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DOI 10.1002/prca.201300049
Proteomics Clin. Appl. 2014, 8, 178–184
REVIEW
Proteomic analysis as a means to approach limbal stem cell biology in a search for stem cell markers Bent Honore´ 1 and Henrik Vorum2∗ 1 2
Department of Biomedicine, Aarhus University, Aarhus, Denmark Department of Ophthalmology, Aalborg University Hospital, Aalborg, Denmark
The cornea consists of three main layers: an outer surface epithelium, the stroma, and the endothelium. A clear cornea is necessary for optimal vision and is maintained and repaired from limbal epithelial stem cells located in the limbus between the cornea and the sclera. Diseases and injury may result in deficiency of the stem cells impairing their ability to renew the corneal epithelium. Patients with limbal stem cell deficiency experience chronic pain and ultimately blindness. Attempts to treat the disease are based on replacement of the stem cells by transplantation or by culturing the stem cells. We here review the proteomic techniques that so far have been used to approach characterization of limbal stem cells and markers to identify them. It is apparent that the field is in a rather inchoate state due to the scarcity and relative inaccessibility of the stem cells. However, the importance of revealing limbal stem cell biology and identifying stem cell biomarkers calls for greater use of emerging methodology. Strategies for future studies are discussed.
Received: June 26, 2013 Revised: October 6, 2013 Accepted: December 2, 2013
Keywords: 2D-PAGE / Limbal stem cells / Stem cell markers
1
Introduction
The surface of the eye ball consists of the sclera, conjuntiva, limbus, and the cornea (Fig. 1). The cornea is the clear part at the front of the eye surrounded by the limbus. The cornea consists of three main layers, an outer surface epithelium, separated by Bowman’s layer from the stroma, which again is separated by Descemet’s membrane from the endothelium. The central corneal epithelium has limited proliferative properties as compared with the peripheral corneal epithelium [1]. At the corneoscleral limbus the limbal stem cells are located in the basal epithelium as originally proposed by Davanger and Evensen [2]. Here, the stem cells may be supplied with factors from the local environment, the so-called stem cell niche [3, 4]. It is proposed that the stem cell niche consists of anatomical and functional dimensions that maintain “stemness.” The niche contains stromal invaginations called the palisades of Vogt [5]. The palisades of Vogt contain a distinct
´ Correspondence: Professor Bent Honore, Department of Biomedicine, Aarhus University, Ole Worms Alle´ 3, DK-8000 Aarhus C, Denmark E-mail: [email protected] Fax: +45-86-13-11-60 Abbreviations: LSCD, limbal stem cell deficiency; SOD2, superoxide dismutase 2 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
vasculature with radially oriented hairpin loops of arteries and veins [5]. This structure is suggested to protect stem cells from trauma and to give access to substances that may diffuse from the nearby vascular network. More recently, additional anatomical structures have been described to be part of the stem cell niche, the limbal epithelial crypt [6, 7], the limbal crypt [8], and focal stromal projections [8]. These anatomical structures are not uniformly spread in the limbal region but mainly localized in the superior region [9]. These structures consist of cells that stain positive for some of the putative limbal stem cell markers [6–8]. A number of observations indicate that the basal membrane in the region of the stem cell niche has a specific composition of the extracellular matrix that differ from that found in other parts of the cornea and that this probably creates the special environment that supports “stemness,” that is, inhibiting differentiation and maintaining the proliferative abilities of the limbal stem cells [4]. The stem cells are responsible for maintaining a healthy cornea. The so-called X, Y, Z hypothesis introduced by Thoft and Friend [10] describes a net movement of cells from the basal limbal epithelium (X) that adds to the net movement of more centrally migrating epithelial cells in the cornea (Y). It is hypothesized that the two components added are equal to the cells that shed at the central cornea (Z). Although the ∗ Additional corresponding author: Professor Henrik Vorum, E-mail: [email protected]
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Figure 1. The surface of the eye ball consists of sclera, conjuntiva, limbus, and cornea. The stem cells are located in the limbus (stippled circle), which is the region between cornea and conjunctiva.
concept with limbus as the sole localization has been challenged some years ago [11], the localization of stem cells in the limbus only is greatly accepted [12, 13] and more recent transcriptomic analysis also support that stem cells are preferentially located in the limbal epithelial crypts [14]. Injury or diseases may result in limbal stem cell deficiency (LSCD) with impairment in the renewal of the corneal epithelium resulting in impaired vision and chronic pain [13]. The disease may be treated medically or surgically (Fig. 2). The medical treatment is used to relieve the symptoms of pain and inflammation by using lubricants, corticosteroids, and autologous serum drops [15]. The medical treatment, however, cannot cure the disease. Surgery is required for this and can be divided in autologous and allogenic transplantation of tissue or stem cells. Allogenic transplantation of limbal corneal grafts from living donors or cadavers is a possibility. However, the disadvantage is that systemic immunosuppression is needed to maintain the graft. Autologous transplatation is possible for unilateral disease, but the amount of tissue is limited and there is a risk of inducing iatrogenic LSCD in the donor eye. It is therefore of interest to isolate and cultivate the cells in order to be able to obtain enough cells for therapy. A number of different types of cells have been cultivated for the purpose, including oral mucosa cells, embryonic stem cells, and stem cells from hair follicles, umbilical cord, and immature dental pulp as reviewed by Osei-Bempong et al. [12]. However, here we will focus on limbal epithelial stem cells. Stem cells are characterized by their undifferentiated phenotype, a small cell size, a high nucleus to cytoplasm ratio, and a low mitotic activity [16, 17]. There is no specific single marker that identifies a stem cell. Instead the expression pattern of a set of proteins is used to characterize stem cells as recently reviewed by a number of authors [12, 16–18]. Examples of protein expression patterns characteristic of stem cells include presence of ABCG2, vimentin, and cytokeratins 5 and 14, and the absence of cytokeratins 3 and 12, connexin 43, and nestin. Thus, in order to properly be able to isolate and cultivate stem cells for therapy a great deal of work is still required to characterize the protein expression pattern of stem cells as well as their biological properties. This would help to optimize the growth conditions for proper expansion of the cells. We here review how proteomic techniques have been used recently to approach characterization of stem cell biology and the identification of putative markers. Some studies have focused on analysis of the proteomic composition of the limbal stem cell, while others have focused on the identi C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
fication of factors that are important for maintaining the stem cells. In the long term this may help to identify the necessary growth conditions. First, we will review the proteomic studies performed to approach a characterization of the stem cells as well as the support layer necessary for the cultivation of the cells and then suggest future studies that may be performed in order to improve our understanding of limbal stem cell biology.
