Enrichment, Characterization, and Proteomic Profiling of Small Extracellular Vesicles Derived from Human Limbal Mesenchymal Stromal Cells and Melanocytes.

Limbal epithelial progenitor cells (LEPC) rely on their niche environment for proper functionality and self-renewal. While extracellular vesicles (EV), specifically small EVs (sEV), have been proposed to support LEPC homeostasis, data on sEV derived from limbal niche cells like limbal mesenchymal stromal cells (LMSC) remain limited, and there are no studies on sEVs from limbal melanocytes (LM). In this study, we isolated sEV from conditioned media of LMSC and LM using a combination of tangential flow filtration and size exclusion chromatography and characterized them by nanoparticle tracking analysis, transmission electron microscopy, Western blot, multiplex bead arrays, and quantitative mass spectrometry. The internalization of sEV by LEPC was studied using flow cytometry and confocal microscopy. The isolated sEVs exhibited typical EV characteristics, including cell-specific markers such as CD90 for LMSC-sEV and Melan-A for LM-sEV. Bioinformatics analysis of the proteomic data suggested a significant role of sEVs in extracellular matrix deposition, with LMSC-derived sEV containing proteins involved in collagen remodeling and cell matrix adhesion, whereas LM-sEV proteins were implicated in other cellular bioprocesses such as cellular pigmentation and development. Moreover, fluorescently labeled LMSC-sEV and LM-sEV were taken up by LEPC and localized to their perinuclear compartment. These findings provide valuable insights into the complex role of sEV from niche cells in regulating the human limbal stem cell niche.

Influence of Organ Culture on the Characteristics of the Human Limbal Stem Cell Niche.

Organ culture storage techniques for corneoscleral limbal (CSL) tissue have improved the quality of corneas for transplantation and allow for longer storage times. Cultured limbal tissue has been used for stem cell transplantation to treat limbal stem cell deficiency (LSCD) as well as for research purposes to assess homeostasis mechanisms in the limbal stem cell niche. However, the effects of organ culture storage conditions on the quality of limbal niche components are less well described. Therefore, in this study, the morphological and immunohistochemical characteristics of organ-cultured limbal tissue are investigated and compared to fresh limbal tissues by means of light and electron microscopy. Organ-cultured limbal tissues showed signs of deterioration, such as edema, less pronounced basement membranes, and loss of the most superficial layers of the epithelium. In comparison to the fresh limbal epithelium, organ-cultured limbal epithelium showed signs of ongoing proliferative activity (more Ki-67+ cells) and exhibited an altered limbal epithelial phenotype with a loss of N-cadherin and desmoglein expression as well as a lack of precise staining patterns for cytokeratin ((CK)14, CK17/19, CK15). The analyzed extracellular matrix composition was mainly intact (collagen IV, fibronectin, laminin chains) except for Tenascin-C, whose expression was increased in organ-cultured limbal tissue. Nonetheless, the expression patterns of cell-matrix adhesion proteins varied in organ-cultured limbal tissue compared to fresh limbal tissue. A decrease in the number of melanocytes (Melan-A+ cells) and Langerhans cells (HLA-DR+, CD1a+, CD18+) was observed in the organ-cultured limbal tissue. The organ culture-induced alterations of the limbal epithelial stem cell niche might hamper its use in the treatment of LSCD as well as in research studies. In contrast, reduced numbers of donor-derived Langerhans cells seem associated with better clinical outcomes. However, there is a need to consider the preferential use of fresh CSL for limbal transplants and to look at ways of improving the limbal stem cell properties of stored CSL tissue.

Characterization of Porcine Ocular Surface Epithelial Microenvironment.

