Human limbal biopsy-derived stromal stem cells prevent corneal scarring (original) (raw)
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Compressed Collagen Enhances Stem Cell Therapy for Corneal Scarring
STEM CELLS Translational Medicine
Stem cells from human corneal stroma (CSSC) suppress corneal stromal scarring in a mouse wound-healing model and promote regeneration of native transparent tissue (PMID:25504883). This study investigated efficacy of compressed collagen gel (CCG) as a vehicle to deliver CSSC for corneal therapy. CSSC isolated from limbal stroma of human donor corneas were embedded in soluble rat-tendon collagen, gelled at 378C, and partially dehydrated to a thickness of 100 mm by passive absorption. The CCG disks were dimensionally stable, easy to handle, and could be adhered securely to de-epithelialized mouse cornea with fibrin-based adhesive. CSSC in CCG maintained >80% viability for >1 week in culture media and could be cryopreserved in 20% fetal bovine serum-10%DMSO in liquid nitrogen. CCG containing as few as 500 CSSC effectively prevented visible scarring and suppressed expression of fibrotic Col3a1 mRNA. CSSC in CCG were more effective at blocking scarring on a per-cell basis than CSSC delivered directly in a fibrin gel as previously described. Collagen-embedded cells retained the ability to suppress corneal scarring after conventional cryopreservation. This study demonstrates use of a common biomaterial that can facilitate storage and handling of stem cells in a manner that may provide off-the-shelf delivery of stem cells as a therapy for corneal scarring. STEM CELLS TRANSLATIONAL MEDICINE 2018;7:487-494 SIGNIFICANCE STATEMENT Corneal scarring is a blinding condition affecting millions world wide. Experimental stem cell therapy can reverse cornea scarring but is currently available only in a medical center with specialized cell culture facilities. This study examines the use of a novel compressed collagen gel to deliver stem cells to the cornea. The gels facilitate handling, storage, and transfer of cells to the eye, and gel-embedded cells exhibit greater potency for corneal regeneration than stem cells alone. Results suggest that a collagen-stem cell corneal bandage could bring sight-restoring therapy to blind individuals for whom corneal transplantation is not available.
Investigative Ophthalmology & Visual Science, 2012
Corneal tissue regeneration is of crucial importance for maintaining normal vision. We aimed to isolate and cultivate human corneal stroma-derived mesenchymal stem-like cells (CSMSCs) from the central part of cadaver corneas and study their phenotype, multipotency, role in immunity and wound healing. The isolated cells grew as monolayers in vitro, expressed mesenchymal-and stemness-related surface markers (CD73, CD90, CD105, CD140b), and were negative for hematopoietic markers as determined by flow cytometry. CSMSCs were able to differentiate in vitro into fat, bone and cartilage. Their gene expression profile was closer to bone marrow-derived MSCs (BMMSCs) than to limbal epithelial stem cells (LESC) as determined by high-throughput screening. The immunosuppressive properties of CSMSCs were confirmed by a mixed lymphocyte reaction (MLR), while they could inhibit proliferation of activated immune cells. Treatment of CSMSCs by pro-inflammatory cytokines and toll-like receptor ligands significantly increased the secreted interleukin-6 (IL-6), interleukin-8 (IL-8) and C-X-C motif chemokine 10 (CXCL-10) levels, as well as the cell surface adhesion molecules. CSMSCs were capable of closing a wound in vitro under different stimuli. These cells thus contribute to corneal tissue homeostasis and play an immunomodulatory and regenerative role with possible implications in future cell therapies for treating sight-threatening corneal diseases. The cornea is the transparent front part of the eye responsible for two-thirds of its refractive power. It serves as a first barrier against external pathogens. Up to 90% of the corneal thickness is composed of corneal stroma, which contains different types of cells packed between regularly stacked and equally spaced collagen fibrils. Viral, fungal, bacterial infections