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Proteomic analyses of cornea and stem cells
The first study performed on human tissue aiming to identify limbal stem cell protein markers by a proteomic approach was performed by Lyngholm et al. [19]. They used a trephine to mark the central 8 and 10 mm of the corneal epithelium. Then it was stippled with 70% ethanol to loosen the epithelium from the stroma. The central 8 mm disc was scraped-off and collected as the central part of the cornea. The fraction containing the 8–10 mm ring of the intermediate epithelium was discarded and finally the limbal fraction located outside of the 10-mm ring was scraped-off and collected as the limbal fraction. By comparing the 2D-PAGE images of central parts of the corneas from cadavers (taken within 48 h post mortem) with the cornea from a living myopic patient they found no apparent degradation in the cadaver corneas. The study was then performed on cadaver corneas. The aim was to indentify proteins upregulated in the limbal cornea, since proteins upregulated here might be candidates to be among stem cell markers. More than 1000 protein spots were detected by silver staining [20] and 32 spots were significantly overexpressed in the central part while 70 spots were significantly overexpressed in the limbal part. Twenty-five different proteins were identified by nano-LC-ESI-Q-TOF-MS/MS [21]. Although keratin identifications are often contaminants in MS studies, the authors identified and included a few keratins, cytokeratin 15, 17, and 19, that are likely not to be contaminants in corneal studies [22]. This was supported by previous studies where cytokeratin 15 and 19 are known to be basal limbal markers [16,23]. The authors further investigated nine of the identified proteins with immunologic methods, Western blotting, and immunohistochemistry and could confirm the differential expression in seven cases. Thus, they found and confirmed overexpression of superoxide dismutase 2 (SOD2), HSP 70 kDa protein 1 (Hsp70), annexin I, cytokeratin 15, cytokeratin 17, S100A8, and ␣-1 antitrypsin in the limbal epithelium www.clinical.proteomics-journal.com
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Figure 2. Treatment options for limbal stem cell deficiency (LSCD). Medical treatment is used to relieve symptoms of pain and inflammation. Surgical treatment is used to cure the disease. Allogenic transplants are obtained from living donors or cadavers. Autologous transplants are taken from healthy eye. Due to limitations of tissue in the healthy eye a number of techniques have been used to cultivate various types of cells for treatment of LSCD.
compared with the central epithelium. Among these SOD2 and cytokeratin 15 were almost exclusively expressed in few cells in the basal limbal epithelium. It was suggested that these two proteins should be added to the panel of existing proteins that characterize basal limbal stem cells and early transient-amplifying cells. Thus, SOD2 and cytokeratin 15 could be markers for limbal stem cells or transient amplifying cells. In a subsequent immunohistochemical study of the early developing human eye, the authors confirmed this observation [24]. They found that SOD2 and cytokeratin 15 were present in the earliest-formed corneal epithelium indicating that the proteins are involved in the initial differentiation and proliferation of the stem cells. From week 14 they found the proteins to be predominantly expressed in the limbal epithe C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
lium [24]. The authors concluded that the change in staining pattern supports that SOD2 and cytokeratin 15 are characteristic of the limbal phenotype and thereby potential limbal stem cell markers. In addition to these proteins suggested to be markers, the authors found Hsp70 overexpressed in the limbal epithelium. The importance of this protein for limbal stem cells has been further highlighted in a subsequent study performed by Ma et al. [25]. In order to analyze factors that influence the growth of human limbocorneal epithelial cells cultivated on amniotic membranes Ma et al. performed a 2D-PAGE based proteomic study on cultivated human limbocorneal epithelial cells [25]. The authors compared stem cells cultivated on dishes precoated with type I collagen with stem cells grown on dishes overlaid with intact amniotic membrane. The cells were www.clinical.proteomics-journal.com
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separated from the dish with dispase and the proteins extracted into lysis buffer. By comparative 2D-PAGE, they revealed 198 proteins of 1133 spots to be at least 1.5 times overexpressed in the stem cells cultivated with amniotic membrane while 231 spots were underexpressed. As a control they also analyzed a separate 2D-gel of proteins extracted in a similar way with dispase from intact amniotic membranes and found no obvious spots. They excised the 15 proteins that were most upregulated in the cells grown on amniotic membrane and identified 13 by MALDI-TOF MS analysis; Hsp70, galectin-7, S100-A9, FABPA, FABP-E, heat shock 27 kDa protein -I, actin, annexin A2, GST P, proteasome subunit beta type 3, cathepsin D precursor, triosephosphate isomerase, and proto-oncogene tyrosine-protein kinase FRG. They further functionally analyzed the role of Hsp70. By plasmid vector-induced overexpression of the transcription factor ⌬Np63␣ they found a subsequent increase in Hsp70 expression and a decrease in expression after silencing the ⌬Np63␣. Furthermore, they found a cytoprotective effect of Hsp70 when limbocorneal epithelial cells were exposed to sublethal UVB irradiation or hydrogen peroxide. Cells with Hsp70 silenced showed a higher degree of apoptosis and cell death. Thus, the authors have shown that culturing of stem cells on amniotic membranes upregulate a number of proteins and that ⌬Np63␣-induced overexpression of Hsp70 are likely to promote stem cell survival through a cytoprotetive effect of Hsp70.