The porcine ocular surface is used as a model of the human ocular surface; however, a detailed characterization of the porcine ocular surface has not been documented. This is due, in part, to the scarcity of antibodies produced specifically against the porcine ocular surface cell types or structures. We performed a histological and immunohistochemical investigation on frozen and formalin-fixed, paraffin-embedded ocular surface tissue from domestic pigs using a panel of 41 different antibodies related to epithelial progenitor/differentiation phenotypes, extracellular matrix and associated molecules, and various niche cell types. Our observations suggested that the Bowman's layer is not evident in the cornea; the deep invaginations of the limbal epithelium in the limbal zone are analogous to the limbal interpalisade crypts of human limbal tissue; and the presence of goblet cells in the bulbar conjunctiva. Immunohistochemistry analysis revealed that the epithelial progenitor markers cytokeratin (CK)15, CK14, p63α, and P-cadherin were expressed in both the limbal and conjunctival basal epithelium, whereas the basal cells of the limbal and conjunctival epithelium did not stain for CK3, CK12, E-cadherin, and CK13. Antibodies detecting marker proteins related to the extracellular matrix (collagen IV, Tenascin-C), cell-matrix adhesion (ß-dystroglycan, integrin α3 and α6), mesenchymal cells (vimentin, CD90, CD44), neurons (neurofilament), immune cells (HLA-ABC; HLA-DR, CD1, CD4, CD14), vasculature (von Willebrand factor), and melanocytes (SRY-homeobox-10, human melanoma black-45, Tyrosinase) on the normal human ocular surface demonstrated similar immunoreactivity on the normal porcine ocular surface. Only a few antibodies (directed against N-cadherin, fibronectin, agrin, laminin α3 and α5, melan-A) appeared unreactive on porcine tissues. Our findings characterize the main immunohistochemical properties of the porcine ocular surface and provide a morphological and immunohistochemical basis useful to research using porcine models. Furthermore, the analyzed porcine ocular structures are similar to those of humans, confirming the potential usefulness of pig eyes to study ocular surface physiology and pathophysiology.

PAX6 Expression Patterns in the Adult Human Limbal Stem Cell Niche.

Paired box 6 (PAX6), a nuclear transcription factor, determines the fate of limbal epithelial progenitor cells (LEPC) and maintains epithelial cell identity. However, the expression of PAX6 in limbal niche cells, primarily mesenchymal stromal cells (LMSC), and melanocytes is scarce and not entirely clear. To distinctly assess the PAX6 expression in limbal niche cells, fresh and organ-cultured human corneoscleral tissues were stained immunohistochemically. Furthermore, the expression of PAX6 in cultured limbal cells was investigated. Immunostaining revealed the presence of PAX6-negative cells which were positive for vimentin and the melanocyte markers Melan-A and human melanoma black-45 in the basal layer of the limbal epithelium. PAX6 staining was not observed in the limbal stroma. Moreover, the expression of PAX6 was observed by Western blot in cultured LEPC but not in cultured LMSC or LM. These data indicate a restriction of PAX6 expression to limbal epithelial cells at the limbal stem cell niche. These observations warrant further studies for the presence of other PAX isoforms in the limbal stem cell niche.

Transcriptomic Landscape and Functional Characterization of Human Induced Pluripotent Stem Cell-Derived Limbal Epithelial Progenitor Cells.

Limbal stem cell deficiency (LSCD) is a complex, multifactorial disease affecting limbal epithelial progenitor cells (LEPC), which are essential for maintaining corneal stability and transparency. Human induced pluripotent stem cell-derived (hiPSC-) LEPC are a promising cell source for the treatment of LSCD. However, their similarity to native tissue-derived (T-) LEPC and their functional characterization has not been studied in detail. Here, we show that hiPSC-LEPC and T-LEPC have rather similar gene expression patterns, colony-forming ability, wound-healing capacity, and melanosome uptake. In addition, hiPSC-LEPC exhibited lower immunogenicity and reduced the proliferation of peripheral blood mononuclear cells compared with T-LEPC. Similarly, the hiPSC-LEPC secretome reduced the proliferation of vascular endothelial cells more than the T-LEPC secretome. Moreover, hiPSC-LEPC successfully repopulated decellularized human corneolimbal (DHC/L) scaffolds with multilayered epithelium, while basal deposition of fibrillary material was observed. These findings suggest that hiPSC-LEPC exhibited functional properties close to native LEPC and that hiPSC-LEPC-DHC/L scaffolds might be feasible for transplantation in patients suffering from LSCD in the future. Although hiPSC-LEPC-based stem cell therapy is promising, the current study also revealed new challenges, such as abnormal extracellular matrix deposition, that need to be overcome before hiPSC-LEPC-based stem cell therapies are viable.

Efficient Isolation and Functional Characterization of Niche Cells from Human Corneal Limbus.

