For the treatment of bilateral limbal stem cell deficiency (LSCD), cell therapy with transplantation of cultivated oral mucosa epithelial cells (COMET) is a promising alternative. Although not yet established, current protocols on the cultivation of oral mucosal epithelial cell (OMECs) sheets are based mainly on substrates and xenobiotic additives that may lead to variable outcomes and undesirable immune responses by the patient. The aim of this study was to characterize OMECs cultivated in xenobiotic-free media (XF) seeded on fibrin gel, in comparison to conventional complex (COM) medium. Oral mucosal biopsies were retrieved from 31 donors. After cultivation in COM or XF medium, OMECs were compared based on growth kinetics, morphology, cell size and viability. Using immunofluorescence and gene expression analyses, the degree of stemness, proliferation and differentiation was evaluated in OMEC cultures. Our findings showed that although OMECs showed a similar morphology and viability, and comparable growth kinetics, immunofluorescence revealed the preservation of stemness (p63 + p40 positivity in cells ≤11 μm) and proliferation in both COM and XF. Gene expression analyses showed that keratin (K)13 and K15 expression levels were significantly higher in XF (adj. p < 0.001), but otherwise COM and XF-treated OMECs had comparable transcriptional profiles in a panel of stemness, proliferation and differentiation genes. These results demonstrate the feasibility of culturing OMECs on fibrin gel without xenogeneic additives, while maintaining their undifferentiated state and preserving stemness. In conclusion, both in terms of results and methodology, the procedures presented here are suitable for implementation in clinical practice.
- Klíčová slova
- Advanced therapy medicinal products, Cell culture, Fibrin glue substrate, Limbal stem cell deficiency, Oral mucosal epithelial cells, Stemness,
- MeSH
- buněčná diferenciace MeSH
- buněčné kultury * MeSH
- deficit limbálních kmenových buněk MeSH
- dospělí MeSH
- epitelové buňky * metabolismus účinky léků MeSH
- fibrin * MeSH
- gely MeSH
- kmenové buňky * metabolismus cytologie MeSH
- kultivační média MeSH
- kultivované buňky MeSH
- lidé středního věku MeSH
- lidé MeSH
- limbus corneae * cytologie patologie metabolismus MeSH
- nemoci rohovky patologie farmakoterapie metabolismus MeSH
- proliferace buněk účinky léků MeSH
- rohovkový epitel metabolismus cytologie účinky léků patologie MeSH
- senioři MeSH
- transplantace kmenových buněk metody MeSH
- ústní sliznice * cytologie MeSH
- viabilita buněk MeSH
- xenobiotika farmakologie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fibrin * MeSH
- gely MeSH
- kultivační média MeSH
- xenobiotika 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
Destruction or dysfunction of limbal epithelial stem cells (LESCs) leads to unilateral or bilateral limbal stem cell deficiency (LSCD). Fifteen years have passed since the first transplantation of ex vivo cultivated oral mucosal epithelial cells (COMET) in humans in 2004, which represents the first use of a cultured non-limbal autologous cell type to treat bilateral LSCD. This review summarizes clinical outcomes from COMET studies published from 2004 to 2019 and reviews results with emphasis on the culture methods by which grafted cell sheets were prepared.
- Klíčová slova
- Cultivated oral mucosal epithelial cell, Limbal stem cell deficiency, Oral mucosal epithelial cells, Tissue regeneration,
- MeSH
- autologní transplantace MeSH
- epitelové buňky MeSH
- kultivované buňky MeSH
- lidé MeSH
- limbus corneae * MeSH
- nemoci rohovky * terapie MeSH
- rohovkový epitel * MeSH
- transplantace buněk MeSH
- transplantace kmenových buněk MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy 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
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
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.