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Proteomic analyses of support factors
The stem cell are localized in the limbus in the stem cell niche, where they are supplied with factors of importance for their growth [3]. Recently, there have been performed proteomic studies addressing the importance of the environment for the growth of stem cells. Two groups have focused on the amniotic membrane as a support medium and one group has analyzed the proteins that are attached to contact lenses used therapeutically for treatment of LSCD. Amniotic membranes have been used for therapeutic purposes for more than a century and recently also been used in the treatment of ocular disorders [26]. The explanation for how it works is under debate. It is suggested to possess antiangiogenic, anti-inflammatory, and antiscarring properties mediated by cytokines [26]. Also, it promotes epithelialization and can be used as a carrier for expansion of corneal epithelial cells. In order to characterize the proteins expressed in transplantation-ready amniotic membranes, Hopkinson et al. [27] have performed studies using 2D-PAGE and MS. They collected amniotic membranes from patients undergoing elective cesarean section and stored them at −80⬚C for more than 6 months before analysis. Then the membranes were thawed, cleaned in saline, and the tissue solubilized in lysis buffer before 2D-PAGE. Protein spots were visualized by silver staining and identified using MALDI-TOF-MS and nano-LC-ESI-Q-TOF-MS/MS. The authors both analyzed the C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
181 amniotic membrane as well as the wash medium and thereby constructed a template reference map of proteins. Examples of interesting proteins identified included thrombospondin1, mimecan, TGF--induced gene human, clone 3, and integrin ␣6. They suggest that these proteins may play important roles for the function of amniotic membranes, for example, thrombospondin-1 inhibits angiogenesis [28], which may be important for maintaining the avascularity of the cornea, mimecan is also present in the cornea [29] and is involved in collagen fibrillogenesis [30], TGF--induced gene human, clone 3 is present in the cornea [31, 32] and is involved in corneal dystrophies [33] and integrin ␣6 belongs to the larger family of integrins that are important for attachment of cells to extracellular matrix proteins and may function as two-way signaling molecules from inside of the cell to the outside [34]. Another study that focused on the importance of amniotic membranes for growth of limbal stem cells was performed by Baharvand et al. [35]. This group compared growth of limbal stem cells on epithelium-denuded aminotic membranes with growth on matrigel as well as on collagen. The cells grown on epithelium-denuded amniotic membranes showed a phenotype similar to in vivo limbal epithelium [35], while those grown on matrigel and collagen exhibited a more differentiated phenotype. They then analyzed the protein expression of epithelium-denuded human amniotic membranes in order to detect abundant proteins that could influence the growth of limbal stem cells by using 2D-PAGE in combination with MALDI-TOF/TOF-MS identification. A number of highly abundant proteins were identified, including lumican, osteoglycin/memican, collagen ␣ type IV, and fibrinogen. Thus, Baharvand et al. [35] analyzing intact amniotic membranes identified other highly abundant proteins than Hopkinson et al. [27] analyzing epithelium-denuded amniotic membranes. However, their study objects were different and further studies are therefore needed in order to elucidate which specific proteins are important for the maintenance of the stem cells. Indeed, Hopkinson et al. [27] highlighted the importance of standardization procedures in order not to compromise desired therapeutic effects of amniotic membranes. A very different approach to identify proteins of importance for maintaining the function of stem cells was performed by Echevarria et al. analyzing proteins attached to contact lenses used therapeutically for treatment of LSCD [36]. The authors had previously observed that epithelial cells adhered to contact lenses that had been used therapeutically after pterygium surgery. Following this observation, they started to culture limbal epithelial cells on the contact lenses in autologous serum [37]. Then they took epithelial biopsies from three patients with LSCD and cultivated cells from the biopsies on contact lenses submerged in autologous serum. The contact lenses were then transferred to the patient’s cornea in order to restore the epithelium [38]. All patients experienced establishment of a stable transparent corneal epithelium and significant improvement of symptoms [38]. The authors then analyzed by MS which proteins could be attached to the lenses and whether the proteins were critical www.clinical.proteomics-journal.com
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for the method to work [36]. The serum proteins identified from the contact lenses depended on the concentration of serum used for incubation. No proteins were detected without serum while 1 or 10% serum each revealed a set of proteins. Amongst the identified proteins were vitronectin, apolipoproteins, serum amyloid proteins, complement, hemoglobin, coagulation factors, and prepromultimerin. The authors then focused on vitronectin for further investigations. They found that vitronectin was localized in the limbal basement membrane where the stem cells are known to be present. Similarly, vitronectin was present in other organs possessing stem cells, for example, human skin and small intestine. The ability of the contact lenses to bind vitronectin depended on the lens type. Lotrafilcon A binds five- to sixfold higher amounts as compared with Balafilcon A lenses and they found that the protein was released again when lenses were incubated in PBS. By incubating limbal epithelial stem cells in vitronectin coated tissue culture dishes, they found that vitronectin in a dose-dependent way enhanced the ability of the cells to form colonies. Moreover, vitronectin increased the transfer of cells from contact lenses onto tissue culture plastic. Furthermore, they could show in an ex vivo model with chemically damaged human corneas from cadavers that vitronectin could be detected at the basement membrane of regenerating cells. Normally this zone does not contain vitronectin. Together their results underline the importance of vitronectin in supporting the growth of limbal stem cells.
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Limbal stem cell proteomics as it stands
Proteomic characterization of limbal stem cell biology is in its infancy with only a few studies performed so far. However, the few studies available present quite interesting observations. Proteins have been added to the list of putative markers for limbal stem cells such as SOD2 and cytokeratin 15 and the cytoprotective role of Hsp70 has been highlighted. In addition, it was shown that amniotic membranes that support the phenotype of stem cells possesses a number of abundant proteins, among which several could be of importance. Moreover, the specific importance of vitronectin for growth of stem cells has also been revealed. However, elucidation of the specific role of individual proteins expressed by the stem cells as well as defining which type of support factors that are important in maintaining the phenotype of stem cells during cultivation still requires substantial research effort.
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Future proteomic studies to reveal stem cell biology and to find markers
The lack of stem cells for therapy of stem cell deficiency stresses the importance of novel proteomic analyses to decipher the biology of the cells and to find suitable markers that may be used to characterize the cells. The analyses performed C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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so far have mainly been based on 2D-PAGE separation with subsequent MS identification of proteins. In future, there is a great need to apply the latest technology to this field in order to progress further. First, the scarcity of cells makes this a particularly difficult task. Another obstacle is the relative inaccessibility of the stem cells due to the localization of the cells in the specialized anatomical structures at the limbus [6–9]. Thus, the application of proteomic techniques to characterize limbal stem cell biology thereby faces huge challenges. No “one-size-fits-all” proteomic strategy [39] is able to reveal this. There is no doubt that a number of techniques need to be applied in parallel. Although “top-down” strategies may ultimately be developed further for high-throughput analyses [40] in the foreseeable future the more traditional “bottomup” procedures would undoubtedly still be the procedures at hand for these analyses. One approach to study limbal stem cells would be to analyze cultivated cells as the stem cells are very limited in number. Usually cultivation is performed with mouse 3T3 fibroblasts as feeder layer or with human amniotic membranes as substrate [41]. However, a very attractive approach would be to take advantage of the recent observation that stem cells may be cultivated and expanded from biopsies on therapeutic contact lenses in serum [37]. This could be applied to cadaver eyes for research purposes or alternative analyses could be performed on the additional biopsies that have been taken out and not used since one other already have worked successfully for therapy. Once the cells have been expanded to confluency MS techniques with instruments having sensitivity in the attomole range [42] should then be applied. Unfortunately, suitable reference cells are not obtained by this approach so a relative quantification method such as ICAT [43] is not applicable. Instead, the strategy could be to perform absolute abundance estimates of the proteins in the stem cells with the use of accurately quantified standard peptides or a standard protein mix, which recently has been shown to work quite well [44]. Ultimately it may become possible to compare the obtained protein expression profile with other cells/tissues with the emerging efforts toward the construction of a human proteomics atlas [45]. Cultivation always involves the risk that the proteome varies with the passages of the cells [46]. However, this would provide an initial set of putative highly expressed proteins beyond those already suggested [12, 14, 16] which subsequently could be further tested as markers. MALDI imaging [47, 48], especially with further improvements in resolution, may be used on, for example, cadaver eyes as a supplement or for more detailed localization of the markers for the stem cells in the limbus region. These analyses may then be further supplemented with immunohistochemical analyses for confirmation of expression and localization of stem cells in the crypts if high-quality antibodies are available. An alternative or supplementary approach would be to use tissue laser capture microdissection [49] of cells from various parts of the limbus supposed to harbor stem cells as it has been done previously for transcriptomic analyses www.clinical.proteomics-journal.com
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from cadaver eyes [14]. These cells may then be analyzed alone with absolute quantification as previously mentioned or together with microdissected cells located outside the limbal area serving as putative reference cells. Then a comparative differential proteomics analysis with highly sensitive LC-MS/MS [42] could be performed using a relative quantification method with labeling like ICAT or iTRAQ [50] or a nonlabeling technique [51]. It would also be possible to use immunolaser capture microdissection [52] using some of the putative markers for stem cells for example, ABCG2 [12, 16] before analysis. As there is no single specific marker available for limbal stem cells yet the choice of antibody may always be questioned. The advantage of using microdissection with or without the use of antibodies would be to avoid the cultivation step that putatively may change the proteomic presentation [46]. One challenge would be to histologically localize the various anatomical structures supposed to harbor stem cells as well as the locations where they are absent. This, however, should be possible since it has previously been performed for transcriptional profiling [14]. Another challenge would be to identify and obtain enough cells for analysis. The ultimate goal with these proteomic analyses would be to isolate homogeneous stem cells as well as corneal epithelial reference cells in order to establish their protein profile. Once this eventually is established by using a combination of methods, it may then be possible to monitor the stem cells under growth and to adjust the conditions to maintain the “proper” protein phenotype of the cells for optimal treatment of stem cell deficiency. We thank Linda Neumann for photographs of the eye. The work in the authors laboratories was supported by the John and Birthe Meyer Foundation, Bagger-Sørensen Fonden, Herta Christensens Fond, Svend Andersen Fonden, and Region Nordjyllands Forskningsfond. The authors have declared no conflict of interest.