The fate decision of limbal epithelial progenitor cells (LEPC) at the human corneal limbus is determined by the surrounding microenvironment with limbal niche cells (LNC) as one of its essential components. Research on freshly isolated LNC which mainly include limbal mesenchymal stromal cells (LMSC) and limbal melanocytes (LM) has been hampered by a lack of efficient protocols to isolate and purify these cells. We devised a protocol for rapid retrieval of pure LMSC, LM and LEPC populations by collagenase digestion of limbal tissue and subsequent fluorescence-activated cell sorting (FACS) using antibodies against CD90 and CD117. The sorted cells were characterized by immunophenotyping and functional assays. The effects of LMSC and LM on LEPC were studied in 3D co-cultures and LEPC differentiation status was assessed by immunohistochemistry. Enzymatic digestion and flow sorting yielded pure populations of LMSC (CD117-CD90+), LM (CD117+CD90-), and LEPC (CD117-CD90-). The LMSC exhibited self-renewal capacity (55.0 ± 4.6 population doublings), expressed mesenchymal stem cell markers (CD73, CD90, CD105, and CD44), and transdifferentiated to adipocytes, osteocytes, or chondrocytes. The LM exhibited self-renewal capacity and sustained melanin production. The sorted LEPC expressed epithelial progenitor markers (CK14, CK19, and CK15) and showed a colony-forming ability. Co-cultivation of LMSC and LM with LEPC resulted in a 4-5-layered stratified epithelium and supported the preservation of a LEPC phenotype, as reflected by increased p63+ and Ki67+ cells and decreased CK12+ cells compared with LEPC monocultures. A highly efficient isolation of pure LM, LMSC, and LEPC populations from a single preparation may allow for direct transcriptomic and proteomic profiling as well as functional studies on native unpassaged LNC, which can be considered as proper equivalents of LNC in vivo. The developed biomimetic 3D co-culture method could provide an experimental model for investigating the functional role of LNC in the limbal stem cell niche.

A Decellularized Human Limbal Scaffold for Limbal Stem Cell Niche Reconstruction.

The transplantation of ex vivo expanded limbal epithelial progenitor cells (LEPCs) on amniotic membrane or fibrin gel is an established therapeutic strategy to regenerate the damaged corneal surface in patients with limbal stem cell deficiency (LSCD), but the long-term success rate is restricted. A scaffold with niche-specific structure and extracellular matrix (ECM) composition might have the advantage to improve long-term clinical outcomes, in particular for patients with severe damage or complete loss of the limbal niche tissue structure. Therefore, we evaluated the decellularized human limbus (DHL) as a biomimetic scaffold for the transplantation of LEPCs. Corneoscleral tissue was decellularized by sodium deoxycholate and deoxyribonuclease I in the presence or absence of dextran. We evaluated the efficiency of decellularization and its effects on the ultrastructure and ECM composition of the human corneal limbus. The recellularization of these scaffolds was studied by plating cultured LEPCs and limbal melanocytes (LMs) or by allowing cells to migrate from the host tissue following a lamellar transplantation ex vivo. Our decellularization protocol rapidly and effectively removed cellular and nuclear material while preserving the native ECM composition. In vitro recellularization by LEPCs and LMs demonstrated the good biocompatibility of the DHL and intrastromal invasion of LEPCs. Ex vivo transplantation of DHL revealed complete epithelialization as well as melanocytic and stromal repopulation from the host tissue. Thus, the generated DHL scaffold could be a promising biological material as a carrier for the transplantation of LEPCs to treat LSCD.

Melanocytes as emerging key players in niche regulation of limbal epithelial stem cells.

