Bilateral limbal stem cell deficiency (LSCD) can be effectively treated with cultivated oral mucosa epithelial cell transplantation (COMET). However, COMET is associated with greater superficial neovascularization than limbal stem cell (LESC) transplantation, the gold standard for unilateral LSCD. To investigate the intrinsic molecular features of cells intended for grafting, we assessed the in vitro expression of genes involved in vascularization and inflammation using real-time quantitative PCR and multifactorial linear models. Oral mucosal epithelial cells (OMECs) and limbal epithelial cells (LECs) were cultured in either conventional (COM) or xenobiotic-free (XF) media on fibrin substrates. Gene expression profiling revealed distinct transcriptional signatures. The pro-angiogenic genes AGR2, ANGPTL2, CRYAB, EREG, JAM3, and S100A4 were significantly higher in LECs (adjusted p < 0.01), whereas FGF2 was higher in OMECs (adjusted p < 0.001). The anti-angiogenic genes TIMP3 and SERPINF1 were higher in LECs (adjusted p < 0.01), while COL18A1 was higher in OMECs (adjusted p < 0.01). OMECs also showed significantly greater expression of the immunoregulatory genes IL1B, IL6, TNF, CXCL10, and IL1RN (adjusted p < 0.01). Cultivation induced phenotypic changes in OMECs, with COM and XF media exerting comparable effects. These results highlight the contribution of inflammatory mediators to neovascularization following COMET.

• Klíčová slova
• Limbal stem cell deficiency, angiogenesis-related genes, gene expression, inflammation regulatory genes, limbal epithelial cells, oral mucosal epithelial cells,
• Publikační typ
• časopisecké články MeSH

Ultrathin electrospun poly (l-lactide-co-dl-lactide) nanofibrous membranes coated with fibronectin were explored as scaffolds for the ex vivo cultivation of limbal epithelial cells (LECs) for the treatment of limbal stem cell deficiency. The developed scaffolds were compared with the "gold-standard" fibrin gel. The resulting membranes composed of nanofibers possessed a very low thickness of 4 μm and allowed very good optical transparency in the wet state. The fibronectin-coated nanofibrous scaffolds demonstrated LEC expansion and successful cultivation similar to that on fibrin gel. Unlike the regular cobblestone epithelial cell morphology on the fibrin gel, the nanofibrous scaffold presented a mostly irregular epithelial morphology with a shift to a mesenchymal phenotype, as confirmed by the upregulation of profibroblastic genes: ACTA2 (p = 0.023), FBLN1 (p < 0.001), and THY1 (p < 0.001). Both culture conditions revealed comparable expression of stem cell markers, including KLF4, ΔNp63α and ABCG2, emphasizing the promise of polylactide-based nanofibrous membranes for further investigations.

• Klíčová slova
• Biomaterial, Limbal epithelial stem cells, Ocular tissue engineering, PDLLA, Tissue sealant,
• Publikační typ
• časopisecké články MeSH

This study aimed to determine the effect of interleukin-13 (IL13) on the stemness, differentiation, proliferation, clonogenicity, and morphology of cultured limbal epithelial cells (LECs). Human limbal explants were used to culture LECs up to the second passage (P0-P2) with or without IL13 (IL13+ and IL13-, respectively). Cells were analyzed by qPCR (for the expression of ΔNp63α, BMI-1, keratin (K) 3, K7, K12, K14, K17, mucin 4, and MKI67) and immunofluorescence staining for p63α. The clonogenic ability was determined by colony-forming assay (CFA), and their metabolic activity was measured by WST-1 assay. The results of the CFA showed a significantly increased clonogenic ability in P1 and P2 cultures when LECs were cultured with IL13. In addition, the expression of putative stem cell markers (ΔNp63α, K14, and K17) was significantly higher in all IL13+ cultures compared to IL13-. Similarly, immunofluorescence analysis showed a significantly higher percentage of p63α positive cells in P2 cultures with IL13 than without it. LECs cultures without IL13 lost their cuboidal morphology with a high nucleocytoplasmic ratio after P1. The use of IL13 also led to significantly higher proliferation in P2, which can be reflected by a higher ability to reach confluence in P2 cultures. On the other hand, IL13 had no effect on corneal epithelial cell differentiation (K3 and K12 expression), and the expression of the conjunctival marker K7 significantly increased in all IL13+ cultures compared to the respective cell culture without IL13. This study showed that IL13 enhanced the stemness of LECs by increasing the clonogenicity and the expression of putative stem cell markers of LECs while maintaining their stem cell morphology. We established IL13 as a culture supplement for LESCs, which increases their stemness potential in culture, even after the second passage, and may lead to the greater success of LESCs transplantation in patients with LSCD.

• MeSH
• buněčná diferenciace MeSH
• buněčné kultury MeSH
• epitelové buňky metabolismus   MeSH
• interleukin-13 metabolismus   MeSH
• kmenové buňky MeSH
• kultivované buňky MeSH
• lidé MeSH
• limbus corneae * MeSH
• rohovkový epitel * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• interleukin-13 MeSH

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
• Názvy látek
• 5-bromouridine triphosphate MeSH Prohlížeč
• 5-fluorouridine MeSH Prohlížeč
• ribozomální DNA MeSH
• RNA ribozomální MeSH
• uridin MeSH
• uridintrifosfát MeSH