Peter Trosan

The Role of Mouse Mesenchymal Stem Cells in Differentiation of Naive T-Cells into Anti-Inflammatory Regulatory T-Cell or Proinflammatory Helper T-Cell 17 Population

Bone marrow-derived mesenchymal stem cells (MSCs) modulate immune response and can produce significant levels of transforming growth factor-β (TGF-β) 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 RORγt 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-β alone or TGF-β together with IL-6. Unstimulated MSCs produce TGF-β, but not IL-6, and the production of TGF-β can be further enhanced by the anti-inflammatory cytokines IL-10 or TGF-β. In the presence of proinflammatory cytokines, MSCs secrete significant levels of IL-6, in addition to a spontaneous production of TGF-β. MSCs producing TGF-β induced preferentially expression of Foxp3 and activation of Tregs, whereas MSC supernatants containing TGF-β together with IL-6 supported RORγt expression and development of Th17 cells. The effects of MSC supernatants were blocked by the inclusion of neutralization monoclonal antibody anti-TGF-β 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 RORγt(+) Th17 cell population and thereby can modulate autoimmune, immunopathological, and transplantation reactions.

The Key Role of Insulin-Like Growth Factor I in Limbal Stem Cell Differentiation and the Corneal Wound-Healing Process

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-β (FGF-β), 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.

Cyclosporine A-loaded and stem cell-seeded electrospun nanofibers for cell-based therapy and local immunosuppression

Cyclosporine A (CsA), a potent immunosuppressive drug with low water solubility, was dissolved in poly(L-lactic acid) (PLA) solution, and nanofibers were fabricated from this mixture by electrospinning technology. The addition of CsA into the PLA solution and the conditions of the electrospinning process did not influence the structure of the nanofibers nor affect the pharmacological activity of CsA. Study of the CsA release behavior in culture medium showed a release for at least 96 h. After the topical application of CsA-loaded nanofibers on skin allografts in vivo, the release was significantly slower and about 35% of the drug was still retained in the nanofibers on day 8. The addition of CsA-loaded nanofibers into cultures of mouse spleen cells stimulated with Concanavalin A selectively inhibited T cell functions; the activity of stimulated macrophages or the growth of non-T-cell populations was not suppressed in the presence of CsA-loaded nanofibers. The covering of skin allografts with CsA-loaded nanofibers significantly attenuated the local production of the proinflammatory cytokines IL-2, IFN-γ and IL-17. These results suggest that CsA-loaded electrospun nanofibers can serve as effective drug carriers for the local/topical suppression of an inflammatory reaction and simultaneously could be used as scaffolds for cell-based therapy.

The healing of alkali-injured cornea is stimulated by a novel matrix regenerating agent (RGTA, CACICOL20): a biopolymer mimicking heparan sulfates reducing proteolytic, oxidative and nitrosative damage.

The efficacy of a chemically modified dextran - heparan sulfate mimicking regenerating agent (RGTA) on the healing of the rabbit cornea injured with alkali was examined. The eyes were injured with 0.15 N NaOH applied on the cornea or with 1.0 N NaOH using a 8 mm diameter filter paper disk. Then RGTA or placebo was applied on the cornea. In the last group of rabbits, corneas injured with the high alkali concentration were left without any treatment for four weeks; subsequently, the corneas were treated with RGTA or placebo. The central corneal thickness was measured using a pachymeter. The corneas were examined morphologically, immunohistochemically and for real time-PCR. Compared to control (unaffected) corneas, following the application of low alkali concentration the expression of urokinase-type plasminogen activator, metalloproteinase 9, nitric oxide synthase and xanthine oxidase was increased in the injured corneal epithelium of placebo-treated eyes, whereas the expression of antioxidant enzymes was reduced. Nitrotyrosine and malondialdehyde stainings appeared in the corneal epithelium. RGTA application suppressed the antioxidant/prooxidant imbalance and reduced the expression of the above-mentioned immunohistochemical markers. The corneal thickness increased after alkali injury, decreased during corneal healing after RGTA treatment faster than after placebo application. Following the injury with the high alkali concentration, corneal inflammation and neovascularization were highly pronounced in placebo-treated corneas, whereas in RGTA-treated corneas they were significantly supressed. When RGTA or placebo application was started later after alkali injury and corneas were ulcerated, subsequent RGTA treatment healed the majority of them. In conclusion, RGTA facilitates the healing of injured corneas via a reduction of proteolytic, oxidative and nitrosative damage.