and injuries caused by physical or chemical agents can all cause corneal scar formation, which eventually leads to vision loss or blindness 1-3. The damage of the corneal epithelial cell layer and the deeper stromal layer invoke a healing process mediated by activation of progenitor cells that are found in the limbal region of the cornea-the limbal epithelial stem cells (LESCs) 3-6. These cells can be found in six limbal crypts ordered in special niches capable of differentiating into transient amplifying cells (TACs) and differentiated corneal epithelial cells (CECs) 7-10. The regeneration of the cornea and the role of CECs play is not fully understood. It is hypothesized that TACs migrate centripetally and superficially during differentiation or, alternatively, the LESCs migrate to the site of injury 9. LESCs can express mesenchymal stem cell (MSC)-like markers on their surface such as CD73, CD90 and CD105 and show potential for clonal expansion, however, these cells are distinct from MSCs 11. LESC deficiency can lead to abnormal epithelial regeneration and visual loss 1,12 , but such deficiency in mice could not stop the corneal epithelial regeneration in the central part of the cornea, suggesting another type of progenitor/stem-like cells plays a role in the wound healing process 13,14. Corneal stroma stem cells have been isolated from the limbal stroma of mice and differentiated into keratocytes, but no evidence exists whether these cells are
Growth, differentiation, and secretory properties of mesenchymal stem cells (MSCs) and limbal ep-ithelial stem cells (LSCs) were compared and their therapeutic potential characterized in a rabbit model of eye injury. Results show that MSCs from bone marrow had a therapeutic effect on healing of the injured corneal surface comparable to that of tissue-specific LSCs.Stem cell-based therapy has become an attractive and promising approach for the treatment of severe injuries or thus-far incurable diseases. However, the use of stem cells is often limited by a shortage of available tissue-specific stem cells; therefore, other sources of stem cells are being investigated and tested. In this respect, mesenchymal stromal/stem cells (MSCs) have proven to be a promising stem cell type. In the present study, we prepared MSCs from bone marrow (BM-MSCs) or adipose tissue (Ad-MSCs) as well as limbal epithelial stem cells Q:2 (LSCs), and their growth, differentiation, and secretory properties were compared. The cells were grown on nanofiber scaffolds and transferred onto the alkali-injured eye in a rabbit model, and their therapeutic potential was characterized. We found that BM-MSCs and tissue-specific LSCs had similar therapeutic effects. Clinical characterization of the healing process, as well as the evaluation of corneal thickness, re-epithelialization, neovascularization, and the suppression of a local inflammatory reaction, were comparable in the BM-MSC-and LSC-treated eyes, but results were significantly better than in injured, untreated eyes or in eyes treated with a nanofiber scaffold alone or with a nanofiber scaffold seeded with Ad-MSCs. Taken together, the results show that BM-MSCs' therapeutic effect on healing of injured corneal surface is comparable to that of tissue-specific LSCs. We suggest that BM-MSCs can be used for ocular surface regeneration in cases when autologous LSCs are absent or difficult to obtain. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:1–13 SIGNIFICANCE Damage of ocular surface represents one of the most common causes of impaired vision or even blindness. Cell therapy, based on transplantation of stem cells, is an optimal treatment. However, if limbal stem cells (LSCs) are not available, other sources of stem cells are tested. Mesenchymal stem cells (MSCs) are a convenient type of cell for stem cell therapy. The therapeutic potential of LSCs and MSCs were compared in an experimental model of corneal injury and healing was observed following chemical injury. MSCs and tissue-specific LSCs had similar therapeutic effects. The results suggest that bone marrow-derived MSCs can be used for ocular surface regeneration in cases when autologous LSCs are absent or difficult to obtain.
