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

To use human limbal explants as an alternative source for generating conjunctival epithelium and to determine the effect of interleukin-13 (IL-13) on goblet cell number, mucin expression, and stemness. Human limbal explants prepared from 17 corneoscleral rims were cultured with or without IL-13 (IL-13+ and IL-13-, respectively) and followed up to passage 2 (primary culture [P0]-P2). Cells were characterized by alcian blue/periodic acid-Schiff (AB/PAS) staining (goblet cells); immunofluorescent staining for p63α (progenitor cells), Ki-67 (proliferation), MUC5AC (mucin, goblet cells), and keratin 7 (K7, conjunctival epithelial and goblet cells); and by quantitative real-time polymerase chain reaction for expression of the p63α (TP63), MUC5AC, MUC4 (conjunctival mucins), K3, K12 (corneal epithelial cells), and K7 genes. Clonogenic ability was determined by colony-forming efficiency (CFE) assay. Using limbal explants, we generated epithelium with conjunctival phenotype and high viability in P0, P1, and P2 cultures under IL-13+ and IL-13- conditions, i.e., epithelium with strong K7 positivity, high K7 and MUC4 expression and the presence of goblet cells (AB/PAS and MUC5AC positivity; MUC5AC expression). p63α positivity was similar in IL-13+ and IL-13- cultures and was decreased in P2 cultures; however, there was increased TP63 expression in the presence of IL-13 (especially in the P1 cultures). Similarly, IL-13 increased proliferative activity in P1 cultures and significantly promoted P0 and P1 culture CFE. IL-13 did not increase goblet cell number in the P0-P2 cultures, nor did it influence MUC5AC and MUC4 expression. By harvesting unattached cells on day 1 of P1 we obtained goblet cell rich subpopulation showing AB/PAS, MUC5AC, and K7 positivity, but with no growth potential. In conclusion, limbal explants were successfully used to develop conjunctival epithelium with the presence of putative stem and goblet cells and with the ability to preserve the stemness of P0 and P1 cultures under IL-13 influence.

• MeSH
• buněčná diferenciace účinky léků   MeSH
• buněčné kultury MeSH
• epitelové buňky cytologie   metabolismus   MeSH
• interleukin-13 metabolismus   farmakologie   MeSH
• kmenové buňky metabolismus   MeSH
• končetiny růst a vývoj   MeSH
• konjunktiva cytologie   metabolismus   MeSH
• lidé MeSH
• limbus corneae růst a vývoj   metabolismus   MeSH
• muciny genetika   MeSH
• pohárkové buňky účinky léků   metabolismus   MeSH
• proliferace buněk účinky léků   MeSH
• vývojová regulace genové exprese MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• interleukin-13 MeSH
• muciny MeSH

The role of epidermal keratinocytes in the early phase of normal unimpaired wound healing has been extensively studied. However, little is known of the cell biological process in the epidermis and the role of keratinocytes in hypertrophic scar formation. This study investigated the possible role of p63 in the early phase of hypertrophic scarring pathogenesis. Nine skin samples were taken from nine patients during plastic surgery operations, as follows: 1. six samples from patients who on account of thyroid disease or other reasons presented risk factors (RFs) for hypertrophic scarring; 2. one sample from a healthy young person (as control); and 3. one sample from the upper eyelid during blepharoplasty and one sample from an elderly patient during breast reduction. All the patients were women, and were followed up clinically for 12 months. Skin specimens were cultured and sectioned, and analysed by histology and immunohistochemistry. In normal skin, nuclear p63 was abundantly expressed by the basal cells, but expressed by very low levels of transient amplifying (TA) keratinocytes covering the surface. TA keratinocytes, immediately after their withdrawal from the stem cell compartment, reduced p63, even though they possessed a proliferative capacity. In some skin, samples with RFs possessed a high level of p63 expression - not only basal stem cells but also four to five rows of parabasal cells. Four of the six skin samples with RFs showed significant epidermal abnormalities through the expression of both p63 and ki-67. Staining for ki-67, a marker for cell proliferation, revealed more increase in the suprabasal than in the basal keratinocyte proliferation rate. These results suggest that the epidermal keratinocytes may have an important role in hypertrophic scarring pathogenesis, using paracrine or epithelial-mesenchymal signalling. At 3, 6, and 12 months post-operation this finding clinically appeared in four patients with RFs.

Le rôle des kératinocytes épidermiques dans les phases précoces de la guérison normale non détériorée des lésions a été abondamment étudié, mais on sait très peu des processus biologiques cellulaires dans l’épiderme et du rôle des kératinocytes dans la formation des cicatrices hypertrophiques. Les Auteurs de cette étude ont investigué le rôle éventuel de p63 dans les phases précoces de la pathogenèse de la cicatrisation hypertrophique. Ils ont effectué neuf prélèvements dans neuf patients pendant des opérations de chirurgie plastique : 1. six prélèvements dans des patientes qui à cause d’une maladie thyroïde ou pour d’autres raisons présentaient des facteurs de risque pour la cicatrisation hypertrophique; 2. un prélèvement dans une personne jeune en bonne santé (comme témoin); et 3. un prélèvement obtenu de la paupière supérieure pendant une blépharoplastie et un prélèvement d’une patiente aînée opérée pour la réduction du sein. Toutes les personnes observées étaient du sexe féminin; elles ont été suivies cliniquement pour 12 mois. Les prélèvements cutanés ont été cultivés et sectionnés, et analysés pour l’histologie et l’immunohistochimie. Dans la peau normale, le p63 nucléaire était abondamment exprimé par les cellules basales, mais exprimé par des niveaux très bas de kératinocytes amplifiants transitoires (AT) qui couvraient la surface. Les kératinocytes AT, immédiatement après avoir abandonné le compartiment des cellules souches, réduisent le p63, même s’ils possèdent la capacité de proliférer. Dans la peau, en quelques cas, les prélèvements avec des facteurs de risque possédaient un niveau élevé d’expression de p63 - non seulement les cellules souches basales mais aussi quatre ou cinq rangs de cellules parabasales. Quatre des six prélèvements cutanés avec des facteurs de risque présentaient des anormalités épidermiques significatives par l’expression soit de p63 soit de ki-67. La coloration pour ki-67, un marqueur pour la prolifération cellulaire, révélait une augmentation majeure dans le taux de prolifération des kératinocytes basaux par rapport à celui des kératinocytes suprabasaux. Ces résultats semblent indiquer qu’il est possible que les kératinocytes épidermiques jouent un rôle important dans la pathogenèse de la cicatrisation hypertrophique, en utilisant la signalisation paracrine ou épithéliale-mésenchymale. Les Auteurs ont observé cliniquement ce résultat 3, 6 et 12 mois après l’opération chirurgicale dans quatre patients qui présentaient des facteurs de risque.

• Klíčová slova
• epidermal, hypertrophic, immunohistochemical, important, keratinocytes, ki-67, p63, pathogenesis, role, staining,
• Publikační typ
• časopisecké články MeSH