Modulation of the early inflammatory microenvironment in the alkali-burned eye by systemically administered interferon-γ-treated mesenchymal stromal cells.

The aim of this study was to investigate the effects of systemically administered bone-marrow-derived mesenchymal stromal cells (MSCs) on the early acute phase of inflammation in the alkali-burned eye. Mice with damaged eyes were either untreated or treated 24 h after the injury with an intravenous administration of fluorescent-dye-labeled MSCs that were unstimulated or pretreated with interleukin-1α (IL-1α), transforming growth factor-β (TGF-β), or interferon-γ (IFN-γ). Analysis of cell suspensions prepared from the eyes of treated mice on day 3 after the alkali burn revealed that MSCs specifically migrated to the damaged eye and that the number of labeled MSCs was more than 30-times higher in damaged eyes compared with control eyes. The study of the composition of the leukocyte populations within the damaged eyes showed that all types of tested MSCs slightly decreased the number of infiltrating lymphoid and myeloid cells, but only MSCs pretreated with IFN-γ significantly decreased the percentage of eye-infiltrating cells with a more profound effect on myeloid cells. Determining cytokine and NO production in the damaged eyes confirmed that the most effective immunomodulation was achieved with MSCs pretreated with IFN-γ, which significantly decreased the levels of the proinflammatory molecules IL-1α, IL-6, and NO. Taken together, the results show that systemically administered MSCs specifically migrate to the damaged eye and that IFN-γ-pretreated MSCs are superior in inhibiting the acute phase of inflammation, decreasing leukocyte infiltration, and attenuating the early inflammatory environment.

Distinct cytokines balance the development of regulatory T cells and interleukin-10-producing regulatory B cells

Regulatory T cells have been well described and the factors regulating their development and function have been identified. Recently, a growing body of evidence has documented the existence of interleukin‐10 (IL‐10) ‐producing B cells, which are called regulatory B10 cells. These cells attenuate autoimmune, inflammatory and transplantation reactions, and the main mechanism of their inhibitory action is the production of IL‐10. We show that the production of IL‐10 by lipopolysaccharide‐stimulated B cells is significantly enhanced by IL‐12 and interferon‐γ and negatively regulated by IL‐21 and transforming growth factor‐β. In addition, exogenous IL‐10 also inhibits B‐cell proliferation and the expression of the IL‐10 gene in lipopolysaccharide‐stimulated B cells. The negative autoregulation of IL‐10 production is supported by the observation that the inclusion of anti‐IL‐10 receptor monoclonal antibody enhances IL‐10 production and the proliferation of activated B cells. The effects of cytokines on IL‐10 production by B10 cells did not correlate with their effects on B‐cell proliferation or on IL‐10 production by T cells or macrophages. The cytokine‐induced changes in IL‐10 production occurred on the level of IL‐10 gene expression, as confirmed by increased or decreased IL‐10 mRNA expression in the presence of a particular cytokine. The regulatory cytokines modulate the number of IL‐10‐producing cells rather than augmenting or decreasing the secretion of IL‐10 on a single‐cell level. Altogether these data show that the production of IL‐10 by B cells is under the strict regulatory control of cytokines and that individual cytokines differentially regulate the development and activity of regulatory T cells and IL‐10‐producing regulatory B cells.

Suppression of IL-10 production by activated B cells via a cell contact-dependent cyclooxygenase-2 pathway upregulated in IFN-γ-treated mesenchymal stem cells.

The immunoregulatory properties of mesenchymal stem cells (MSCs) have been well documented in various models in vitro and in vivo. Furthermore, a population of regulatory B cells (Bregs) that produce relatively high concentrations of IL-10 has been recently described. To study the relationship between MSCs and Bregs, we analyzed the effects of MSCs on IL-10 production by lipopolysaccharide (LPS)-activated mouse B cells. The production of IL-10 by B cells remained preserved in the presence of MSCs and was even significantly enhanced by IFN-γ. However, the production of IL-10 was strongly suppressed in cultures containing MSCs and IFN-γ. Preincubation of MSCs, but not of B cells, with IFN-γ induced the suppression of IL-10 secretion in cultures containing MSCs and B cells. The supernatants from IFN-γ-treated MSCs had no inhibitory effect, and the suppression of IL-10 production was abrogated if the MSCs and B cells were separated in a transwell system. Analysis of the gene expression of IFN-γ- or IFN-γ and LPS-treated MSCs revealed a strong upregulation of genes for indoleamine-2,3-dioxygenase (IDO), cyclooxygenase-2 (Cox-2) and programmed cell death-ligand 1 (PD-L1). While the inhibition of IDO activity or the inclusion of the neutralization monoclonal antibody anti-PD-L1 did not abrogate the suppression, indomethacin, an inhibitor of Cox-2, completely inhibited the MSC-mediated suppression of IL-10 production. Accordingly, the production of IL-10 by B cells was inhibited by exogenous prostaglandin E2. The results thus suggest that IFN-γ-treated MSCs strongly inhibit IL-10 production by activated B cells by a mechanism requiring cell contact and involving the Cox-2 pathway.

