| 1. |
- Aabel, Peder, et al.
(författare)
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Transcription and microRNA Profiling of Cultured Human Tympanic Membrane Epidermal Keratinocytes
- 2018
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Ingår i: Journal of the Association for Research in Otolaryngology. - : SPRINGER. - 1525-3961 .- 1438-7573. ; 19:3, s. 243-260
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Tidskriftsartikel (refereegranskat)abstract
- The human tympanic membrane (TM) has a thin outer epidermal layer which plays an important role in TM homeostasis and ear health. The specialised cells of the TM epidermis have a different physiology compared to normal skin epidermal keratinocytes, displaying a dynamic and constitutive migration that maintains a clear TM surface and assists in regeneration. Here, we characterise and compare molecular phenotypes in keratinocyte cultures from TM and normal skin. TM keratinocytes were isolated by enzymatic digestion and cultured in vitro. We compared global mRNA and microRNA expression of the cultured cells with that of human epidermal keratinocyte cultures. Genes with either relatively higher or lower expression were analysed further using the biostatistical tools g:Profiler and Ingenuity Pathway Analysis. Approximately 500 genes were found differentially expressed. Gene ontology enrichment and Ingenuity analyses identified cellular migration and closely related biological processes to be the most significant functions of the genes highly expressed in the TM keratinocytes. The genes of low expression showed a marked difference in homeobox (HOX) genes of clusters A and C, giving the TM keratinocytes a strikingly low HOX gene expression profile. An in vitro scratch wound assay showed a more individualised cell movement in cells from the tympanic membrane than normal epidermal keratinocytes. We identified 10 microRNAs with differential expression, several of which can also be linked to regulation of cell migration and expression of HOX genes. Our data provides clues to understanding the specific physiological properties of TM keratinocytes, including candidate genes for constitutive migration, and may thus help focus further research.
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| 2. |
- Jackson, Catherine, et al.
(författare)
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Effect of Storage Temperature on Cultured Epidermal Cell Sheets Stored in Xenobiotic-Free Medium
- 2014
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:8, s. e105808-
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Tidskriftsartikel (refereegranskat)abstract
- Cultured epidermal cell sheets (CECS) are used in regenerative medicine in patients with burns, and have potential to treat limbal stem cell deficiency (LSCD), as demonstrated in animal models. Despite widespread use, short-term storage options for CECS are limited. Advantages of storage include: flexibility in scheduling surgery, reserve sheets for repeat operations, more opportunity for quality control, and improved transportation to allow wider distribution. Studies on storage of CECS have thus far focused on cryopreservation, whereas refrigeration is a convenient method commonly used for whole skin graft storage in burns clinics. It has been shown that preservation of viable cells using these methods is variable. This study evaluated the effect of different temperatures spanning 4 degrees C to 37 degrees C, on the cell viability, morphology, proliferation and metabolic status of CECS stored over a two week period in a xenobiotic-free system. Compared to non-stored control, best cell viability was obtained at 24 degrees C (95.2 +/- 9.9%); reduced cell viability, at approximately 60%, was demonstrated at several of the temperatures (12 degrees C, 28 degrees C, 32 degrees C and 37 degrees C). Metabolic activity was significantly higher between 24 degrees C and 37 degrees C, where glucose, lactate, lactate/glucose ratios, and oxygen tension indicated increased activation of the glycolytic pathway under aerobic conditions. Preservation of morphology as shown by phase contrast and scanning electron micrographs was best at 12 degrees C and 16 degrees C. PCNA immunocytochemistry indicated that only 12 degrees C and 20 degrees C allowed maintenance of proliferative function at a similar level to non-stored control. In conclusion, results indicate that 12 degrees C and 24 degrees C merit further investigation as the prospective optimum temperature for short-term storage of cultured epidermal cell sheets.
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| 3. |
- Ong, Huan Ting, et al.
(författare)
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Paracrine Activity from Adipose-Derived Stem Cells on In Vitro Wound Healing in Human Tympanic Membrane Keratinocytes
- 2017
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Ingår i: Stem Cells and Development. - : MARY ANN LIEBERT, INC. - 1547-3287 .- 1557-8534. ; 26:6, s. 405-418
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Tidskriftsartikel (refereegranskat)abstract
- Stem cell therapies for tympanic membrane repair have shown initial experimental success using mesenchymal stem cells in rat models to promote healing; however, the mechanisms providing this benefit are not known. We investigated in vitro the paracrine effects of human adipose-derived stem cells (ADSCs) on wound healing mechanisms for human tympanic membrane-derived keratinocytes (hTM) and immortalized human keratinocytes (HaCaT). ADSC conditioned media (CMADSC) were assessed for paracrine activity on keratinocyte proliferation and migration, with hypoxic conditions for ADSC culture used to generate contrasting effects on cytokine gene expression. Keratinocytes cultured in CMADSC showed a significant increase in cell number compared to serum-free cultures and further significant increases in hypoxic CMADSC. Assessment of ADSC gene expression on a cytokine array showed a range of wound healing cytokines expressed and under stringent hypoxic and serum-free conditions was upregulated (VEGF A, MMP9, Tissue Factor, PAI-1) or downregulated (CXCL5, CCL7, TNF-). Several of these may contribute to the activity of conditioned media on the keratinocytes with potential applications in TM perforation repair. VEGFA protein was confirmed by immunoassay to be increased in conditioned media. Together with gene regulation associated with hypoxia in ADSCs, this study has provided several strong leads for a stem cell-derived approach to TM wound healing.
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