the promoter of TGF-β2 as refied from Meller et al67 with permission of Br J Ophthalmol.) ported,94 but also in those of TGF-β3, TGF-βRI, TGF-βRII and TGF-βRIII, but predominantly found in the stromal matrix. uniquely absent in that of TGF-β1. This pattern is identical to The biological effects of NGF are mediated by a low the aforementioned pattern of inhibition of TGF-β and TGFaffinity receptor (p75NTR), and a high-affinity, tyrosine βR transcripts by AM (Figure 3). We are currently testing the kinase transducing receptor (TrkA).83-85 It is believed that hypothesis that AM downregulates transcription of TGF-β signaling through TrkA promotes cell survival,86,87 while and TGF-βR genes by activating the Akt-FKHRL1 pathway signaling through p75NTR promotes apoptosis and difin cultured human corneal fibroblasts. It remains to be deterferentiation.88 Immunostaining of the normal human mined whether NGF-TrkA signaling linked to TGF-β signalcorneoscleral button showed that a strongly positive TrkA ing is also utilized by AM to exert its anti-inflammatory acstaining is localized at the basal epithelial cell layer of nortion to PMNs or immune cells as mentioned above. mal corneal and limbal epithelia, with the highest intenTherefore, not only the AM provides the means to genersity noted in the stem cell-containing limbus (Figure 8). In contrast, positive staining of p75NTR is localized in the full-thickness of the corneal epithelium, but limited to the superficial layers of the limbus. This finding suggests that the limbal epithelial stem cells (located at the limbal basal epithelial layer) preferentially utilize TrkA, but not p75NTR, to sustain its unique anti-apoptotic survival. To prove that NGF signaling indeed plays an important role in maintaining expansion of limbal epithelial stem cells, we compared the epithelial outgrowth using limbal explant cultures on intact AM.67-69 As shown in Figure 9, the control culture showed vivid outgrowth in all six wells after two weeks of culturing on AM. Nevertheless, the experimental culture treated with K252a, a small MW specific inhibitor for TrkAmediated phosphorylation,89 resulted in markedly-inhibited epithelial outgrowth in all six wells. These results confirm that the NGF signaling exerted by AM is involved in promotFigure 8. Expression of TrkA and p75NTR by normal human corneolimbal epithelia. Immunostaining showed strong TrkA staining expansion of limbal epithelial stem cells. ing confined to basal and some suprabasal epithelial layers of the Signaling triggered by NGF-TrkA leads to one intracellulimbus and the peripheral and central cornea. In contrast, p75NTR 90 lar signaling pathway operated by PI3K-Akt-FKHRL1, staining was seen only in superficial epithelial layers of the limbus and the full-thickness layers of the peripheral and the central corwhich has been shown to be a dominant pathway governing nea. (Modified from Touhami et al37 with permission of Invest cell survival in both neuronal and non-neuronal cells (see Ophthalmol Vis Sci.) review91). NGF-TrkA signaling also leads to another mitoge184
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Figure 9. Outgrowth rate of HLE expanded on intact AM treated with or without K252a. In the course of 3 weeks, addition of K252a significantly suppressed explant outgrowth on AM. (Modified from Touhami et al37 with permission of Invest Ophthalmol Vis Sci.)
ate a therapeutic agent for today’s therapy, but this tissue may play an invaluable role in the struggle for the definition of the natural niche, and thereby its study should facilitate future medical drug therapy, with or without AM. Further studies determining the exact molecular mechanism underlying AM effects will undoubtedly unravel additional applications in reconstruction and engineering of both ocular and nonocular tissues in the burgeoning field of regenerative medicine. REFERENCES 1. Dua HS, Gomes JA, King AJ, Maharajan VS. The amniotic membrane in ophthalmology. Surv Ophthalmol 2004;49:51-77 2. Kim JC, Tseng SC. Transplantation of preserved human amniotic membrane for surface reconstruction in severely damaged rabbit corneas. Cornea 1995;14:473-84 3. Kruse FE, Joussen AM, Rohrschneider K, et al. Cryopreserved human amniotic membrane for ocular surface reconstruction. Graefe’s Arch Clin Exp Ophthalmol 2000;238:68-75 4. Holland EJ, Schwartz GS. The evolution of epithelial transplantation for severe ocular surface disease and a proposed classification system. Cornea 1996;15:549-56 5. Kruse FE, Rohrschneider K, Volcker HE. [Techniques for reconstruction of the corneal surface by transplantation of preseved human amniotic membrane]. Ophthalmologe 1999;96:673-8 6. Sippel KC, Ma JJ, Foster CS. Amniotic membrane surgery. Curr Opin Ophthalmol 2001;12:269-81 7. Tseng SCG, Tsubota K. Amniotic membrane transplantation for ocular surface reconstruction, in Holland EJ, Mannis MJ (eds). Ocular surface disease. NY, Berlin, Heidelberg: Springer; 2002, pp 226-31 8. Bouchard CS, John T: Amniotic membrane transplantation in the management of severe ocular surface disease: Indications and outcomes. Ocular Surface 2004;2:201-11 9. Mast BA, Diegelmann RF, Krummel TM, Cohen IK. Scarless wound healing in mammalian fetus. Surg Gyencol Obstet
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