PURPOSE: Limbal melanocytes (LMel) represent essential components of the corneal epithelial stem cell niche and are known to protect limbal epithelial stem/progenitor cells (LEPCs) from UV damage by transfer of melanosomes. Here, we explored additional functional roles for LMel in niche homeostasis, immune regulation and angiostasis. METHODS: Human corneoscleral tissues were morphologically analyzed in normal, inflammatory and wound healing conditions. The effects of LMel on LEPCs were analyzed in direct and indirect co-culture models using electron microscopy, immunocytochemistry, qRT-PCR, Western blotting and functional assays; limbal mesenchymal stromal cells and murine embryonic 3T3 fibroblasts served as controls. The immunophenotype of LMel was assessed by flow cytometry before and after interferon-γ stimulation, and their immunomodulatory properties were analyzed by mixed lymphocytes reaction, monocyte adhesion assays and cytometric bead arrays. Their angiostatic effects on human umbilical cord endothelial cells (HUVECs) were evaluated by proliferation, migration, and tube formation assays. RESULTS: LMel and LEPCs formed structural units in the human limbal stem cell niche in situ, which could be functionally replicated, including melanosome transfer, by co-cultivation in vitro. LMel supported LEPCs during clonal expansion and during epithelial wound healing by stimulating proliferation and migration, and suppressed their differentiation through direct contact and paracrine effects. Under inflammatory conditions, LMel were increased in numbers and upregulated expression of ICAM-1 and MHC II molecules (HLA-DR), but lacked expression of HLA-G, -DP, -DQ and costimulatory molecules CD80 and CD86. They were also found to be potent suppressors of alloreactive T- cell proliferation and cytokine secretion, which largely depended on direct cell-cell interaction. Moreover, the LMel secretome exerted angiostatic activity by inhibiting vascular endothelial cell proliferation and capillary network formation. CONCLUSION: These findings suggest that LMel are not only professional melanin-producing cells, but exert various non-canonical functions in limbal niche homeostasis by regulating LEPC maintenance, immune responses, and angiostasis. Their potent regulatory, immunomodulatory and anti-angiogenic properties may have important implications for future regenerative cell therapies.

A decellularized human corneal scaffold for anterior corneal surface reconstruction.

Allogenic transplants of the cornea are prone to rejection, especially in repetitive transplantation and in scarred or highly vascularized recipient sites. Patients with these ailments would particularly benefit from the possibility to use non-immunogenic decellularized tissue scaffolds for transplantation, which may be repopulated by host cells in situ or in vitro. So, the aim of this study was to develop a fast and efficient decellularization method for creating a human corneal extracellular matrix scaffold suitable for repopulation with human cells from the corneal limbus. To decellularize human donor corneas, sodium deoxycholate, deoxyribonuclease I, and dextran were assessed to remove cells and nuclei and to control tissue swelling, respectively. We evaluated the decellularization effects on the ultrastructure, optical, mechanical, and biological properties of the human cornea. Scaffold recellularization was studied using primary human limbal epithelial cells, stromal cells, and melanocytes in vitro and a lamellar transplantation approach ex vivo. Our data strongly suggest that this approach allowed the effective removal of cellular and nuclear material in a very short period of time while preserving extracellular matrix proteins, glycosaminoglycans, tissue structure, and optical transmission properties. In vitro recellularization demonstrated good biocompatibility of the decellularized human cornea and ex vivo transplantation revealed complete epithelialization and stromal repopulation from the host tissue. Thus, the generated decellularized human corneal scaffold could be a promising biological material for anterior corneal reconstruction in the treatment of corneal defects.

Isolation and enrichment of melanocytes from human corneal limbus using CD117 (c-Kit) as selection marker.

Limbal melanocytes (LM) are located in the basal epithelial layer of the corneoscleral limbus and interact with adjacent limbal epithelial progenitor cells. The exploration of their biological role in the maintenance of the limbal stem cell niche has been limited by the difficulty of LM isolation and cultivation. Here, we report on a facile protocol for the efficient isolation and enrichment of pure populations of human LMs by fluorescence-activated cell sorting (FACS) using antibodies raised against the cell surface marker CD117 (c-Kit). The enriched LMs retain self-renewal capacity and sustained melanin production, and are suitable to study the potential of LMs in stem cell-based corneal tissue engineering.

Immunomodulatory properties of bone marrow mesenchymal stem cells.

Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent progenitor cells of mesodermal origin possessing multilineage differentiation potential and ease of expansion in vitro. Over the years, these cells have gained attention owing to their potential in cell-based therapies in treating various diseases. In particular, the wide spectrum of immunoregulatory/immunomodulatory role of MSCs in various clinical conditions has gained immense attention. The immunomodulatory properties of BM-MSCs are mediated by either cell-cell contact (interactions with various immune cells in a context-dependent manner), paracrine mode of action or extracellular vesicles, making them a potential option as immunosuppressants/immunomodulators in treating various clinical conditions. A plethora of studies have demonstrated that MSCs do so by exhibiting a profound effect on various immune cells for example they can inhibit the proliferation of T cells, B cells, and natural killer cells; modulate the activities of dendritic cells and induce regulatory T cells both in vitro and in vivo. In this review we aim at briefly elucidating the characteristics of BM-MSCs, specifically addressing the current understanding on the hypoimmunogeneticity and immunomodulatory properties of the same with specific reference to their interactions with B cells, T cells, Dendritic cells and natural killer cells. We also aim at reviewing the secretory profile and their role in some clinical conditions that have shown promising outcomes.

