Two house mouse subspecies, Mus musculus domesticus and Mus musculus musculus, form a hybrid zone in Europe and represent a suitable model for inferring the genes contributing to isolation barriers between parental taxa. Despite long-term intensive studies of this hybrid zone, we still know relatively little about the causes and mechanisms maintaining the 2 taxa as separate subspecies; therefore, to gain insight into this process, we developed 8 wild-derived inbred house mouse strains. In order to produce strains as pure domesticus or musculus genomes as possible, the individuals used to establish the breeding colony for the 3 domesticus and 2 of the musculus strains were captured in the Czech Republic from wild populations at extreme western and eastern edges of the subspecific contact zone, respectively. The remaining 3 musculus strains were bred from mice captured about 250 km east of the hybrid zone. Genetic analysis based on 361 microsatellite loci showed that 82% of these markers are diagnostic for either the musculus or the domesticus strains. In order to demonstrate the potential utility of this genetic differentiation in such strains, phenotypic variation was scored for 2 strains from opposite edges of the hybrid zone and significant differences in morphology, reproductive performance, in vitro immune responses, mate choice based on urinary signals, and aggressiveness were found. In addition, the 3 strains derived from musculus populations far from the hybrid zone display significant differences in polymorphism in hybrid male sterility when crossed with the laboratory strains C57BL/6 or C57BL/10, which have a predominantly domesticus genome. Although further studies will be necessary to demonstrate intersubspecific differences, all analyses presented here indicate that these newly developed house mouse strains represent a powerful tool for elucidating the genetic basis of isolation barriers in hybrid zones and for studying speciation in general.

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.

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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.

Biomedical applications such as wound dressing for skin regeneration, stem cell transplantation, or drug delivery require special demands on the three-dimensional porous scaffolds. Besides the biocompatibility and mechanical properties, the morphology is the most important attribute of the scaffold. Specific surface area, volume, and size of the pores have considerable effect on cell adhesion, growth, and proliferation. In the case of incorporated biologically active substances, their release is also influenced by the internal structure of nanofibers. Although many scientific papers are focused on the preparation of nanofibers and evaluation of biological tests, the morphological characterization was described just briefly as service methods. The aim of this paper is to summarize the methods applicable for morphological characterization of nanofibers and supplement it by the results of our research. Needleless electrospinning technique was used to prepare nanofibers from polylactide, poly(ε-caprolactone), gelatin, and polyamide. Scanning electron microscopy was used to evaluate the fiber diameters and to reveal eventual artifacts in the nanofibrous structure. Nitrogen adsorption/desorption measurements were employed to measure the specific surface areas. Mercury porosimetry was used to determine total porosities and compare pore size distributions of the prepared samples.

Electrospun gelatin and poly-ε-caprolactone (PCL) nanofibers were prepared using needleless technology and their biocompatibility and therapeutic efficacy have been characterized in vitro in cell cultures and in an experimental model of a skin wound. Human dermal fibroblasts, keratinocytes and mesenchymal stem cells seeded on the nanofibers revealed that both nanofibers promoted cell adhesion and proliferation. The effect of nanofibers on wound healing was examined using a full thickness wound model in rats and compared with a standard control treatment with gauze. Significantly faster wound closure was found with gelatin after 5 and 10 days of treatment, but no enhancement with PCL nanofibers was observed. Histological analysis revealed enhanced epithelialisation, increased depth of granulation tissue and increased density of myofibroblasts in the wound area with gelatin nanofibers. The results show that gelatin nanofibers produced by needleless technology accelerate wound healing and may be suitable as a scaffold for cell transfer and skin regeneration.