The human amniotic membrane (HAM) is widely used for its wound healing effect in clinical practice, as a feeder for the cell cultivation, or a source of cells to be used in cell therapy. The aim of this study was to find effective and safe enzymatic HAM de-epithelialization method leading to harvesting of both denuded undamaged HAM and viable human amniotic epithelial cells (hAECs). The efficiency of de-epithelialization using TrypLE Express, trypsin/ ethylenediaminetetraacetic (EDTA), and thermolysin was monitored by hematoxylin and eosin staining and by the measurement of DNA concentration. The cell viability was determined by trypan blue staining. Scanning electron microscopy and immunodetection of collagen type IV and laminin α5 chain were used to check the basement membrane integrity. De-epithelialized hAECs were cultured and their stemness properties and proliferation potential was assessed after each passage. The HAM was successfully de-epithelialized using all three types of reagents, but morphological changes in basement membrane and stroma were observed after the thermolysin application. About 60% of cells remained viable using trypsin/EDTA, approximately 6% using TrypLE Express, and all cells were lethally damaged after thermolysin application. The hAECs isolated using trypsin/EDTA were successfully cultured up to the 5th passage with increasing proliferation potential and decreased stem cell markers expression (NANOG, SOX2) in prolonged cell culture. Trypsin/EDTA technique was the most efficient for obtaining both undamaged denuded HAM and viable hAECs for consequent culture.

• MeSH
• amnion cytologie   metabolismus   patologie   MeSH
• DNA analýza   izolace a purifikace   MeSH
• EDTA chemie   MeSH
• epitelové buňky cytologie   metabolismus   patologie   MeSH
• kolagen typu IV metabolismus   MeSH
• kultivované buňky MeSH
• laminin metabolismus   MeSH
• lidé MeSH
• mikroskopie elektronová rastrovací MeSH
• nanog metabolismus   MeSH
• proliferace buněk MeSH
• reepitalizace MeSH
• transkripční faktory SOXB1 metabolismus   MeSH
• trypsin metabolismus   MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• EDTA MeSH
• kolagen typu IV MeSH
• laminin alpha5 MeSH Prohlížeč
• laminin MeSH
• nanog MeSH
• transkripční faktory SOXB1 MeSH
• trypsin MeSH

Retinal degenerative diseases, which include age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, and glaucoma, mostly affect the elderly population and are the most common cause of decreased quality of vision or even blindness. So far, there is no satisfactory treatment protocol to prevent, stop, or cure these disorders. A great hope and promise for patients suffering from retinal diseases is represented by stem cell-based therapy that could replace diseased or missing retinal cells and support regeneration. In this respect, mesenchymal stem cells (MSCs) that can be obtained from the particular patient and used as autologous cells have turned out to be a promising stem cell type for treatment. Here we show that MSCs can differentiate into cells expressing markers of retinal cells, inhibit production of pro-inflammatory cytokines by retinal tissue, and produce a number of growth and neuroprotective factors for retinal regeneration. All of these properties make MSCs a prospective cell type for cell-based therapy of age-related retinal degenerative diseases.

• Klíčová slova
• age-related retinal degenerative diseases, mesenchymal stem cells, stem cell therapy,
• MeSH
• buněčná diferenciace genetika   fyziologie   MeSH
• degenerace retiny metabolismus   terapie   MeSH
• mezenchymální kmenové buňky cytologie   metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• nemoci retiny metabolismus   terapie   MeSH
• prospektivní studie MeSH
• transplantace kmenových buněk metody   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

PURPOSE: The present study aims to prepare poly(D,L-lactic acid) (PLA) nanofibers loaded by the immunosuppressant cyclosporine A (CsA, 10 wt%). Amphiphilic poly(ethylene glycol)s (PEG) additives were used to modify the hydrophobic drug release kinetics. METHODS: Four types of CsA-loaded PLA nanofibrous carriers varying in the presence and molecular weight (MW) of PEG (6, 20 and 35 kDa) were prepared by needleless electrospinning. The samples were extracted for 144 h in phosphate buffer saline or tissue culture medium. A newly developed and validated LC-MS/MS method was utilized to quantify the amount of released CsA from the carriers. In vitro cell experiments were used to evaluate biological activity. RESULTS: Nanofibers containing 15 wt% of PEG showed improved drug release characteristics; significantly higher release rates were achieved in initial part of experiment (24 h). The highest released doses of CsA were obtained from the nanofibers with PEG of the lowest MW (6 kDa). In vitro experiments on ConA-stimulated spleen cells revealed the biological activity of the released CsA for the whole study period of 144 h and nanofibers containing PEG with the lowest MW exhibited the highest impact (inhibition). CONCLUSIONS: The addition of PEG of a particular MW enables to control CsA release from PLA nanofibrous carriers. The biological activity of CsA-loaded PLA nanofibers with PEG persists even after 144 h of previous extraction. Prepared materials are promising for local immunosuppression in various medical applications.