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DOI 10.1002/prca.201300049
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REVIEW
Proteomic analysis as a means to approach limbal stem cell biology in a search for stem cell markers Bent Honore´ 1 and Henrik Vorum2∗ 1 2
Department of Biomedicine, Aarhus University, Aarhus, Denmark Department of Ophthalmology, Aalborg University Hospital, Aalborg, Denmark
The cornea consists of three main layers: an outer surface epithelium, the stroma, and the endothelium. A clear cornea is necessary for optimal vision and is maintained and repaired from limbal epithelial stem cells located in the limbus between the cornea and the sclera. Diseases and injury may result in deficiency of the stem cells impairing their ability to renew the corneal epithelium. Patients with limbal stem cell deficiency experience chronic pain and ultimately blindness. Attempts to treat the disease are based on replacement of the stem cells by transplantation or by culturing the stem cells. We here review the proteomic techniques that so far have been used to approach characterization of limbal stem cells and markers to identify them. It is apparent that the field is in a rather inchoate state due to the scarcity and relative inaccessibility of the stem cells. However, the importance of revealing limbal stem cell biology and identifying stem cell biomarkers calls for greater use of emerging methodology. Strategies for future studies are discussed.
Received: June 26, 2013 Revised: October 6, 2013 Accepted: December 2, 2013
Keywords: 2D-PAGE / Limbal stem cells / Stem cell markers
1
Introduction
The surface of the eye ball consists of the sclera, conjuntiva, limbus, and the cornea (Fig. 1). The cornea is the clear part at the front of the eye surrounded by the limbus. The cornea consists of three main layers, an outer surface epithelium, separated by Bowman’s layer from the stroma, which again is separated by Descemet’s membrane from the endothelium. The central corneal epithelium has limited proliferative properties as compared with the peripheral corneal epithelium [1]. At the corneoscleral limbus the limbal stem cells are located in the basal epithelium as originally proposed by Davanger and Evensen [2]. Here, the stem cells may be supplied with factors from the local environment, the so-called stem cell niche [3, 4]. It is proposed that the stem cell niche consists of anatomical and functional dimensions that maintain “stemness.” The niche contains stromal invaginations called the palisades of Vogt [5]. The palisades of Vogt contain a distinct
´ Correspondence: Professor Bent Honore, Department of Biomedicine, Aarhus University, Ole Worms Alle´ 3, DK-8000 Aarhus C, Denmark E-mail: [email protected] Fax: +45-86-13-11-60 Abbreviations: LSCD, limbal stem cell deficiency; SOD2, superoxide dismutase 2 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
vasculature with radially oriented hairpin loops of arteries and veins [5]. This structure is suggested to protect stem cells from trauma and to give access to substances that may diffuse from the nearby vascular network. More recently, additional anatomical structures have been described to be part of the stem cell niche, the limbal epithelial crypt [6, 7], the limbal crypt [8], and focal stromal projections [8]. These anatomical structures are not uniformly spread in the limbal region but mainly localized in the superior region [9]. These structures consist of cells that stain positive for some of the putative limbal stem cell markers [6–8]. A number of observations indicate that the basal membrane in the region of the stem cell niche has a specific composition of the extracellular matrix that differ from that found in other parts of the cornea and that this probably creates the special environment that supports “stemness,” that is, inhibiting differentiation and maintaining the proliferative abilities of the limbal stem cells [4]. The stem cells are responsible for maintaining a healthy cornea. The so-called X, Y, Z hypothesis introduced by Thoft and Friend [10] describes a net movement of cells from the basal limbal epithelium (X) that adds to the net movement of more centrally migrating epithelial cells in the cornea (Y). It is hypothesized that the two components added are equal to the cells that shed at the central cornea (Z). Although the ∗ Additional corresponding author: Professor Henrik Vorum, E-mail: [email protected]
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Figure 1. The surface of the eye ball consists of sclera, conjuntiva, limbus, and cornea. The stem cells are located in the limbus (stippled circle), which is the region between cornea and conjunctiva.
concept with limbus as the sole localization has been challenged some years ago [11], the localization of stem cells in the limbus only is greatly accepted [12, 13] and more recent transcriptomic analysis also support that stem cells are preferentially located in the limbal epithelial crypts [14]. Injury or diseases may result in limbal stem cell deficiency (LSCD) with impairment in the renewal of the corneal epithelium resulting in impaired vision and chronic pain [13]. The disease may be treated medically or surgically (Fig. 2). The medical treatment is used to relieve the symptoms of pain and inflammation by using lubricants, corticosteroids, and autologous serum drops [15]. The medical treatment, however, cannot cure the disease. Surgery is required for this and can be divided in autologous and allogenic transplantation of tissue or stem cells. Allogenic transplantation of limbal corneal grafts from living donors or cadavers is a possibility. However, the disadvantage is that systemic immunosuppression is needed to maintain the graft. Autologous transplatation is possible for unilateral disease, but the amount of tissue is limited and there is a risk of inducing iatrogenic LSCD in the donor eye. It is therefore of interest to isolate and cultivate the cells in order to be able to obtain enough cells for therapy. A number of different types of cells have been cultivated for the purpose, including oral mucosa cells, embryonic stem cells, and stem cells from hair follicles, umbilical cord, and immature dental pulp as reviewed by Osei-Bempong et al. [12]. However, here we will focus on limbal epithelial stem cells. Stem cells are characterized by their undifferentiated phenotype, a small cell size, a high nucleus to cytoplasm ratio, and a low mitotic activity [16, 17]. There is no specific single marker that identifies a stem cell. Instead the expression pattern of a set of proteins is used to characterize stem cells as recently reviewed by a number of authors [12, 16–18]. Examples of protein expression patterns characteristic of stem cells include presence of ABCG2, vimentin, and cytokeratins 5 and 14, and the absence of cytokeratins 3 and 12, connexin 43, and nestin. Thus, in order to properly be able to isolate and cultivate stem cells for therapy a great deal of work is still required to characterize the protein expression pattern of stem cells as well as their biological properties. This would help to optimize the growth conditions for proper expansion of the cells. We here review how proteomic techniques have been used recently to approach characterization of stem cell biology and the identification of putative markers. Some studies have focused on analysis of the proteomic composition of the limbal stem cell, while others have focused on the identi C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
fication of factors that are important for maintaining the stem cells. In the long term this may help to identify the necessary growth conditions. First, we will review the proteomic studies performed to approach a characterization of the stem cells as well as the support layer necessary for the cultivation of the cells and then suggest future studies that may be performed in order to improve our understanding of limbal stem cell biology.