Laminin-511-E8 promotes efficient in vitro expansion of human limbal melanocytes.

Limbal melanocytes, located in the basal epithelial layer of the corneoscleral limbus, represent essential components of the corneal epithelial stem cell niche, but, due to difficulties in their isolation and cultivation, their biological roles and potential for stem cell-based tissue engineering approaches have not been comprehensively studied. Here, we established a protocol for the efficient isolation and cultivation of pure populations of human limbal melanocytes, which could be expanded at high yield by using recombinant laminin (LN)-511-E8 as culture substrate. Co-cultivation of limbal melanocytes with limbal epithelial stem/progenitor cells on fibrin hydrogels pre-incubated with LN-511-E8 resulted in multilayered stratified epithelial constructs within ten days. By reproducing physiological cell-cell and cell-matrix interactions of the native niche environment, these biomimetic co-culture systems provide a promising experimental model for investigating the functional roles of melanocytes in the limbal stem cell niche and their suitability for developing advanced epithelial grafts for ocular surface surface reconstruction.

Isolation and ex vivo Expansion of Human Limbal Epithelial Progenitor Cells.

Limbal stem cell transplantation has been used successfully to treat patients with limbal stem cell deficiency all over the world. However, long term clinical results often proved less satisfactory due to the low quality of the graft or inadequate properties of transplanted cells. To enhance the ex vivo expansion of human limbal epithelial stem or progenitor cells (LEPC) by preserving stem cell phenotype and to improve subsequent transplantation efficiency, cell-matrix interactions ex vivo should mimic the condition in vivo. The laminin isoforms preferentially expressed in the limbal niche can be used as a culture matrix for epithelial tissue engineering. We recently published the expansion of LEPC on various laminin isoforms and observed that laminin alpha 5-derived matrices support the efficient expansion of LEPC compared to tissue culture plates and other laminin isoforms by preserving stem/progenitor cell phenotype. Here, we describe an optimized protocol for the isolation of LEPC from cadaveric corneal limbal tissue by collagenase digestion and efficient expansion of LEPC using recombinant human laminin-511 E8 fragment (LN-511E8) as culture substrate.

Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells.

Understanding transcription factor (TF) regulation of limbal epithelial stem/progenitor cells (LEPCs) may aid in using non-ocular cells to regenerate the corneal surface. This study aimed to identify and characterize TF genes expressed specifically in LEPCs isolated from human donor eyes by laser capture microdissection. Using a profiling approach, preferential limbal expression was found for SoxE and SoxF genes, particularly for Sox9, which showed predominantly cytoplasmic localization in basal LEPCs and nuclear localization in suprabasal and corneal epithelial cells, indicating nucleocytoplasmic translocation and activation during LEPC proliferation and differentiation. Increased nuclear localization of Sox9 was also observed in activated LEPCs following clonal expansion and corneal epithelial wound healing. Knockdown of SOX9 expression in cultured LEPCs by RNAi led to reduced expression of progenitor cell markers, e.g. keratin 15, and increased expression of differentiation markers, e.g. keratin 3. Furthermore, SOX9 silencing significantly suppressed the proliferative capacity of LEPCs and reduced levels of glycogen synthase kinase 3 beta (GSK-3ß), a negative regulator of Wnt/ß-catenin signaling. Sox9 expression, in turn, was significantly suppressed by treatment of LEPCs with exogenous GSK-3ß inhibitors and enhanced by small molecule inhibitors of Wnt signaling. Our results suggest that Sox9 and Wnt/ß-catenin signaling cooperate in mutually repressive interactions to achieve a balance between quiescence, proliferation and differentiation of LEPCs in the limbal niche. Future molecular dissection of Sox9-Wnt interaction and mechanisms of nucleocytoplasmic shuttling of Sox9 may aid in improving the regenerative potential of LEPCs and the reprogramming of non-ocular cells for corneal surface regeneration.

