1 Department of Biology , Islamic Azad University , Iran
2 Department of Research and Development , Pasteur Institute of Iran , Iran
4 Skin and Stem Cell Research Center , Tehran University of Medical Sciences , Iran
Open J Pharma Sci
Article Type : Research Article
Mesenchymal stem cells; Atopic dermatitis; Subcutaneous; IgE; Mast cells.
Isolation and culture of hAT-MSCs
Human adipose tissues were obtained by simple liposuction from freshly excised human fat tissue with informed consent. Subcutaneous adipose tissues were digested with 0/1% collagenase (type I, Gibco) under gentle agitation for 30 minutes at 370 C. The digested tissues were filtered through a 100 µm nylon mesh to remove cellular debris and were centrifuged at 2,000 rpm for 10 min to obtain a pellet. The pellet was resuspended cells in DMEM (Invitrogen)-based media containing 0,2 mM ascorbic acid and 15% Fetal bovine serum (FBS). The cell suspension was re-centrifuged at 2,000 rpm for 10 min. The supernatant was discarded and the cell pellet was collected. The cell fraction was cultured overnight at 370 C 5% CO2 in DMEM- based media containing 0,2mM ascorbic acid and 15% FBS. After 24h, the cell adhesion was checked under an inverted microscope, and non-adherent cells were removed by washing with phosphate-buffered saline (PBS). The cells were maintained for 2-3 days until confluent (passage 0). When the cells reached 90% confluency, they were used in this study. Given that hAT-MSCs isolated from 8 different donors were used after verification of characteristics for MSCs by observing the surface markers and differentiation capabilities.
1- Fluoro-2, 4-dinitrobenzene (DNFB) from merckInc (Germany). N-terminal amino acids of proteins and peptides. PBS, PS, Fungizone, DMEM, FBS, L_Glutamine, Dispase, Trypsine, Collagenase, DMSO, ELISA kit, MTT assay kit, CD44, CD90, CD34, CD45, Hypotonic ammonium chloride, Sodium dodecyl sulfate, Osteoblast differentiation medium, Chondroblast differentiation medium, toluidine blue, H&E, Alizarin red, Ketamine, were purchased from sigma Aldrich.
NC/Nga mice (male, 8wk old) were used for the experiments. Animal care and all experimental procedures were approved by and followed the regulations of the Institute of Laboratory Animal Resources. They were obtained from Pasteur Institute and group housed in wire mesh cages under specific pathogen free conditions in the animal care facility.
AD model induction in NC/Nga mice
Atopic dermatitis (AD) was induced as previously method described with some modifications [27, 28, and 29]. We used 8 mice per group. Briefly, the upper backs of the mice were shaved with a clipper. Sodium dodecyl sulfate (4%, 150 µl/head) was treated on the shaved dorsal skin including surfaces of ears to achieve skin barrier disruption. After 3-4 hours, in a volume of 150 µl DNFB (Merck, Inc) was topically applied. 1-Fluoro-2, 4-dinitrobenzene (DNFB) was treated 3 times a week for 3-week intervals (i.e., days 1, 3, 6, 9, 12, 15, 18, 21 and 24). Thereafter, hAT-MSCs (0/75 × 105 or 0/375 × 105 cells/200 µl normal saline) were subcutaneously injected into mice on days 32, 40, 48, and 56 (figure 1A). After sacrifice on day 64, sera and skin biopsy specimens were obtained to detect the concentration of total IgE using a commercial ELISA kit (Zell Bio) or to evaluate histopathological lesions, respectively. Mast cells in skins were determined by toluidine blue.
Image j analysis to assess lesions area
To investigate changes in lesions surface area, and measure the percentage of lesions reduction, on different days (8, 16, after treatment) during the treatment period, the surface of the lesions using image j software with cm2 unit. The surface of the lesions was first photographed with a digital camera under the same conditions, then using image j software, the size of the lesions was calculated with high accuracy.
Skin samples were collected, fixed in 10% formalin followed by consecutive tissue processing steps including alcohol-xylene changes, and embedding in paraffin. Sections of 5 µm thicknesses were prepared and stained with H&E or toluidine blue. Mast cell infiltration was determined by toluidine blue staining.
Statistical analysis was performed using Prism5 and excels software (Microsoft Office 2013). For quantitative data analysis, One Way ANOVA in case of cluster comparison was applied. P <0.05 was considered statistically significant.
Subcutaneous administration of hAT-MSCs reduces the symptoms of DNFB- induced atopic dermatitis in mice.
We first investigated whether the xenogeneic administration of hAT-MSCs could exert a therapeutic effect against DNFB-induced murine AD. To assess the therapeutic effects, two different doses (low dose: 0/375 × 105 ;high dose:0/75 × 105 ) of hAT-MSCs were injected subcutaneously at day 32 when AD was fully induced (figure 1A). Phosphate buffer saline (PBS) was infused as a cell control group. None of the mice that received hAT-MSCs showed any adverse events or lethality. Interestingly, subcutaneous administration of high dose hAT-MSCs significantly reduced the clinical severity of AD mice.
The hAT-MSCs suppress AD in the mouse model.
We aimed to exclude the xenogeneic rejection response in this AD mouse model. Therefore, we used human adipose tissue-derived mesenchymal stem cells. It is now generally accepted that MSCs are hypo immunogenic as they do not express MHC class II activity . We used the DNFB-induced AD mice models that were subcutaneously sensitized with 150 µl of DNFB on days 1, 3, 6, 9, 12, 15, 18, 21 and 24. The hAT-MSCs (0/375 × 105 or 0/75 × 105) were then subcutaneously injected 4 times for 4weeks (Figure 1A). Decreased cell infiltration in the skin was observed in mice treated with both high dose and low dose hAT-MSCs compared with that in the PBS control group (Figure 1B). The severity score of skin lesions was significantly decreased by hAT-MSCs (Figure 1C).
Figure 1: Therapeutic effect of s.c. injected hAT-MSCs in AD mice. (AD)
Atopic dermatitis was induced by the repetitive application of 1-Fluoro-2, 4-dinitrobenzene (DNFB ) on day 24, after the onset of disease. Two different doses of hAT-MSCs or phosphate buffer saline (PBS) were injected subcutaneously (s.c).
(A) Scheme of AD induction and cell injection.
(B) Photographs of skin gross lesions were taken for pathological evaluation
(C) Lesions surface area on different days (8, 16, after treatment) during the recovery period on control and treatment groups.
Mast cells were identified by toluidine blue. These cells were also significantly decreased by hAT-MSCs (Figure 2B and 2C). The total and DNFB- specific IgE levels were significantly decreased in sera of mice treated with both high dose and low dose hAT-MSCs. These results indicate therapeutic effects on AD by xenogeneic hAT-MSCs in two doses, high and low.