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Proteomic analyses of cornea and stem cells
The first study performed on human tissue aiming to identify limbal stem cell protein markers by a proteomic approach was performed by Lyngholm et al. [19]. They used a trephine to mark the central 8 and 10 mm of the corneal epithelium. Then it was stippled with 70% ethanol to loosen the epithelium from the stroma. The central 8 mm disc was scraped-off and collected as the central part of the cornea. The fraction containing the 8–10 mm ring of the intermediate epithelium was discarded and finally the limbal fraction located outside of the 10-mm ring was scraped-off and collected as the limbal fraction. By comparing the 2D-PAGE images of central parts of the corneas from cadavers (taken within 48 h post mortem) with the cornea from a living myopic patient they found no apparent degradation in the cadaver corneas. The study was then performed on cadaver corneas. The aim was to indentify proteins upregulated in the limbal cornea, since proteins upregulated here might be candidates to be among stem cell markers. More than 1000 protein spots were detected by silver staining [20] and 32 spots were significantly overexpressed in the central part while 70 spots were significantly overexpressed in the limbal part. Twenty-five different proteins were identified by nano-LC-ESI-Q-TOF-MS/MS [21]. Although keratin identifications are often contaminants in MS studies, the authors identified and included a few keratins, cytokeratin 15, 17, and 19, that are likely not to be contaminants in corneal studies [22]. This was supported by previous studies where cytokeratin 15 and 19 are known to be basal limbal markers [16,23]. The authors further investigated nine of the identified proteins with immunologic methods, Western blotting, and immunohistochemistry and could confirm the differential expression in seven cases. Thus, they found and confirmed overexpression of superoxide dismutase 2 (SOD2), HSP 70 kDa protein 1 (Hsp70), annexin I, cytokeratin 15, cytokeratin 17, S100A8, and ␣-1 antitrypsin in the limbal epithelium www.clinical.proteomics-journal.com
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Figure 2. Treatment options for limbal stem cell deficiency (LSCD). Medical treatment is used to relieve symptoms of pain and inflammation. Surgical treatment is used to cure the disease. Allogenic transplants are obtained from living donors or cadavers. Autologous transplants are taken from healthy eye. Due to limitations of tissue in the healthy eye a number of techniques have been used to cultivate various types of cells for treatment of LSCD.
compared with the central epithelium. Among these SOD2 and cytokeratin 15 were almost exclusively expressed in few cells in the basal limbal epithelium. It was suggested that these two proteins should be added to the panel of existing proteins that characterize basal limbal stem cells and early transient-amplifying cells. Thus, SOD2 and cytokeratin 15 could be markers for limbal stem cells or transient amplifying cells. In a subsequent immunohistochemical study of the early developing human eye, the authors confirmed this observation [24]. They found that SOD2 and cytokeratin 15 were present in the earliest-formed corneal epithelium indicating that the proteins are involved in the initial differentiation and proliferation of the stem cells. From week 14 they found the proteins to be predominantly expressed in the limbal epithe C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
lium [24]. The authors concluded that the change in staining pattern supports that SOD2 and cytokeratin 15 are characteristic of the limbal phenotype and thereby potential limbal stem cell markers. In addition to these proteins suggested to be markers, the authors found Hsp70 overexpressed in the limbal epithelium. The importance of this protein for limbal stem cells has been further highlighted in a subsequent study performed by Ma et al. [25]. In order to analyze factors that influence the growth of human limbocorneal epithelial cells cultivated on amniotic membranes Ma et al. performed a 2D-PAGE based proteomic study on cultivated human limbocorneal epithelial cells [25]. The authors compared stem cells cultivated on dishes precoated with type I collagen with stem cells grown on dishes overlaid with intact amniotic membrane. The cells were www.clinical.proteomics-journal.com
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separated from the dish with dispase and the proteins extracted into lysis buffer. By comparative 2D-PAGE, they revealed 198 proteins of 1133 spots to be at least 1.5 times overexpressed in the stem cells cultivated with amniotic membrane while 231 spots were underexpressed. As a control they also analyzed a separate 2D-gel of proteins extracted in a similar way with dispase from intact amniotic membranes and found no obvious spots. They excised the 15 proteins that were most upregulated in the cells grown on amniotic membrane and identified 13 by MALDI-TOF MS analysis; Hsp70, galectin-7, S100-A9, FABPA, FABP-E, heat shock 27 kDa protein -I, actin, annexin A2, GST P, proteasome subunit beta type 3, cathepsin D precursor, triosephosphate isomerase, and proto-oncogene tyrosine-protein kinase FRG. They further functionally analyzed the role of Hsp70. By plasmid vector-induced overexpression of the transcription factor ⌬Np63␣ they found a subsequent increase in Hsp70 expression and a decrease in expression after silencing the ⌬Np63␣. Furthermore, they found a cytoprotective effect of Hsp70 when limbocorneal epithelial cells were exposed to sublethal UVB irradiation or hydrogen peroxide. Cells with Hsp70 silenced showed a higher degree of apoptosis and cell death. Thus, the authors have shown that culturing of stem cells on amniotic membranes upregulate a number of proteins and that ⌬Np63␣-induced overexpression of Hsp70 are likely to promote stem cell survival through a cytoprotetive effect of Hsp70.
