Numerous studies show that various genes in all kinds of organisms are transcribed discontinuously, i.e. in short bursts or pulses with periods of inactivity between them. But it remains unclear whether ribosomal DNA (rDNA), represented by multiple copies in every cell, is also expressed in such manner. In this work, we synchronized the pol I activity in the populations of tumour derived as well as normal human cells by cold block and release. Our experiments with 5-fluorouridine (FU) and BrUTP confirmed that the nucleolar transcription can be efficiently and reversibly arrested at +4°C. Then using special software for analysis of the microscopic images, we measured the intensity of transcription signal (incorporated FU) in the nucleoli at different time points after the release. We found that the ribosomal genes in the human cells are transcribed discontinuously with periods ranging from 45 min to 75 min. Our data indicate that the dynamics of rDNA transcription follows the undulating pattern, in which the bursts are alternated by periods of rare transcription events.
- MeSH
- buněčné jadérko genetika MeSH
- epitelové buňky metabolismus MeSH
- genetická transkripce * MeSH
- HeLa buňky MeSH
- kinetika MeSH
- lidé středního věku MeSH
- lidé MeSH
- limbus corneae cytologie MeSH
- mrtvola MeSH
- nízká teplota MeSH
- ribozomální DNA genetika MeSH
- ribozomy genetika MeSH
- RNA ribozomální genetika MeSH
- senioři MeSH
- software MeSH
- transfekce MeSH
- uridin analogy a deriváty imunologie metabolismus MeSH
- uridintrifosfát analogy a deriváty imunologie metabolismus MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- senioři MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Human limbal epithelial cells (LECs) intended for treatment of limbal stem cell deficiency are commonly cultivated on a 3T3 feeder layer with complex culture medium supplemented with fetal bovine serum (FBS). However, FBS is a xenogeneic component containing poorly characterised constituents and exhibits quantitative and qualitative lot-to-lot variations. Human limbal explants were plated on untreated or fibrin coated plastic plates and cultured in two non-xenogeneic media (supplemented with either human serum or platelet lysate only). Our aim was to find out whether the characteristics of harvested LEC cultures are comparable to those of LEC cultivated in the gold standard - FBS-supplemented complex medium. The growth kinetics, cell proliferation, differentiation, stemness maintenance, apoptosis and contamination by other cell types were evaluated and compared among these conditions. In all of them LECs were successfully cultivated. Stemness was preserved in both xeno-free media. However, cells cultured with human serum on the fibrin-coated plates had the highest growth rate and cell proliferation and very low fibroblast-like cell contamination. These data suggest that xeno-free cell culture conditions can replace the traditional FBS-supplemented medium and thereby provide a safer protocol for ex vivo cultured limbal stem cell transplants.
- MeSH
- biokompatibilní potahované materiály MeSH
- biologické markery metabolismus MeSH
- buněčná diferenciace MeSH
- buněčné kultury MeSH
- dárci tkání MeSH
- dospělí MeSH
- fibrin farmakologie MeSH
- kultivační média MeSH
- kultivované buňky MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé středního věku MeSH
- lidé MeSH
- limbus corneae cytologie metabolismus MeSH
- počet buněk MeSH
- podkladové buňky MeSH
- proliferace buněk fyziologie MeSH
- rohovkový epitel cytologie metabolismus MeSH
- senioři MeSH
- sérum * MeSH
- trombocyty * MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- senioři MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie 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
The injured or otherwise damaged cornea is healed by limbal stem cells (LSC). If the limbus where LSC reside is also damaged or nonfunctional, the cornea cannot heal properly and this defect leads to impaired vision that can result in blindness. The only way to treat total LSC deficiency is by transplantation of limbal tissue or a transfer of LSC. Recently, mesenchymal stem cells (MSC) have been shown as another promising source of stem cells for corneal healing and regeneration. Here, we describe a protocol for the use of polyamide 6/12 nanofiber scaffolds for the growth of MSC and LSC, and for their transfer onto a mechanically damaged ocular surface in the experimental mouse model.
- MeSH
- buněčné kultury MeSH
- CD antigeny metabolismus MeSH
- dospělé kmenové buňky metabolismus transplantace MeSH
- kultivované buňky MeSH
- lidé MeSH
- limbus corneae cytologie MeSH
- mezenchymální kmenové buňky fyziologie MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- nanovlákna chemie MeSH
- nylony chemie MeSH
- separace buněk MeSH
- tkáňové podpůrné struktury chemie MeSH
- transplantace mezenchymálních kmenových buněk * MeSH
- tvar buňky MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
PURPOSE: The purpose of this study was to investigate the expression of cytokeratin (CK) 8 in the corneoconjunctival epithelium. METHODS: In 17 cadaveric corneoscleral discs and 3 other discs, the presence of CK8 alone or CK8, together with CK3, CK15, vimentin, and integrin α6, was investigated by using indirect immunohistochemistry on radial cryosections. Four corneoscleral discs stored in organ culture were used for the preparation of tangential sections of the limbus and for the isolation of limbal epithelial cells and their subsequent cultivation. CK8 expression was examined by RT-PCR in the corneal, limbal, and conjunctival epithelium. RESULTS: Sixty percent of the cadaveric corneoscleral samples and all samples stored in organ culture revealed positivity for CK8 in the basal epithelial layer of the limbus. Positive basal cells formed a single line or separated clusters. The signal for CK8 became weaker toward the surface of the limbal epithelium. The colocalization of CK8 with vimentin and CK15 in the limbus was also found. CK3 showed only occasional positivity in some of the surface limbal cells. The expression of integrin α6 in the basal membrane was absent or decreased under the CK8-positive clusters. Cell cultures revealed strong positivity for CK8 in approximately 80% of the cultured cells, and CK8 expression in the cornea, limbus, and conjunctiva was determined by RT-PCR. CONCLUSIONS: The study demonstrates the strong expression of CK8 in limbal epithelial basal cells, which is maintained during the differentiation and migration of the limbal cells toward the central corneal epithelium.
- MeSH
- buněčné kultury MeSH
- dospělí MeSH
- epitelové buňky metabolismus MeSH
- exprese genu MeSH
- fluorescenční mikroskopie MeSH
- fluorescenční protilátková technika nepřímá MeSH
- integrin alfa6 metabolismus MeSH
- keratin-15 metabolismus MeSH
- keratin-3 metabolismus MeSH
- keratin-8 genetika metabolismus MeSH
- konjunktiva cytologie metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- limbus corneae cytologie metabolismus MeSH
- messenger RNA genetika MeSH
- mladiství MeSH
- orgánové kultury - kultivační techniky MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- rohovkový epitel cytologie metabolismus MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- vimentin metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- buněčná diferenciace MeSH
- kaprolaktam analogy a deriváty chemie MeSH
- kmenové buňky cytologie MeSH
- kultivované buňky MeSH
- limbus corneae cytologie MeSH
- mezenchymální kmenové buňky cytologie MeSH
- myši MeSH
- nanovlákna MeSH
- polymery chemie MeSH
- poranění oka terapie MeSH
- proliferace buněk MeSH
- rohovkový epitel cytologie MeSH
- tkáňové inženýrství MeSH
- tkáňové podpůrné struktury MeSH
- transplantace kmenových buněk MeSH
- transplantace mezenchymálních kmenových buněk MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH