Limbal niche cells are a potent resource of adult mesenchymal progenitors

Abstract Limbal niche cells located in the limbal Palisades of Vogt are mesenchymal stem cells that reside next to limbal basal epithelial cells. Limbal niche cells are progenitors that express embryonic stem cell markers such as Nanog, Nestin, Oct4, Rex1, Sox2 and SSEA4, mesenchymal cell markers such as CD73, CD90 and CD105, and angiogenesis markers such as Flk‐1, CD31, CD34, VWF, PDGFRβ and α‐SMA, but negative for CD45. In addition, the stemness of limbal niche cells can be maintained during their cell culture in a three‐dimension environment. Furthermore, expanded limbal niche cells have the capability to undergo adipogenesis, chondrogenesis, osteogenesis and endogenesis in vitro, indicating that they are in fact a valuable resource of adult progenitors. Furthermore studies on how the limbal niche cells regulate the aforementioned stemness and corneal fate decision are warranted, as those investigations will shed new light on how mesenchymal progenitors reverse limbal stem cell deficiency and lead to new methods for limbal niche cell treatment.


| HUMAN LIMBUS
Anatomically the limbal stem cell (SC) niche is located in the region while homogeneously expressing CK15 that extends to conjunctival basal epithelial cells. 10 N-cadherin can also be expressed by putative stem cells or progenitors as well as melanocytes in the human limbal epithelial SC niche. 11 Cx43, on the other hand, is well demonstrated to be a negative marker of limbal SC, but found positive in epithelial cells in LEC. 1 However, it remains unclear how the aforementioned SC and molecules modulate the quiescent and/or active stage of limbal epithelial SC.

| ISOLATION AND EXPANSION OF HUMAN LIMBAL NICHE CELLS
The limbal stem cell niche is a structure of the ocular surface that is characterized by high specification, organization and clinical significance (Reviewed in 12). Limbal stromal niche cells expressing SC markers can be isolated and expanded without differentiation and maintain clonal growth of limbal epithelial progenitors. 13 Culture of such cells on denuded amniotic membrane can maintain native niche cells 14 and quiescence. 15 Several types of cells enriched in the limbal niche may serve as niche cells, including melanocytes, 11 blood vascular endothelial cells 3 and neural cells. 16 Although the presence of niche cells (NC) is implicated by the aforementioned studies, it has not been identified until recently when a novel isolation method using collagenase A was used. 17 This method helps to identify putative NC as small Vim positive stromal cells in the form of cell clusters. 17,18 In addition, expansion of the niche/stem cell population isolated from collagenase A digestion is more efficient than single cell suspension by trypsin/EDTA digestion based on the number of p63a positive cells, 19 suggesting that an intact cell-cell contact help to maintain LSC in an undifferentiated state in vitro during expansion. Compared to the method of dispase digestion which removes intact sheets of limbal epithelium 20 but not all limbal epithelial progenitors, collagenase A digestion removes the entire progenitors in the limbal basal area which express ESC markers such as Nanog, Oct4 and Sox2. 18 In addition, collagenase A digestion has been performed without shaking, as shaking is not as effective in the concept of releasing progenitor cells. 21 Modified embryonic stem cell medium (MESCM) is considered an optimal medium for expansion of limbal niche cells. MESCM is comprised of DMEM/F-12 (1:1) but supplemented with 10% knockout serum, 5 lg/mL insulin, 5 lg/mL transferrin, 5 ng/mL sodium selenite, 4 ng/mL bFGF and 10 ng/mL hLIF, which is similar to another widely used medium containing DMEM-F12, knockout serum, basic fibroblast growth factor and leukaemia inhibitory factor (LIF) which produces a cell phenotype closest to that of a pluripotent stem cells. 22 In fact, such stem cells in the limbal niche have been confirmed (for review, see 23 and 24). Maintenance of close association between the epithelial and niche cells lead to clonal growth in a lowcalcium, serum-free medium while disruption of this linkage with trypsin/EDTA abolishes clone growth unless cocultured with 3T3 feeder layers. 17 Interestingly, Bhartiya et al 25  The phenotype of such potent cells includes a very small size (3-5 lm), high nucleo-cytoplasmic ratio, and expression of Oct-4, SSEA-4, and other pluripotent markers such as Nanog, Sox-2, Rex-1 and Tert. 25 Similarly, on epithelial-denuded amniotic membrane, maintenance of such linkage may cause extensive epithelial outgrowth, which can be attenuated by trypsin/EDTA treatment. 17 Epithelial outgrowth from the clusters obtained from collagenase digestion may be significantly larger. In addition, single cells obtained from clonal growth may generate more holoclones when cultured on 3T3 fibroblast feeder layers than those obtained from dispase digestion, 17 suggesting that these cells are indeed young progenitors. Interestingly, human corneal stromal progenitors exhibit survival capacity following isolation from a stored organ. 26 Furthermore, mesenchymal stromal progenitors cultivated from the limbus display immunosuppressive qualities in addition to their established non-immunogenic profile and stimulate limbal epithelial cell growth in vitro across species boundaries. 27 Using a novel method of serial passages on a 2D Matrigel system, we further isolated and expanded LNC to up to 12 passages with 33 population doublings and characterized them as angiogenesis progenitors when cultured in embryonic stem cell medium (ESCM) with fibroblast growth factor-basic (bFGF) and LIF. 28 The cells are thought to be angiogenesis progenitors due of up-regulation of these specific progenitor markers when the cells are reseeded in 3D Matrigel. 28 These cells may be differentiated into vascular endothelial cells along with pericytes for stabilization of tube network generated by human umbilical vein endothelial cells (HUVEC). 28 Interestingly, stromal cells which express a number of angiogenesis markers are located perivascularly, next to epithelial cells in the limbal basal area. 29 If cultured in a 3D Matrigel system, the stromal cells obtained by dispase/collagenase digestion but not the stromal cells at the limbal residual area may form the spheres with angiogenesis progenitors to maintain vascular networks. 29 Such sphere-forming cells from peripheral cornea demonstrate the ability to repopulate the ocular surface, expressing stem cell markers such as DNp63a, ABCG2, ABCB5 as well as the basal limbal and putative niche marker notch 1. 30 Similar to the cells obtained by collagenase digestion, the cells obtained from dispase/collagenase digestion may also proliferate when cultured on 2D Matrigel for 12 passages. As a result, the spindle cells may express the specific markers of MSC and angiogenesis, have higher ability to form colonies, and retain higher efficiency of tri-lineage differentiation, when compared to the stromal cells isolated by dispase/collagenase digestion from the limbal residual area when cultured in DMEM containing 10% fetal bovine serum (FBS). 29 Although limbal NC or HUVEC may reunite with LEPC to generate the spheres with higher expression of CK15, C/EBPd and DNp63a, limbal NC but not HUVEC can attenuate over-expression of CK12 keratin in LEPC. 28 Those findings raise the hypothesis that a vascular niche also exist in the human limbal niche.
The above findings also suggest that we may devise another novel method of separating limbal basal epithelial cells into two compartments, that is, one removed solely by dispase and the other that dispase cannot remove. The latter can be obtained by collagenase A digestion.