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Proteomic analyses of support factors
The stem cell are localized in the limbus in the stem cell niche, where they are supplied with factors of importance for their growth [3]. Recently, there have been performed proteomic studies addressing the importance of the environment for the growth of stem cells. Two groups have focused on the amniotic membrane as a support medium and one group has analyzed the proteins that are attached to contact lenses used therapeutically for treatment of LSCD. Amniotic membranes have been used for therapeutic purposes for more than a century and recently also been used in the treatment of ocular disorders [26]. The explanation for how it works is under debate. It is suggested to possess antiangiogenic, anti-inflammatory, and antiscarring properties mediated by cytokines [26]. Also, it promotes epithelialization and can be used as a carrier for expansion of corneal epithelial cells. In order to characterize the proteins expressed in transplantation-ready amniotic membranes, Hopkinson et al. [27] have performed studies using 2D-PAGE and MS. They collected amniotic membranes from patients undergoing elective cesarean section and stored them at −80⬚C for more than 6 months before analysis. Then the membranes were thawed, cleaned in saline, and the tissue solubilized in lysis buffer before 2D-PAGE. Protein spots were visualized by silver staining and identified using MALDI-TOF-MS and nano-LC-ESI-Q-TOF-MS/MS. The authors both analyzed the C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
181 amniotic membrane as well as the wash medium and thereby constructed a template reference map of proteins. Examples of interesting proteins identified included thrombospondin1, mimecan, TGF--induced gene human, clone 3, and integrin ␣6. They suggest that these proteins may play important roles for the function of amniotic membranes, for example, thrombospondin-1 inhibits angiogenesis [28], which may be important for maintaining the avascularity of the cornea, mimecan is also present in the cornea [29] and is involved in collagen fibrillogenesis [30], TGF--induced gene human, clone 3 is present in the cornea [31, 32] and is involved in corneal dystrophies [33] and integrin ␣6 belongs to the larger family of integrins that are important for attachment of cells to extracellular matrix proteins and may function as two-way signaling molecules from inside of the cell to the outside [34]. Another study that focused on the importance of amniotic membranes for growth of limbal stem cells was performed by Baharvand et al. [35]. This group compared growth of limbal stem cells on epithelium-denuded aminotic membranes with growth on matrigel as well as on collagen. The cells grown on epithelium-denuded amniotic membranes showed a phenotype similar to in vivo limbal epithelium [35], while those grown on matrigel and collagen exhibited a more differentiated phenotype. They then analyzed the protein expression of epithelium-denuded human amniotic membranes in order to detect abundant proteins that could influence the growth of limbal stem cells by using 2D-PAGE in combination with MALDI-TOF/TOF-MS identification. A number of highly abundant proteins were identified, including lumican, osteoglycin/memican, collagen ␣ type IV, and fibrinogen. Thus, Baharvand et al. [35] analyzing intact amniotic membranes identified other highly abundant proteins than Hopkinson et al. [27] analyzing epithelium-denuded amniotic membranes. However, their study objects were different and further studies are therefore needed in order to elucidate which specific proteins are important for the maintenance of the stem cells. Indeed, Hopkinson et al. [27] highlighted the importance of standardization procedures in order not to compromise desired therapeutic effects of amniotic membranes. A very different approach to identify proteins of importance for maintaining the function of stem cells was performed by Echevarria et al. analyzing proteins attached to contact lenses used therapeutically for treatment of LSCD [36]. The authors had previously observed that epithelial cells adhered to contact lenses that had been used therapeutically after pterygium surgery. Following this observation, they started to culture limbal epithelial cells on the contact lenses in autologous serum [37]. Then they took epithelial biopsies from three patients with LSCD and cultivated cells from the biopsies on contact lenses submerged in autologous serum. The contact lenses were then transferred to the patient’s cornea in order to restore the epithelium [38]. All patients experienced establishment of a stable transparent corneal epithelium and significant improvement of symptoms [38]. The authors then analyzed by MS which proteins could be attached to the lenses and whether the proteins were critical www.clinical.proteomics-journal.com
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for the method to work [36]. The serum proteins identified from the contact lenses depended on the concentration of serum used for incubation. No proteins were detected without serum while 1 or 10% serum each revealed a set of proteins. Amongst the identified proteins were vitronectin, apolipoproteins, serum amyloid proteins, complement, hemoglobin, coagulation factors, and prepromultimerin. The authors then focused on vitronectin for further investigations. They found that vitronectin was localized in the limbal basement membrane where the stem cells are known to be present. Similarly, vitronectin was present in other organs possessing stem cells, for example, human skin and small intestine. The ability of the contact lenses to bind vitronectin depended on the lens type. Lotrafilcon A binds five- to sixfold higher amounts as compared with Balafilcon A lenses and they found that the protein was released again when lenses were incubated in PBS. By incubating limbal epithelial stem cells in vitronectin coated tissue culture dishes, they found that vitronectin in a dose-dependent way enhanced the ability of the cells to form colonies. Moreover, vitronectin increased the transfer of cells from contact lenses onto tissue culture plastic. Furthermore, they could show in an ex vivo model with chemically damaged human corneas from cadavers that vitronectin could be detected at the basement membrane of regenerating cells. Normally this zone does not contain vitronectin. Together their results underline the importance of vitronectin in supporting the growth of limbal stem cells.