Role of human corneal stroma-derived mesenchymal-like stem cells in immunity and wound healing
Acta Ophthalmologica, 2012
Corneal tissue regeneration is of crucial importance for maintaining normal vision. We aimed to isolate and cultivate human corneal stroma-derived mesenchymal stem-like cells (CSMSCs) from the central part of cadaver corneas and study their phenotype, multipotency, role in immunity and wound healing. The isolated cells grew as monolayers in vitro, expressed mesenchymal-and stemness-related surface markers (CD73, CD90, CD105, CD140b), and were negative for hematopoietic markers as determined by flow cytometry. CSMSCs were able to differentiate in vitro into fat, bone and cartilage. Their gene expression profile was closer to bone marrow-derived MSCs (BMMSCs) than to limbal epithelial stem cells (LESC) as determined by high-throughput screening. The immunosuppressive properties of CSMSCs were confirmed by a mixed lymphocyte reaction (MLR), while they could inhibit proliferation of activated immune cells. Treatment of CSMSCs by pro-inflammatory cytokines and toll-like receptor ligands significantly increased the secreted interleukin-6 (IL-6), interleukin-8 (IL-8) and C-X-C motif chemokine 10 (CXCL-10) levels, as well as the cell surface adhesion molecules. CSMSCs were capable of closing a wound in vitro under different stimuli. These cells thus contribute to corneal tissue homeostasis and play an immunomodulatory and regenerative role with possible implications in future cell therapies for treating sight-threatening corneal diseases. The cornea is the transparent front part of the eye responsible for two-thirds of its refractive power. It serves as a first barrier against external pathogens. Up to 90% of the corneal thickness is composed of corneal stroma, which contains different types of cells packed between regularly stacked and equally spaced collagen fibrils. Viral, fungal, bacterial infections and injuries caused by physical or chemical agents can all cause corneal scar formation, which eventually leads to vision loss or blindness 1-3. The damage of the corneal epithelial cell layer and the deeper stromal layer invoke a healing process mediated by activation of progenitor cells that are found in the limbal region of the cornea-the limbal epithelial stem cells (LESCs) 3-6. These cells can be found in six limbal crypts ordered in special niches capable of differentiating into transient amplifying cells (TACs) and differentiated corneal epithelial cells (CECs) 7-10. The regeneration of the cornea and the role of CECs play is not fully understood. It is hypothesized that TACs migrate centripetally and superficially during differentiation or, alternatively, the LESCs migrate to the site of injury 9. LESCs can express mesenchymal stem cell (MSC)-like markers on their surface such as CD73, CD90 and CD105 and show potential for clonal expansion, however, these cells are distinct from MSCs 11. LESC deficiency can lead to abnormal epithelial regeneration and visual loss 1,12 , but such deficiency in mice could not stop the corneal epithelial regeneration in the central part of the cornea, suggesting another type of progenitor/stem-like cells plays a role in the wound healing process 13,14. Corneal stroma stem cells have been isolated from the limbal stroma of mice and differentiated into keratocytes, but no evidence exists whether these cells are
Experimental Eye Research, 2019
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PloS one, 2017
Epithelial and stromal stem cells are required to maintain corneal transparency. The aim of the study was to develop a new method to isolate and grow both corneal stromal (SSC) and epithelial limbal (LSC) stem cells from small human limbal biopsies under culture conditions in accordance with safety requirements mandatory for clinical use in humans. Superficial limbal explants were retrieved from human donor corneo-scleral rims. Human limbal cells were dissociated by digestion with collagenase A, either after epithelial scraping or with no scraping. Isolated cells were cultured with Essential 8 medium (E8), E8 supplemented with EGF (E8+) or Green's medium with 3T3 feeder-layers. Cells were characterized by immunostaining, RT-qPCR, colony forming efficiency, sphere formation, population doubling, second harmonic generation microscopy and differentiation potentials. LSC were obtained from unscraped explants in E8, E8+ and Green's media and were characterized by colony formation...
Limbal Epithelial and Mesenchymal Stem Cell Therapy for Corneal Regeneration
Current Eye Research, 2019
Corneal pathologies are a major cause of blindness and visual impairment, especially in the developing world. However, not only is there a global shortage of donor corneal tissue, but a significant proportion of these blinding pathologies also carry an unfavourable long-term prognosis for conventional corneal transplantation. In the last few decades, there has been a spurt of research on developing alternate approaches to address corneal blindness, including stem cell therapy. After the discovery of epithelial stem cells at the limbus, successful cell-based approaches to treat severe ocular surface disease were developed and have subsequently become widely practised across the world. More recently, mesenchymal stem cells were identified near the epithelial stem cells at the limbus, providing a unique opportunity to develop regenerative therapies for both corneal epithelial and stromal pathologies. This review firstly emphasises on qualifying limbal stem cells as either epithelial or mesenchymal and then summarises all the existing knowledge on both cell types and their individual roles in corneal regeneration. The review describes the history, indications, techniques, and outcomes of the different methods of limbal epithelial stem cell transplantation and elaborates on the potential applications of limbal mesenchymal stem cell therapy.
