Magdalena Krulova

Treatment of Ocular Surface Injuries by Limbal and Mesenchymal Stem Cells Growing on Nanofiber Scaffolds

Stem cell (SC) therapy represents a promising approach to treat a wide variety of injuries, inherited diseases, or acquired SC deficiencies. One of the major problems associated with SC therapy remains the absence of a suitable matrix for SC growth and transfer. We describe here the growth and metabolic characteristics of mouse limbal stem cells (LSCs) and mesenchymal stem cells (MSCs) growing on 3D nanofiber scaffolds fabricated from polyamide 6/12 (PA6/12). The nanofibers were prepared by the original needleless electrospun Nanospider technology, which enables to create nanofibers of defined diameter, porosity, and a basis weight. Copolymer PA6/12 was selected on the basis of the stability of its nanofibers in aqueous solutions, its biocompatibility, and its superior properties as a matrix for the growth of LSCs, MSCs, and corneal epithelial and endothelial cell lines. The morphology, growth properties, and viability of cells grown on PA6/12 nanofibers were comparable with those grown on plastic. LSCs labeled with the fluorescent dye PKH26 and grown on PA6/12 nanofibers were transferred onto the damaged ocular surface, where their seeding and survival were monitored. Cotransfer of LSCs with MSCs, which have immunosuppressive properties, significantly inhibited local inflammatory reactions and supported the healing process. The results thus show that nanofibers prepared from copolymer PA6/12 represent a convenient scaffold for growth of LSCs and MSCs and transfer to treat SC deficiencies and various ocular surface injuries.

Nitric oxide as a regulatory and effector molecule in the immune system

Nitric oxide (NO) as a small ubiquitous molecule influencing a great variety of biological processes in the organism. Within the immune system, increased levels of NO were observed in various immunopathological situations, inflammatory reactions and during the response to transplantation and tumour antigens. It appears that NO can influence various facets of immune response. We studied involvement and the role of NO in immune response to skin allograft in mice. The production of NO at the site of graft rejection correlated well with the kinetic of rejection reaction and with the fate of the allograft. Graft infiltrating macrophages were identified as a principal cell population producing NO and the production of NO by macrophages was dependent on the presence of activated CD4(+) T cells. Survival of skin allografts was significantly prolonged by the treatment of graft recipients with 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), a specific inhibitor of inducible NO synthase (iNOS). These results suggest a role for NO as the effector cytotoxic molecule involved in the graft rejection. Experiments in vitro demonstrated that NO, in addition to its effector function, acts as a modulator of cytokine production. Spleen cells stimulated with alloantigens in the presence of AMT or S-ethylisothiourea (EIT), an another selective iNOS inhibitor, produced considerably more interleukin (IL)-4 and IL-10 than the cells stimulated in the absence of iNOS inhibitors. The production of Th1 cytokines IL-2 and interferon (IFN)-gamma was not enhanced by the inhibition of NO synthesis. The results altogether show that NO can act in transplantation reactions as an immunomodulator on cytokine production level and as an effector molecule involved in the graft destruction.

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.

A rapid separation of two distinct populations of mouse corneal epithelial cells with limbal stem cell characteristics by centrifugation on percoll gradient.

PURPOSE To detect and isolate cells with stem cell (SC) characteristics in the limbus of the mouse. METHODS Limbal tissues from BALB/c mice were trypsin-dissociated and separated on the gradient Percoll (Fluka, Buchs, Switzerland). Several fractions were isolated and characterized by real-time PCR for the presence of limbal SC markers and differentiation markers of corneal epithelial cells by flow cytometry for the determination of the side-population (SP) phenotype and growth properties in vitro. RESULTS Cells retained in the lightest fraction (40% Percoll) and in the densest fraction (80% Percoll) of the gradient were both enriched for populations with a high expression of the SC markers ABCG2 and Lgr5 and also expressing the SP phenotype. However, the lightest fraction (representing approximately 12% of total limbal cells) contained cells with the strongest spontaneous proliferative capacity and expressed the corneal epithelial differentiation marker K12. In contrast the densest fraction (<7% of original cells) was K12 negative and contained small nonspontaneously proliferating cells, which instead were positive for p63. Unexpectedly, cells from this fraction had the highest proliferative activity when cultured on a 3T3 feeder cell monolayer. CONCLUSIONS These findings demonstrate the presence of two distinct populations of corneal epithelial cells with limbal SC characteristics, based on differential expression of the keratin-specific marker K12 and transcription factor p63, and suggest a difference in developmental stage of the two populations, with the K12(-)p63(+) population being closer to the primitive limbal SC.

Immunoregulatory Properties of Mouse Limbal Stem Cells

Stem cells have been demonstrated in nearly all adult mammalian tissues and play a vital role in their physiological renewal and healing after injury. Due to their irreplaceable role in tissue repair, these cells had to develop mechanisms protecting them from deleterious inflammatory immune reactions and ensuring their increased resistance to various apoptosis-inducing agents. In this study, we demonstrate that a population of mouse limbal cells highly enriched for cells expressing markers and charateristics of limbal stem cells (LSCs) suppresses in a dose-dependent manner the proliferation of lymphocytes elicited by mitogens or TCR-triggering and significantly inhibits the production of proinflammatory cytokines by activated T cells. The suppression was mediated by soluble factor(s) and did not affect early cell activation. LSCs were even more suppressive than mesenchymal stem cells or natural regulatory T cells. In addition, the cells expressing markers and characteristics of LSC had significantly higher levels of mRNA for Fas ligand and for the antiapoptotic molecules Mcl-1, XIAP, and survivin than other limbal cell populations. LSCs were also more resistant to staurosporin-induced apoptotic cell death and to cell-mediated cytotoxic reaction than other limbal cells. Collectively, these results suggest that SC isolated from fresh adult limbal tissue possess immunomodulatory properties and inhibit proinflammatory immune reactions. Simultaneously, these cells express high levels of mRNA for antiapoptotic molecules, which can protect them against cell-mediated cytotoxic reactions and various apoptosis-inducing agents.