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Limbal stem cell proteomics as it stands
Proteomic characterization of limbal stem cell biology is in its infancy with only a few studies performed so far. However, the few studies available present quite interesting observations. Proteins have been added to the list of putative markers for limbal stem cells such as SOD2 and cytokeratin 15 and the cytoprotective role of Hsp70 has been highlighted. In addition, it was shown that amniotic membranes that support the phenotype of stem cells possesses a number of abundant proteins, among which several could be of importance. Moreover, the specific importance of vitronectin for growth of stem cells has also been revealed. However, elucidation of the specific role of individual proteins expressed by the stem cells as well as defining which type of support factors that are important in maintaining the phenotype of stem cells during cultivation still requires substantial research effort.
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Future proteomic studies to reveal stem cell biology and to find markers
The lack of stem cells for therapy of stem cell deficiency stresses the importance of novel proteomic analyses to decipher the biology of the cells and to find suitable markers that may be used to characterize the cells. The analyses performed C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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so far have mainly been based on 2D-PAGE separation with subsequent MS identification of proteins. In future, there is a great need to apply the latest technology to this field in order to progress further. First, the scarcity of cells makes this a particularly difficult task. Another obstacle is the relative inaccessibility of the stem cells due to the localization of the cells in the specialized anatomical structures at the limbus [6–9]. Thus, the application of proteomic techniques to characterize limbal stem cell biology thereby faces huge challenges. No “one-size-fits-all” proteomic strategy [39] is able to reveal this. There is no doubt that a number of techniques need to be applied in parallel. Although “top-down” strategies may ultimately be developed further for high-throughput analyses [40] in the foreseeable future the more traditional “bottomup” procedures would undoubtedly still be the procedures at hand for these analyses. One approach to study limbal stem cells would be to analyze cultivated cells as the stem cells are very limited in number. Usually cultivation is performed with mouse 3T3 fibroblasts as feeder layer or with human amniotic membranes as substrate [41]. However, a very attractive approach would be to take advantage of the recent observation that stem cells may be cultivated and expanded from biopsies on therapeutic contact lenses in serum [37]. This could be applied to cadaver eyes for research purposes or alternative analyses could be performed on the additional biopsies that have been taken out and not used since one other already have worked successfully for therapy. Once the cells have been expanded to confluency MS techniques with instruments having sensitivity in the attomole range [42] should then be applied. Unfortunately, suitable reference cells are not obtained by this approach so a relative quantification method such as ICAT [43] is not applicable. Instead, the strategy could be to perform absolute abundance estimates of the proteins in the stem cells with the use of accurately quantified standard peptides or a standard protein mix, which recently has been shown to work quite well [44]. Ultimately it may become possible to compare the obtained protein expression profile with other cells/tissues with the emerging efforts toward the construction of a human proteomics atlas [45]. Cultivation always involves the risk that the proteome varies with the passages of the cells [46]. However, this would provide an initial set of putative highly expressed proteins beyond those already suggested [12, 14, 16] which subsequently could be further tested as markers. MALDI imaging [47, 48], especially with further improvements in resolution, may be used on, for example, cadaver eyes as a supplement or for more detailed localization of the markers for the stem cells in the limbus region. These analyses may then be further supplemented with immunohistochemical analyses for confirmation of expression and localization of stem cells in the crypts if high-quality antibodies are available. An alternative or supplementary approach would be to use tissue laser capture microdissection [49] of cells from various parts of the limbus supposed to harbor stem cells as it has been done previously for transcriptomic analyses www.clinical.proteomics-journal.com
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from cadaver eyes [14]. These cells may then be analyzed alone with absolute quantification as previously mentioned or together with microdissected cells located outside the limbal area serving as putative reference cells. Then a comparative differential proteomics analysis with highly sensitive LC-MS/MS [42] could be performed using a relative quantification method with labeling like ICAT or iTRAQ [50] or a nonlabeling technique [51]. It would also be possible to use immunolaser capture microdissection [52] using some of the putative markers for stem cells for example, ABCG2 [12, 16] before analysis. As there is no single specific marker available for limbal stem cells yet the choice of antibody may always be questioned. The advantage of using microdissection with or without the use of antibodies would be to avoid the cultivation step that putatively may change the proteomic presentation [46]. One challenge would be to histologically localize the various anatomical structures supposed to harbor stem cells as well as the locations where they are absent. This, however, should be possible since it has previously been performed for transcriptional profiling [14]. Another challenge would be to identify and obtain enough cells for analysis. The ultimate goal with these proteomic analyses would be to isolate homogeneous stem cells as well as corneal epithelial reference cells in order to establish their protein profile. Once this eventually is established by using a combination of methods, it may then be possible to monitor the stem cells under growth and to adjust the conditions to maintain the “proper” protein phenotype of the cells for optimal treatment of stem cell deficiency. We thank Linda Neumann for photographs of the eye. The work in the authors laboratories was supported by the John and Birthe Meyer Foundation, Bagger-Sørensen Fonden, Herta Christensens Fond, Svend Andersen Fonden, and Region Nordjyllands Forskningsfond. The authors have declared no conflict of interest.
6
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