- Klíčová slova
- Explant cultures, Fibrin, Human platelet lysate, Human serum, Limbal epithelial stem cells, Limbal stem cell deficiency,
- 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
- Názvy látek
- biokompatibilní potahované materiály MeSH
- biologické markery MeSH
- fibrin MeSH
- kultivační média MeSH
- MeSH
- kmenové buňky metabolismus patologie MeSH
- lidé MeSH
- limbus corneae metabolismus patologie MeSH
- nemoci rohovky genetika metabolismus patologie MeSH
- rohovkový epitel metabolismus patologie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- dopisy MeSH
- práce podpořená grantem MeSH
Patients with limbal stem cell deficiency (LSCD) often experience pain and photophobia due to recurrent epithelial defects and chronic inflammation of the cornea. Successfully restoring a healthy corneal surface in these patients by transplantation of ex vivo expanded human limbal epithelial cells (LECs) may alleviate these symptoms and significantly improve their quality of life. The clinical outcome of transplantation is known to be influenced by the quality of transplanted cells. Presently, several different protocols for cultivation and transplantation of LECs are in use. However, no consensus on an optimal protocol exists. The aim of this study was to examine the effect of culture medium and carrier on the morphology, staining of selected keratins and global gene expression in ex vivo cultured LECs. Limbal biopsies from cadaveric donors were cultured for three weeks on human amniotic membrane (HAM) or on tissue culture coated plastic (PL) in either a complex medium (COM), containing recombinant growth factors, hormones, cholera toxin and fetal bovine serum, or in medium supplemented only with human serum (HS). The expanded LECs were examined by light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) for keratins K3, K7, K8, K12, K13, K14, K15 and K19, as well as microarray and qRT-PCR analysis. The cultured LECs exhibited similar morphology and keratin staining on LM, TEM and IHC examination, regardless of the culture condition. The epithelium was multilayered, with cuboidal basal cells and flattened superficial cells. Cells were attached to each other by desmosomes. Adhesion complexes were observed between basal cells and the underlying carrier in LECs cultured on HAM, but not in LECs cultured on PL. GeneChip Human Gene 2.0 ST microarray (Affymetrix) analysis revealed that 18,653 transcripts were ≥2 fold up or downregulated (p ≤ 0.05). Cells cultured in the same medium (COM or HS) showed more similarities in gene expression than cells cultured on the same carrier (HAM or PL). When each condition was compared to HAM/COM, no statistical difference was found in the transcription level of the selected genes associated with keratin expression, stemness, proliferation, differentiation, apoptosis, corneal wound healing or autophagy. In conclusion, the results indicate that ex vivo cultures of LECs on HAM and PL, using culture media supplemented with COM or HS, yield tissues with similar morphology and keratin staining. The gene expression appears to be more similar in cells cultured in the same medium (COM or HS) compared to cells cultured on the same carrier (HAM or PL).
- Klíčová slova
- Cornea, Explant culture, Human serum, Limbal epithelium, Limbal stem cell deficiency, Stem cell, Transplantation,
- MeSH
- biopsie MeSH
- imunohistochemie MeSH
- keratiny biosyntéza genetika MeSH
- kultivační média MeSH
- kultivované buňky MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé středního věku MeSH
- lidé MeSH
- limbus corneae metabolismus ultrastruktura MeSH
- nemoci rohovky genetika patologie chirurgie MeSH
- regulace genové exprese * MeSH
- RNA genetika MeSH
- rohovkový epitel metabolismus ultrastruktura MeSH
- senioři MeSH
- transmisní elektronová mikroskopie MeSH
- transplantace rohovky * MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
- Názvy látek
- keratiny MeSH
- kultivační média MeSH
- RNA MeSH
The Purpose of present study was to investigate the effect of riboflavin/ultraviolet-A-induced collagen cross-linking (CXL) on central cornea, limbus and intraocular pressure (IOP). This was an animal experimental study. The right corneas of 10 rabbits were ultraviolet-A irradiated (3 mW/cm2 for 30 minutes) after de-epithelialization and instillation of 0.1% riboflavin / 20% Dextran drops. Left corneas served as controls. Samples were examined histologically one month postoperatively. Before and after treatment, IOP measurements were recorded bilaterally. At central cornea of eyes underwent CXL keratocyte repopulation, normal arrangement of collagen fibres and a statistically significant change in fibres diameter were detected, compared to controls. At limbus area, there were not any significant histological differences after CXL. There was no statistically significant difference between pre- and postoperative IOP in all eyes.
- Klíčová slova
- Cornea, Corneal Crosslinking, Intraocular pressure, Limbus,
- MeSH
- fotosenzibilizující látky farmakologie MeSH
- kolagen chemie ultrastruktura MeSH
- králíci MeSH
- limbus corneae účinky léků účinky záření chirurgie ultrastruktura MeSH
- nitrooční tlak * účinky léků účinky záření MeSH
- reagencia zkříženě vázaná farmakologie MeSH
- riboflavin farmakologie MeSH
- rohovka účinky léků účinky záření chirurgie ultrastruktura MeSH
- ultrafialové záření * MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fotosenzibilizující látky MeSH
- kolagen MeSH
- reagencia zkříženě vázaná MeSH
- riboflavin MeSH
UNLABELLED: Regeneration of corneal epithelium is secured by a population of limbal stem cells (LSC), which are located in the basal part of the limbal epithelium. Deficiency in LSC leads to chronic inflammation, scarring and conjunctivization of cornea. Therapy of LSC deficiency consists in transplantation of limbal tissue, cultivated limbal epithelium or more recently in tranplantation of autologous cells including mesenchymal stem cells, oral mucosal epithelial cells or hair follicle-derived stem cells. A significant progress has been achieved in the field of cell therapy and also in the development of convenient scaffolds for the growth and transfer of cells on damaged cornea. KEY WORDS: ocular surface damage, stem cells, cell therapy.