Distinct regulatory roles of transforming growth factor-β and interleukin-4 in the development and maintenance of natural and induced CD4+ CD25+ Foxp3+ regulatory T cells

The development and function of CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) are strictly regulated by cytokines. Here we show that transforming growth factor‐β (TGF‐β) and interleukin‐4 (IL‐4) play a crucial and antagonistic role in the development of Tregs. Additionally, these cytokines also have distinct effects on the maintenance of natural (nTregs) and antigen‐induced (iTregs) Tregs. Using double‐staining and tracking of proliferation of purified and carboxyflourescein succinimidyl ester (CFSE)‐labelled mouse T‐cell subpopulations we demonstrated that CD4+ CD25+ Foxp3+ iTregs develop upon alloantigenic stimulation in the presence of TGF‐β exclusively from CD4+ CD25− Foxp3− precursors. Both the induction of Foxp3 expression and Treg proliferation were prevented when the cells were stimulated in the presence of IL‐4. By contrast, nTregs did not proliferate in the presence of the antigen and TGF‐β, and partially lost their Foxp3 expression. IL‐4 not only prevented the development of iTregs, but also down‐regulated the level of Foxp3 mRNA and decreased the number of Foxp3+ cells in a population of iTregs. Further analyses proved that IL‐4 decreased the expression of Foxp3 only in a population of iTregs, whereas it substantially supported the survival of nTregs. Functional experiments showed that Tregs induced in the presence of alloantigen and TGF‐β inhibited, on a per‐cell basis, cell proliferation comparably to nTregs, and their suppressive capacity was not modulated by IL‐4. These data suggest that TGF‐β and IL‐4 differentially regulate the development of Tregs and distinctly sustain Foxp3 expression and the number of nTregs and iTregs, but have no influence on the suppressive activity of Tregs on a per‐cell basis.

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.

Graft survival and cytokine production profile after limbal transplantation in the experimental mouse model

Limbal transplantation or limbal stem cell (LSC) transfer represents the only way to treat severe ocular surface damage or LSC deficiency. However, limbal allografts are promptly rejected in spite of extensive immunosuppressive therapy. To characterize immune response after limbal transplantation, we established an experimental model of limbal transplantation in the mouse. Syngeneic, allogeneic and xenogeneic (rat) limbal grafts were grafted orthotopically in BALB/c mice and graft survival was evaluated. The presence of graft donor cells and the expression of IL-2, IL-4, IL-10, IFN-γ and inducible nitric oxide synthase (iNOS) mRNA in the grafts were detected by real-time PCR. While syngeneic grafts survived permanently, allografts were rejected in 9.0±1.8 days and xenografts in 6.5±1.1 days. The manifestation of clinical symptoms of rejection correlated with the disappearance of donor cells in the graft and in the recipient cornea. Intragraft expression of iNOS mRNA and distinct expression patterns of Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) cytokines were detected during rejection of limbal allografts and xenografts. The limbal graft rejection was prevented with anti-CD4, but not anti-CD8 monoclonal antibody therapy. The results indicate that limbal grafts do not enjoy immune privilege of the eye and are promptly rejected by Th1 (allografts) or by a combined Th1 and Th2 (xenografts) type of immune response involving CD4+ cells and iNOS expression. Targeting this pathway may be an effective way to prevent and treat limbal graft rejection.

Mesenchymal Stem Cells Attenuate the Adverse Effects of Immunosuppressive Drugs on Distinct T Cell Subopulations

Immunosuppressive drugs are widely used to treat undesirable immune reaction, however their clinical use is often limited by harmful side effects. The combined application of immunosuppressive agents with mesenchymal stem cells (MSCs) offers a promising alternative approach that enables the reduction of immunosuppressive agent doses and simultaneously maintains or improves the outcome of therapy. The present study aimed to determinate the effects of immunosuppressants on individual T cell subpopulations and to investigate the efficacy of MSC-based treatment combined with immunosuppressive drugs. We tested the effect of five widely used immunosuppressants with different action mechanisms: cyclosporine A, mycophenolate mofetil, rapamycin, and two glucocorticoids - prednisone and dexamethasone in combination with MSCs on mouse CD4+ and CD8+ lymphocyte viability and activation, Th17 (RORγt+), Th1 (T-bet+), Th2 (GATA-3+) and Treg (Foxp3+) cell proportion and on the production of corresponding key cytokines (IL-17, IFNγ, IL-4 and IL-10). We showed that MSCs modulate the actions of immunosuppressants and in combination with immunosuppressive drugs display distinct effect on cell activation and balance among different T lymphocytes subpopulations and exert a suppressive effect on proinflammatory T cell subsets while promoting the functions of anti-inflammatory Treg lymphocytes. The results indicated that MSC-based therapy could be a powerful strategy to attenuate the negative effects of immunosuppressive drugs on the immune system.