Corneal regeneration x Stem cell-based therapy ABSTRACTStem 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 (LSCs), and their growth, differentiation, and secretory properties were compared. The cells were grown on nanofiber scaffolds and transferred onto the alkaliinjured 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 tissuespecific 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

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Bone marrow-derived mesenchymal stem cells (MSCs) modulate immune response and can produce significant levels of transforming growth factor-b (TGF-b) and interleukin-6 (IL-6). These 2 cytokines represent the key factors that reciprocally regulate the development and polarization of naive T-cells into regulatory T-cell (Treg) population or proinflammatory T helper 17 (Th17) cells. In the present study we demonstrate that MSCs and their products effectively regulate expression of transcription factors Foxp3 and RORgt and control the development of Tregs and Th17 cells in a population of alloantigen-activated mouse spleen cells or purified CD4 + CD25-T-cells. The immunomodulatory effects of MSCs were more pronounced when these cells were stimulated to secrete TGF-b alone or TGF-b together with IL-6. Unstimulated MSCs produce TGF-b, but not IL-6, and the production of TGF-b can be further enhanced by the anti-inflammatory cytokines IL-10 or TGF-b. In the presence of proinflammatory cytokines, MSCs secrete significant levels of IL-6, in addition to a spontaneous production of TGF-b. MSCs producing TGF-b induced preferentially expression of Foxp3 and activation of Tregs, whereas MSC supernatants containing TGF-b together with IL-6 supported RORgt expression and development of Th17 cells. The effects of MSC supernatants were blocked by the inclusion of neutralization monoclonal antibody anti-TGF-b or anti-IL-6 into the culture system. The results showed that MSCs represent important players that reciprocally regulate the development and differentiation of uncommitted naive T-cells into anti-inflammatory Foxp3 + Tregs or proinflammatory RORgt + Th17 cell population and thereby can modulate autoimmune, immunopathological, and transplantation reactions.

Limbal stem cells (LSC), which reside in the basal layer of the limbus, are thought to be responsible for corneal epithelial healing after injury. When the cornea is damaged, LSC start to proliferate, differentiate, and migrate to the site of injury. To characterize the signaling molecules ensuring communication between the cornea and LSC, we established a mouse model of mechanical corneal damage. The central cornea or limbal tissue was excised at different time intervals after injury, and the expression of genes in the explants was determined. It was observed that a number of genes for growth and differentiation factors were significantly upregulated in the cornea rapidly after injury. The ability of these factors to regulate the differentiation and proliferation of limbal cells was tested. It was found that the insulin-like growth factor-I (IGF-I), which is rapidly overexpressed after injury, enhances the expression of IGF receptor in limbal cells and induces the differentiation of LSC into cells expressing the corneal cell marker, cytokeratin K12, without any effect on limbal cell proliferation. In contrast, the epidermal growth factor (EGF) and fibroblast growth factor-b (FGF-b), which are also produced by the damaged corneal epithelium, supported limbal cell proliferation without any effect on their differentiation. Other factors did not affect limbal cell differentiation or proliferation. Thus, IGF-I was identified as the main factor stimulating the expression of IGF receptors in limbal cells and inducing the differentiation of LSC into cells expressing corneal epithelial cell markers. The proliferation of these cells was supported by EGF and FGF.

The aim of this study was to examine whether mesenchymal stem cells (MSCs) and/or corneal limbal epithelial stem cells (LSCs) influence restoration of an antioxidant protective mechanism in the corneal epithelium and renewal of corneal optical properties changed after alkali burns. The injured rabbit corneas (with 0.25 N NaOH) were untreated or treated with nanofiber scaffolds free of stem cells, with nanofiber scaffolds seeded with bone marrow MSCs (BM-MSCs), with adipose tissue MSCs (Ad-MSCs), or with LSCs. On day 15 following the injury, after BM-MSCs or LSCs nanofiber treatment (less after Ad-MSCs treatment) the expression of antioxidant enzymes was restored in the regenerated corneal epithelium and the expressions of matrix metalloproteinase 9 (MMP9), inducible nitric oxide synthase (iNOS), α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), and vascular endothelial factor (VEGF) were low. The central corneal thickness (taken as an index of corneal hydration) increased after the injury and returned to levels before the injury. In injured untreated corneas the epithelium was absent and numerous cells revealed the expressions of iNOS, MMP9, α-SMA, TGF-β1, and VEGF. In conclusion, stem cell treatment accelerated regeneration of the corneal epithelium, restored the antioxidant protective mechanism, and renewed corneal optical properties.

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Nowadays, the focus in medicine on molecular genetics has resulted in a disregard for the physical basis of treatment even though many diseases originate from changes in cellular mechanics. Perturbations of the cellular nanomechanics promote pathologies, including cardiovascular disease and cancer. Furthermore, whilst the biological and therapeutic effects of magnetic fields are a well-established fact, to date the underlying mechanisms remain obscure. Here, we show that oscillating high-gradient magnetic field (HGMF) and mechanical vibration affect adipogenic differentiation of mesenchymal stem cells by the transmission of mechanical stress to the cell cytoskeleton, resulting in F-actin remodelling and subsequent down-regulation of adipogenic genes adiponectin, PPARγ, and AP2. Our findings propose an insight into the regulation of cellular nanomechanics, and provide a basis for better controlled down-regulation of stem cell adipogenesis by HGMF, which may facilitate the development of challenging therapeutic strategies suitable for the remote control of biological systems.