The Favorable Effect of Mesenchymal Stem Cell Treatment on the Antioxidant Protective Mechanism in the Corneal Epithelium and Renewal of Corneal Optical Properties Changed after Alkali Burns

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.

Targeted neural differentiation of murine mesenchymal stem cells by a protocol simulating the inflammatory site of neural injury.

Damaged neural tissue is regenerated by neural stem cells (NSCs), which represent a rare and difficult‐to‐culture cell population. Therefore, alternative sources of stem cells are being tested to replace a shortage of NSCs. Here we show that mouse adipose tissue‐derived mesenchymal stem cells (MSCs) can be effectively differentiated into cells expressing neuronal cell markers. The differentiation protocol, simulating the inflammatory site of neural injury, involved brain tissue extract, fibroblast growth factor, epidermal growth factor, supernatant from activated splenocytes and electrical stimulation under physiological conditions. MSCs differentiated using this protocol displayed neuronal cell morphology and expressed genes for neuronal cell markers, such as neurofilament light (Nf‐L), medium (Nf‐M) and heavy (Nf‐H) polypeptides, synaptophysin (SYP), neural cell adhesion molecule (NCAM), glutamic acid decarboxylase (GAD), neuron‐specific nuclear protein (NeuN), βIII‐tubulin (Tubb3) and microtubule‐associated protein 2 (Mtap2), which are absent (Nf‐L, Nf‐H, SYP, GAD, NeuN and Mtap2) or only slightly expressed (NCAM, Tubb3 and Nf‐M) in undifferentiated cells. The differentiation was further enhanced when the cells were cultured on nanofibre scaffolds. The neural differentiation of MSCs, which was detected at the level of gene expression, was confirmed by positive immunostaining for Nf‐L protein. The results thus show that the simulation of conditions in an injured neural tissue and inflammatory environment, supplemented with electrical stimulation under physiological conditions and cultivation of cells on a three‐dimensional (3D) nanofibre scaffold, form an effective protocol for the differentiation of MSCs into cells with neuronal markers. Copyright

Transfer of mesenchymal stem cells and cyclosporine A on alkali-injured rabbit cornea using nanofiber scaffolds strongly reduces corneal neovascularization and scar formation.

The aim of this study was to examine whether nanofiber scaffolds seeded with rabbit bone marrow mesenchymal stem cells (MSCs nanofibers) transferred onto the damaged corneal surface and covered with cyclosporine A (CsA)-loaded nanofiber scaffolds (CsA nanofibers) enable healing of the rabbit cornea injured with 1N NaOH. The healing of damaged corneas was examined morphologically, immunohistochemically and biochemically on day 24 after the injury. Compared to untreated injured corneas, where corneal ulceration or large corneal thinning or even perforation were developed, injured corneas treated with drug free nanofibers healed without profound disturbances in a majority of cases, although with fibrosis and scar formation. In injured corneas treated with CsA nanofibers or MSCs nanofibers, the development of scar formation was reduced. Best healing results were obtained with a combination of MSCs and CsA nanofibers (MSCs-CsA nanofibers). Corneas healed with highly restored transparency. Neovascularization highly expressed in untreated injured corneas and reduced in corneas treated with CsA nanofibers or MSCs nanofibers, was suppressed in corneas treated with MSCs-CsA nanofibers. The levels of matrix metalloproteinase 9, inducible nitric oxide synthase, interleukin 6, α-smooth muscle actin, tumor growth factor β and vascular endothelial growth factor were significantly decreased in these corneas as compared to untreated corneas, where the levels of the above mentioned markers were high. In conclusion, MSCs-CsA nanofibers were effective in the treatment of severe alkali-induced corneal injury.