• Klíčová slova
• LC-MS/MS, cyclosporine A, drug release kinetics, poly(D,L-lactic acid) nanofibers, poly(ethylene glycol),
• MeSH
• buněčné linie MeSH
• cyklosporin aplikace a dávkování   chemie   MeSH
• hydrofobní a hydrofilní interakce MeSH
• imunosupresiva aplikace a dávkování   chemie   MeSH
• kinetika MeSH
• kultivační média MeSH
• lidé MeSH
• nanovlákna chemie   MeSH
• nosiče léků MeSH
• polyestery chemie   MeSH
• polyethylenglykoly chemie   MeSH
• povrchové vlastnosti MeSH
• slezina cytologie   MeSH
• techniky tkáňových kultur MeSH
• uvolňování léčiv MeSH
• velikost částic MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cyklosporin MeSH
• imunosupresiva MeSH
• kultivační média MeSH
• nosiče léků MeSH
• poly(lactide) MeSH Prohlížeč
• polyestery MeSH
• polyethylenglykoly MeSH

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.

• MeSH
• alkálie MeSH
• antioxidancia terapeutické užití   MeSH
• buněčná diferenciace účinky léků   MeSH
• chemické popálení enzymologie   genetika   patologie   terapie   MeSH
• imunohistochemie MeSH
• králíci MeSH
• limbus corneae cytologie   MeSH
• matrixová metaloproteinasa 9 metabolismus   MeSH
• mezenchymální kmenové buňky cytologie   účinky léků   MeSH
• ochranné látky farmakologie   terapeutické užití   MeSH
• pachymetrie rohovky MeSH
• regulace genové exprese účinky léků   MeSH
• rohovkový epitel patologie   MeSH
• superoxiddismutasa metabolismus   MeSH
• synthasa oxidu dusnatého, typ II metabolismus   MeSH
• transformující růstový faktor beta genetika   metabolismus   MeSH
• transplantace mezenchymálních kmenových buněk * MeSH
• tukové buňky cytologie   účinky léků   MeSH
• vaskulární endoteliální růstový faktor A metabolismus   MeSH
• zákal rohovky komplikace   terapie   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• publikace stažené z tisku MeSH
• Názvy látek
• alkálie MeSH
• antioxidancia MeSH
• matrixová metaloproteinasa 9 MeSH
• ochranné látky MeSH
• superoxiddismutasa MeSH
• synthasa oxidu dusnatého, typ II MeSH
• transformující růstový faktor beta MeSH
• vaskulární endoteliální růstový faktor A MeSH

UNLABELLED: 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 (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. 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 was 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.

• Klíčová slova
• Alkali-injured ocular surface, Corneal regeneration, Limbal stem cells, Mesenchymal stem cells, Stem cell-based therapy,
• MeSH
• biologické markery metabolismus   MeSH
• buněčná a tkáňová terapie metody   MeSH
• buněčná diferenciace MeSH
• buňky kostní dřeně cytologie   fyziologie   MeSH
• chemické popálení patologie   terapie   MeSH
• epitelové buňky cytologie   fyziologie   transplantace   MeSH
• exprese genu MeSH
• fyziologická neovaskularizace MeSH
• králíci MeSH
• limbus corneae krevní zásobení   zranění   MeSH
• mezenchymální kmenové buňky cytologie   fyziologie   MeSH
• primární buněčná kultura MeSH
• proliferace buněk MeSH
• reepitalizace fyziologie   MeSH
• rohovkový epitel krevní zásobení   zranění   MeSH
• tkáňové podpůrné struktury MeSH
• transplantace mezenchymálních kmenových buněk * MeSH
• tuková tkáň cytologie   fyziologie   MeSH
• tukové buňky cytologie   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• biologické markery MeSH

Ocular surface defects represent one of the most common causes of impaired vision or even blindness. For treatment, keratoplasty represents the first choice. However, if corneal defects are more extensive and associated with a limbal stem cell (LSC) deficiency, corneal transplantation is not a sufficient therapeutic procedure and only viable approach to treatment is the transplantation of LSCs. When the LSC deficiency is a bilateral disorder, autologous LSCs are not available. The use of allogeneic LSCs requires strong immunosuppression, which leads to side-effects, and the treatment is not always effective. The alternative and perspective approach to the treatment of severe ocular surface injuries and LSC deficiency is offered by the transplantation of autologous mesenchymal stem cells (MSCs). These cells can be obtained from the bone marrow or adipose tissue of the particular patient, grow well in vitro and can be transferred, using an appropriate scaffold, onto the damaged ocular surface. Here they exert beneficial effects by possible direct differentiation into corneal epithelial cells, by immunomodulatory effects and by the production of numerous trophic and growth factors. Recent experiments utilizing the therapeutic properties of MSCs in animal models with a mechanically or chemically injured ocular surface have yielded promising results and demonstrated significant corneal regeneration, improved corneal transparency and a rapid healing process associated with the restoration of vision. The use of autologous MSCs thus represents a promising therapeutic approach and offers hope for patients with severe ocular surface injuries and LSC deficiency.