| Vascular/SC niche
The vascular niche refers to a microenvironment that is generated by vascular endothelial cells (VEC), pericytes and bone marrow (BM) cells. 31 The unique perivascular localization of the central neural system, the bone marrow and the testis suggests that vascular niche plays a critical role within the SC niche. [32][33][34] Vascular endothelium can also support bone marrow SC in the central nervous system, the muscle and the pancreatic islets. 35 The mechanisms involved include the physical contact and the paracrine factors such as bFGF, IGFBP2, angiopoietin-1 (Ang1), BMP4, DHH, PEDF, NDEF, FGF4 and SDF-1. 32 34 BM can also improve the proliferation and differentiation of human MSC in vitro. 36 For instance, collagen IV may promote the differentiation potential of ESC to EPC 37 whereas laminin can promote insulin gene expression and proliferation in beta islet cells, and beta1-integrin is required for the beta islet cell response to laminin's signals from VEC. 38  Multi-potential MSC derived from human tissues, including adipose tissue and skeletal muscle, appear to be derived nearly exclusively from perivascular SMC-pericytes. 44 Pericytes represent a unique subtype of perivascular cells with multi-lineage developmental features and various angiogenic functions. 48 As the ancestor of MSC, pericytes can not only give rise to MSC, but also contribute indirectly to tissue regeneration, possibly by promotion of angiogenesis and elimination of inflammation of endogenous progenitor cells. Therefore, pericytes could be applied as alternative therapeutic cells in replacement of MSC for regenerative medicine. 49 In order to select the right cell type, there are clues for the relation between the marker pattern and the multipotent potential of such cells. For example, PDGFRb-expressing cells are perivascular cells distinct from mature pericytes due to their ability to differentiate into mature pericytes and to support vascular tube stability and survival in vitro. 50 Most pericytes in vivo are a-SMA negative 51 except those located near arterioles are routinely a-SMA positive. 52 Interestingly, less than 5% of freshly isolated capillary pericytes express a-SMA, but nearly 100% express this marker in 7 days in culture with serum. 53 Human and rat brain pericytes are positive for stem cell markers such as NG2 and nestin. 43 CD146 can be used in isolating pericytes expressing PDGFR-b, a-SMA and NG2 from human skeletal muscle, pancreases, placenta, heart, skin, lung, brain, eye, gut, bone marrow and umbilical cord. 44 Although some markers are commonly used for progenitor cell identification, such as a-SMA, PDGFR-b, NG2, RGS5, aminopeptidase A and N5-7, those markers are not universal, but rather specific to the developmental stage, tissue bed, and even species. 54 ACTA2, SMAA, SM22, MYOCD, MYH11, SMMHC and CNN1 could be used to identify the presence of SMC. 46 The in vitro data support the concept that in the correct environment, CNS pericytes may differentiate to MSC and then differentiate to bone, adipocytes, smooth muscle, and VEC when cultured in a medium containing 10% FBS and 50 ng/mL vascular endothelial growth factor (VEGF). 52  | 3317 important angiogenesis progenitor cells identified to be positive for Flk-1, CD34, CD31, CD73, CD90, CD105, CD144 and negative for CD10, CD13, CD29, CD44. CD31 is the key marker of EPC and mature VEC, identified as a regulator of adhesion, migration and activation. 57 Flk1 is the earliest marker of angioblast precursors, specifically for a subset of cells that migrate into the extraembryonic yolk sac to form the vascular plexus during the murine development. 58 Foetal mouse lung mesenchymal cells with the highest Flk-1 expression can be differentiated into endothelium more efficiently. 59 EPC could be isolated from bone marrow, blood 60 and non-BM adult tissues, including skeletal muscle, adipose, spleen, liver, intestine and myocardium. 61 In the bone marrow, EPC derive from bone marrow stem cells and exist as residential progenitor cells. Such stem cells in limbal stroma are in fact multi-functional niche cells 23 and interact with limbal epithelial cells to exert their functions. 24,62 Limbal ESC obtained from young donors may produce better stem cells for clinical therapies. 63 Sphere forming cells are also a promising cell type for stem cell repopulations 30,64 and native LNC may promote the expansion of LEPC. 19 Human corneal stromal SC may survive in stored organ or culture, 26 although biomechanics exhibit a powerful effect on LNC and their growth and differentiation. 12 Human corneal stromal SC may prevent corneal scarring. 65 Stem cells and niche cells may interact with cell adhesive molecules to exert their functions. 66 Corneal stromal SC can even be induced to functional corneal endothelium by activation of Wnt signalling. 67 Human corneal stromal SC can also support limbal epithelial cells in vitro. 68 However, it is unclear whether EPC could serve as SC niche cells or serve as limbal niche cells.