Laminin-511 and -521-based matrices for efficient ex vivo-expansion of human limbal epithelial progenitor cells.

Optimization of culture conditions for human limbal epithelial stem/progenitor cells (LEPC) that incorporate the in vivo cell-matrix interactions are essential to enhance LEPC ex vivo-expansion and transplantation efficiency. Here, we investigate the efficacy of laminin (LN) isoforms preferentially expressed in the limbal niche as culture matrices for epithelial tissue engineering. Analyses of expression patterns of LN chains in the human limbal niche provided evidence for enrichment of LN-α2, -α3, -α5, -ß1, -ß2, -ß3, -γ1, -γ2 and -γ3 chains in the limbal basement membrane, with LN-α5 representing a signature component specifically produced by epithelial progenitor cells. Recombinant human LN-521 and LN-511 significantly enhanced in vitro LEPC adhesion, migration and proliferation compared to other isoforms, and maintained phenotype stability. The bioactive LN-511-E8 fragment carrying only C-terminal domains showed similar efficacy as full-length LN-511. Functional blocking of α3ß1 and α6ß1 integrins suppressed adhesion of LEPC to LN-511/521-coated surfaces. Cultivation of LEPC on fibrin-based hydrogels incorporating LN-511-E8 resulted in firm integrin-mediated adhesion to the scaffold and well-stratified epithelial constructs, with maintenance of a progenitor cell phenotype in their (supra)basal layers. Thus, the incorporation of chemically defined LN-511-E8 into biosynthetic scaffolds represents a promising approach for xeno-free corneal epithelial tissue engineering for ocular surface reconstruction.

Evaluation of a 345 nm Femtosecond Laser for Corneal Surgery with Respect to Intraocular Radiation Hazard.

PURPOSE: We report our findings from a preclinical safety study designed to assess potential side effects of corneal ultraviolet femtosecond laser treatment on lens and retina. METHODS: Refractive lenticules (-5 dpt) with a diameter of 6 mm were created in the right cornea of eight Dutch Belted rabbits. Radiant exposure was 0.5 J/cm² in two animals and 18 J/cm² in six animals. The presence of lens opacities was assessed prior to and up to six months following laser application using Scheimpflug images (Pentacam, Oculus) and backscatter analysis (Opacity Lensmeter 702, Interzeag). Ganzfeld flash and flicker electroretinogram (ERG) recordings were obtained from both eyes prior to and up to six weeks following laser application. At the study endpoint, retinas were examined by light microscopy. RESULTS: Independent of energy dose applied, no cataract formation could be observed clinically or with either of the two objective methods used. No changes in ERG recordings over time and no difference between treated and untreated eye were detected. Histologically, retinal morphology was preserved and retinal pigment epithelium as well as photoreceptor inner and outer segments appeared undamaged. Quantitative digital image analysis did not reveal cell loss in inner or outer nuclear layers. CONCLUSIONS: Our analysis confirms theoretical considerations suggesting that ultraviolet femtosecond laser treatment of the cornea is safe for intraocular tissues. Transmitted light including stray light induces no photochemical effects in lens or retina at energy levels much higher than required for the clinical purpose. These conclusions cannot be applied to eyes with pre-existing retinal damage, as these may be more vulnerable to light.

Molecular characterization of prospectively isolated multipotent mesenchymal progenitors provides new insight into the cellular identity of mesenchymal stem cells in mouse bone marrow.

Despite great progress in the identification of mesenchymal stem cells (MSCs) from bone marrow (BM), our knowledge of their in vivo cellular identity remains limited. We report here that cells expressing the transcription factor Ebf2 in adult BM display characteristics of MSCs. The Ebf2(+) cells are highly clonal and physiologically quiescent. In vivo lineage-tracing experiments, single cell clone transplantations, and in vitro differentiation assays revealed their self-renewal and multilineage differentiation capacity. Gene expression analysis of the freshly sorted Ebf2(+) cells demonstrated the expression of genes previously reported to be associated with MSCs and the coexpression of multiple lineage-associated genes at the single-cell level. Thus, Ebf2 expression is not restricted to committed osteoblast progenitor cells but rather marks a multipotent mesenchymal progenitor cell population in adult mouse BM. These cells do not appear to completely overlap the previously reported MSC populations. These findings provide new insights into the in vivo cellular identity and molecular properties of BM mesenchymal stem and progenitor cells.

