Publications by authors named "Peder Aabel"

5 Publications

  • Page 1 of 1

Transcription and microRNA Profiling of Cultured Human Tympanic Membrane Epidermal Keratinocytes.

J Assoc Res Otolaryngol 2018 06 5;19(3):243-260. Epub 2018 Apr 5.

Ear, Nose and Throat Department, Division of Surgery, Akershus University Hospital, Lørenskog, Norway.

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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June 2018

Paracrine Activity from Adipose-Derived Stem Cells on In Vitro Wound Healing in Human Tympanic Membrane Keratinocytes.

Stem Cells Dev 2017 03 21;26(6):405-418. Epub 2016 Nov 21.

1 Ear Science Institute Australia , Nedlands, Australia .

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 (CM) 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 CM showed a significant increase in cell number compared to serum-free cultures and further significant increases in hypoxic CM. 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-α). 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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March 2017

Effect of storage temperature on cultured epidermal cell sheets stored in xenobiotic-free medium.

PLoS One 2014 29;9(8):e105808. Epub 2014 Aug 29.

Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway; Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway.

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°C to 37°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°C (95.2±9.9%); reduced cell viability, at approximately 60%, was demonstrated at several of the temperatures (12°C, 28°C, 32°C and 37°C). Metabolic activity was significantly higher between 24°C and 37°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°C and 16°C. PCNA immunocytochemistry indicated that only 12°C and 20°C allowed maintenance of proliferative function at a similar level to non-stored control. In conclusion, results indicate that 12°C and 24°C merit further investigation as the prospective optimum temperature for short-term storage of cultured epidermal cell sheets.
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June 2015

Antioxidants Improve the Viability of Stored Adult Retinal Pigment Epithelial-19 Cultures.

Ophthalmol Ther 2014 Dec 29;3(1-2):49-61. Epub 2014 Mar 29.

Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.

Introduction: There is increasing evidence that retinal pigment epithelium (RPE) can be used to treat age-related macular degeneration, one of the leading causes of blindness worldwide. However, the best way to store RPE to enable worldwide distribution is unknown. We investigated the effects of supplementing our previously published storage method with seven additives, attempting to improve the number of viable adult retinal pigment epithelial (ARPE)-19 cells after storage.

Materials And Methods: ARPE-19 cells were cultured on multiwell plates before being stored for 1 week at 16 °C. Unsupplemented Minimal Essential Medium (MEM) (control) and a total of seven individual additives (DADLE ([D-Ala(2), D-Leu(5)]-encephalin), capsazepine, docosahexaenoic acid (DHA), resveratrol, quercetin, simvastatin and sulforaphane) at three to four concentrations in MEM were tested. The individual effect of each additive on cell viability was analyzed with a microplate fluorometer. Cell phenotype was investigated by both microplate fluorometer and epifluorescence microscopy, and morphology by scanning electron microscopy.

Results: Supplementation of the storage medium with DADLE, capsazepine, DHA or resveratrol significantly increased the number of viable cells by 86.1% ± 41.9%, 67.9% ± 24.7%, 36.5% ± 10.3% and 21.1% ± 6.4%, respectively, compared to cells stored in unsupplemented MEM. DHA and resveratrol significantly reduced caspase-3 expression, while expression of RPE65 was maintained across groups.

Conclusion: The number of viable ARPE-19 cells can be increased by the addition of DADLE, capsazepine, DHA or resveratrol to the storage medium without perturbing apoptosis or differentiation.
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December 2014

Optimization of Storage Temperature for Cultured ARPE-19 Cells.

J Ophthalmol 2013 22;2013:216359. Epub 2013 Oct 22.

Department of Medical Biochemistry, Oslo University Hospital, Kirkeveien 166, P.O. Box 4956, Nydalen, 0424 Oslo, Norway.

Purpose. The establishment of future retinal pigment epithelium (RPE) replacement therapy is partly dependent on the availability of tissue-engineered RPE cells, which may be enhanced by the development of suitable storage methods for RPE. This study investigates the effect of different storage temperatures on the viability, morphology, and phenotype of cultured RPE. Methods. ARPE-19 cells were cultured under standard conditions and stored in HEPES-buffered MEM at nine temperatures (4°C, 8°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C, and 37°C) for seven days. Viability and phenotype were assessed by a microplate fluorometer and epifluorescence microscopy, while morphology was analyzed by scanning electron microscopy. Results. The percentage of viable cells preserved after storage was highest in the 16°C group (48.7% ± 9.8%; P < 0.01 compared to 4°C, 8°C, and 24°C-37°C; P < 0.05 compared to 12°C). Ultrastructure was best preserved at 12°C, 16°C, and 20°C. Expression of actin, ZO-1, PCNA, caspase-3, and RPE65 was maintained after storage at 16°C compared to control cells that were not stored. Conclusion. Out of nine temperatures tested between 4°C and 37°C, storage at 12°C, 16°C, and 20°C was optimal for maintenance of RPE cell viability, morphology, and phenotype. The preservation of RPE cells is critically dependent on storage temperature.
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November 2013