SOURCE OF MESENCHYMAL PROGE NITORS
MSC clusters represent an important component of the limbal-niche. 29 MSC have great potential for regenerative medicine due to their high plasticity, self-renewal, specific immune response and the ability for genetic modification. Therefore, the growing demand for cell-based therapy necessitates a massive production of MSC. According to the minimum criteria set by the International Society for Cellular Therapy, MSC should be (i) adhere to plastic, (ii) express of specific surface antigens such as CD73, CD90 and CD105 positive, CD45 and CD34 negative, and (iii) have multi-lineage differentiation potential, for example, to adipocyte, chondrocytes and osteocyte. 69 Although first isolated from the bone marrow, 70 MSC could virtually be obtained from all adult tissues. 39,71 MSC are located in perivascular sites of many tissues such as in the bone marrow, 43 in adipose tissue, placenta and skeletal muscle 44 and in the dental pulp. 72 Functional corneal endothelial cells could be derived from corneal stroma stem cells of neural crest origin by retinoic acid and activation of Wnt/b-Catenin Signalling. 67 MSC could also be derived from the human limbal niche. 28,29 The cell origin of the MSC, that is, the progenitor cells of MSC, has not been very well-defined. However, recent evidence indicates that the progenitor cells of MSC lie in a parivascular niche and that MSC derive from the pericytes, or perivascular progenitor cells. 39,44 Pericytes and MSC express the cell markers in their native state, for example, a-SMA, CD44, CD73, CD90 and CD105, NG2, PDGFR-b and stromal progenitor antigen-1 (STRO-1) but not CD45, CD31. 44,[72][73][74][75] In in vitro culture, pericytes display an MSC-like phenotype, with respect to morphology, clonal growth, self-renewal and tri-lineage differentiation to different tissues such as bone, cartilage, fat, 44 nervous tissue 43 and skeletal muscle. 73,76 Meanwhile, MSC could be induced into EPC by VEGF, expressing Flk-1, CD34 and CD31. 77 37 Pericytes isolated from CNS may differentiate to MSC and then differentiate to bone, adipocytes, smooth muscle and VEC with 10% fetal calf serum and 50 ng/mL VEGF. 52 The influence of VEGF and PDGF on the outcome of angiogenesis progenitor expansion from the human limbus is not clear. BMP4 synergizes with LIF to maintain self-renewal by activating JAK/Stat3 signalling in mouse ES cells in the presence of serum. 85 In the absence of serum, BMP synergizes with LIF to maintain self-renewal in mouse ES cells through induction of gene expression of ID genes. 86 In an in vitro reunion model of LEPC and LNC in 3D Matrigel, LEPC+LNC spheres shows their ability of significantly higher clonal growth and dramatic lower differentiation in corneal epithelium. 87 In such a culture, nuclear translocation of pSmad1/5/8 is observed in LEPC, but not in LNC. Such a nuclear translocation of pSmad1/5/8 is associated with activation of canonical BMP signalling. 87 In addition, b-catenin was stabilized in the perinuclear cytoplasm in LEPC and correlated with up-regulation of Wnt7A and FZD5 preferentially expressed by LEPC. 87  Recently, a mice model was established to study the mechanisms of limbal stem cell deficiency (LSCD), suggesting that the phenotype of LSCD in the mouse model was maintained for more than 3 months. 88 A rabbit model was also established for potential use of limbal MSC to treat corneal disorders. 27