• MeSH
• autologní transplantace MeSH
• biologické modely MeSH
• buněčná diferenciace MeSH
• buňky kostní dřeně cytologie   metabolismus   MeSH
• CD antigeny metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mezenchymální kmenové buňky cytologie   metabolismus   MeSH
• mezibuněčné signální peptidy a proteiny metabolismus   MeSH
• nanovlákna * MeSH
• nemoci rohovky chirurgie   MeSH
• pohyb buněk MeSH
• transplantace kmenových buněk metody   MeSH
• transplantace mezenchymálních kmenových buněk metody   MeSH
• tuková tkáň cytologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CD antigeny MeSH
• mezibuněčné signální peptidy a proteiny MeSH

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.

• MeSH
• buněčná diferenciace genetika   MeSH
• epidermální růstový faktor biosyntéza   metabolismus   MeSH
• fibroblastové růstové faktory biosyntéza   metabolismus   MeSH
• hojení ran fyziologie   MeSH
• insulinu podobný růstový faktor I biosyntéza   metabolismus   MeSH
• keratin-12 biosyntéza   MeSH
• kmenové buňky metabolismus   MeSH
• limbus corneae cytologie   metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• proliferace buněk MeSH
• receptor IGF typ 1 biosyntéza   metabolismus   MeSH
• rohovkový epitel * cytologie   zranění   metabolismus   MeSH
• signální transdukce MeSH
• stanovení celkové genové exprese MeSH
• upregulace MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• epidermální růstový faktor MeSH
• fibroblastové růstové faktory MeSH
• insulinu podobný růstový faktor I MeSH
• keratin-12 MeSH
• receptor IGF typ 1 MeSH

Polyvinyl alcohol nanofibers incorporating the wide spectrum antibiotic gentamicin were prepared by Nanospider™ needleless technology. A polyvinyl alcohol layer, serving as a drug reservoir, was covered from both sides by polyurethane layers of various thicknesses. The multilayered structure of the nanofibers was observed using scanning electron microscopy, the porosity was characterized by mercury porosimetry, and nitrogen adsorption/desorption measurements were used to determine specific surface areas. The stability of the gentamicin released from the electrospun layers was proved by high-performance liquid chromatography (HPLC) and inhibition of bacterial growth. Drug release was investigated using in vitro experiments with HPLC/MS quantification, while the antimicrobial efficacy was evaluated on Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa. Both experiments proved that the released gentamicin retained its activity and showed that the retention of the drug in the nanofibers was prolonged with the increasing thickness of the covering layers.

• Klíčová slova
• drug release, electrospinning, gentamicin, morphology, multilayered structure, nanofibers,
• MeSH
• antibakteriální látky aplikace a dávkování   chemie   MeSH
• difuze MeSH
• elektrochemie metody   MeSH
• gentamiciny aplikace a dávkování   MeSH
• grampozitivní bakterie účinky léků   fyziologie   MeSH
• léky s prodlouženým účinkem aplikace a dávkování   chemie   MeSH
• nanokapsle chemie   ultrastruktura   MeSH
• rotace MeSH
• testování materiálů MeSH
• velikost částic MeSH
• viabilita buněk účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• gentamiciny MeSH
• léky s prodlouženým účinkem MeSH
• nanokapsle MeSH

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.

• MeSH
• buněčná diferenciace imunologie   MeSH
• buněčné kultury MeSH
• cytokiny biosyntéza   MeSH
• imunita MeSH
• kultivační média speciální farmakologie   MeSH
• mezenchymální kmenové buňky fyziologie   MeSH
• myši MeSH
• parakrinní signalizace imunologie   MeSH
• regulační T-lymfocyty imunologie   MeSH
• T-lymfocyty imunologie   MeSH
• zánět imunologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokiny MeSH
• kultivační média speciální MeSH

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.

• MeSH
• biokompatibilní materiály * MeSH
• hojení ran * MeSH
• lidé MeSH
• nanovlákna * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biokompatibilní materiály * MeSH