Regenerative approaches as alternatives to donor allografting for restoration of corneal function.

A range of alternatives to human donor tissue for corneal transplantation are being developed to address the shortfall of good quality tissues as well as the clinical conditions for which allografting is contraindicated. Classical keratoprostheses, commonly referred to as artificial corneas, are being used clinically to replace minimal corneal function. However, they are used only as last resorts, as they are associated with significant complications, such as extrusion/rejection, glaucoma, and retinal detachment. The past few years have seen significant developments in technologies designed to replace part or the full thickness of damaged or diseased corneas with materials that encourage regeneration to different extents. This review describes selected examples of these corneal substitutes, which range from cell-based regenerative strategies to keratoprostheses with regenerative capabilities via tissue-engineered scaffolds pre-seeded with stem cells. It is unlikely that one corneal substitute will be best for all indications, but taken together, the various approaches may soon be able to supplement the supply of human donor corneas for transplantation or allow restoration of diseased or damaged corneas that cannot be treated by currently available techniques.

Gene expression profile of epithelial cells and mesenchymal cells derived from limbal explant culture.

PURPOSE: Limbal stem cell deficiency is a challenging clinical problem and the current treatment involves replenishing the depleted limbal stem cell (LSC) pool by either limbal tissue transplantation or use of cultivated limbal epithelial cells (LEC). Our experience of cultivating the LEC on denuded human amniotic membrane using a feeder cell free method, led to identification of mesenchymal cells of limbus (MC-L), which showed phenotypic resemblance to bone marrow derived mesenchymal stem cells (MSC-BM). To understand the transcriptional profile of these cells, microarray experiments were carried out. METHODS: RNA was isolated from cultured LEC, MC-L and MSC-BM and microarray experiments were carried out by using Agilent chip (4x44 k). The microarray data was validated by using Realtime and semiquntitative reverse transcription polymerase chain reaction. RESULTS: The microarray analysis revealed specific gene signature of LEC and MC-L, and also their complementary role related to cytokine and growth factor profile, thus supporting the nurturing roles of the MC-L. We have also observed similar and differential gene expression between MC-L and MSC-BM. CONCLUSIONS: This study represents the first extensive gene expression analysis of limbal explant culture derived epithelial and mesenchymal cells and as such reveals new insight into the biology, ontogeny, and in vivo function of these cells.

Isolation, characterization and differentiation potential of rat bone marrow stromal cells.

BACKGROUND: Bone marrow mesenchymal cells have been identified as a source of pluripotent stem cells with varying degrees of plasticity in humans. However, there are a few reports on rat-derived cells, which could be good models for the research purpose. We describe here a simple method of establishing the rat bone marrow stromal cells by the principle of adhesion and document their phenotype along with their differentiation potential to other lineages. MATERIALS AND METHODS: Rat bone marrow stromal cells were isolated by three methods: direct plastic adherence, ficoll hypaque separation and a combination of both. The stromal cells obtained by these methods were characterized by fluorescent activating cell sorting (FACS) for established hematopoietic and non-hematopoietic markers. The cells obtained by combination method (combination of ficoll density gradient centrifugation and plastic adherence) were cultured and serially passaged. Transcriptional confirmation was done by reverse transcription polymerase chain reaction (RT-PCR) for vimentin and collagen type 1 alpha 1. Attempts were made to differentiate the marrow stromal cells into adipocytes, osteocytes and neuronal like cells. RESULTS: Bone marrow samples from 10 rats yielded 4-5 million bone marrow mononuclear cells /ml per femur. Of the three methods tested, a combination method yielded good growth of spindle cells. The cells obtained by combined method showed high percentage of positivity for vimentin, fibronectin and CD90 and negative for hematopoietic markers. Further, RT-PCR confirmed vimentin and collagen type - 1 alpha 1 expression. Oil red O staining and Alizarin red staining confirmed adipocytic and osteogenic differentiation. On immunocytochemical analysis, the cells expressed nestin, beta-tubulin III, neurofilament and synaptophysin. CONCLUSION: Adequate quantities of rat marrow stromal cell cultures can be established by a simple method based on adhesion properties. Their phenotypic characteristics and plasticity support the evidence that they are mesenchymal stem cells with a distinct tendency for neural lineage.