Multipotent Stem Cells in Human Corneal Stroma
Stem Cells, 2005
Keratocytes of the corneal stroma secrete a specialized extracellular matrix essential for vision. These quiescent cells exhibit limited capacity for self-renewal and after cell division become fibroblastic, secreting nontransparent tissue. This study sought to identify progenitor cells for human keratocytes. Near the corneal limbus, stromal cells expressed ABCG2, a protein present in many adult stem cells. The ABCG2-expressing cell population was isolated as a side population (SP) by cell sorting after exposure to Hoechst 33342 dye. The SP cells exhibited clonal growth and continued to express ABCG2 and also PAX6, product of a homeobox gene not expressed in adult keratocytes. Cloned SP cells cultured in medium with fibroblast growth factor-2 lost ABCG2 and PAX6 expression and upregulated several molecular markers of keratocytes, including keratocan, aldehyde dehydrogenase 3A1, and keratan sulfate. Cloned corneal SP cells under chondrogenic conditions produced matrix staining with toluidine blue and expressed cartilage-specific markers: collagen II, cartilage oligomatrix protein, and aggrecan. Exposure of cloned SP cells to neurogenic culture medium upregulated mRNA and protein for glial fibrillary acidic protein, neuro filament protein, and betatubulin II. These results demonstrate the presence of a population of cells in the human corneal stroma expressing stem cell markers and exhibiting multipotent differentiation potential. These appear to be the first human cells identified with keratocyte progenitor potential. Further analysis of these cells will aid elucidation of molecular mechanisms of corneal development, differentiation, and wound healing. These cells may be a resource for bioengineering of corneal stroma and for cell-based therapeutics.
Concise review: the coming of age of stem cell treatment for corneal surface damage
Stem cells translational medicine, 2014
The cornea is a vital component of the eye because it provides approximately 70% of the refraction and focusing of incoming light. Being the outermost surface of the eye, it faces continuous stress from dryness, photodamage, infection, and injury; however, like the skin, the cornea regularly refreshes itself by shedding its epithelial cells, which are readily replaced, keeping the ocular surface stable and functional. This regular turnover of the corneal epithelial cells occurs through the stem cells in the limbus, an annular ring of a tissue surrounding the cornea, separating it from the sclera and the conjunctival membrane. The loss of this reserve of stem cells leads to a condition called limbal stem cell deficiency. Treatment for this disorder has evolved from transplanting whole limbal tissues to the affected eye to transplanting laboratory cultured limbal cells. This procedure is called cultivated limbal epithelial transplantation (CLET). Since its start in 1997, more than 1,0...
Corneal Stem Cells as a Source of Regenerative Cell-Based Therapy
Stem Cells International
In the past few years, intensive research has focused on corneal stem cells as an unlimited source for cell-based therapy in regenerative ophthalmology. Today, it is known that the cornea has at least two types of stem cells: limbal epithelial stem cells (LESCs) and corneal stromal stem cells (CSSCs). LESCs are used for regeneration of corneal surface, while CSSCs are used for regeneration of corneal stroma. Until now, various approaches and methods for isolation of LESCs and CSSCs and their successful transplantation have been described and tested in several preclinical studies and clinical trials. This review describes in detail phenotypic characteristics of LESCs and CSSCs and discusses their therapeutic potential in corneal regeneration. Since efficient and safe corneal stem cell-based therapy is still a challenging issue that requires continuous cooperation between researchers, clinicians, and patients, this review addresses the important limitations and suggests possible strat...