J Korean Soc Transplant.  2013 Dec;27(4):174-184. 10.4285/jkstn.2013.27.4.174.

Effects of Human Adipose-Tissue Derived Stem Cell Infusion on the Immunological Consequences in Skin Allograft Mice

• Lee SC
• Sul H
• Lee SM
• Kim SJ

• KMID: 1841126
• DOI: http://doi.org/10.4285/jkstn.2013.27.4.174

Abstract

BACKGROUND
Many in vitro experiments have demonstrated the immunosuppressive properties of mesenchymal stem cells (MSCs). However, such properties have not yet been fully established in an in vivo setting. The purpose of this study was to determine immunosuppressive and anti-inflammatory properties of MSCs in a preclinical animal model in order to pave the way for replacement of conventional immunosuppressive therapy.
METHODS
Male C57BL/6 mice and male BALB/c mice were chosen as skin graft donors and recipients, respectively. After performance of full-thickness skin transplantation on the back of mice, adipose tissue derived stem cells (1.0x10(6)/0.1 mL) stained with 4, 6-diamidino-2-phenylindole were transplanted into adipose tissue derived stem cell (ASC)-infused mice and phosphate buffered saline (PBS; 0.1 mL) was infused into PBS-infused mice. Immunological properties and graft survival were accessed and compared.
RESULTS
The serum levels of proinflammatory interleukin (IL)-6 showed a decrease in ASC-infused mice compared to PBS-infused mice (P<0.005). In addition, interferon-lambda, IL-10, and tumor necrosis factor-alpha mRNA levels in the skin graft showed a decrease in ASC-infused mice, although without statistical significance. In ASC-infused mice, donor specific hyporesponsiveness was identified in a mixed lymphocyte reaction assay at 30 days after transplantation. In addition, ASC-infusion resulted in markedly prolonged skin allograft survival compared with PBS-infusion (P<0.001).
CONCLUSIONS
Administration of ASC not only induced anti-inflammation and immunosuppression, but also resulted in prolonged graft survival, suggestive of their potent immunosuppressive properties. Therefore, conduct of further and more exquisite studies will be required in order to determine the role of MSC in the solid organ transplantation field in order to avoid adverse effects and toxicities caused by chemical immunosuppressive regimens.

Keyword

Adipose tissue-derived stem cells; Immunological tolerance; Mesenchymal stromal cells; Skin transplantation; Immunosuppression

MeSH Terms

Adipose Tissue
Animals
Graft Survival
Humans*
Immunosuppression
Interleukin-10
Interleukins
Lymphocyte Culture Test, Mixed
Male
Mesenchymal Stromal Cells
Mice*
Models, Animal
Organ Transplantation
RNA, Messenger
Skin Transplantation
Skin*
Stem Cells*
Tissue Donors
Transplantation, Homologous*
Transplants
Tumor Necrosis Factor-alpha
Interleukin-10
Interleukins
RNA, Messenger
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1. The expression of cell surface markers in adipose tissue-derived stem cells (ASCs). (A) Representative illustrations of flow cy-tometry demonstrating that ASC did not express hematopoietic stem cell marker (CD31, CD34), and (B) did express mesenchymal stem cell marker (CD90).

• 

  Fig. 2. Comparison of the serum levels of proinflammatory cy-tokine interleukin (IL)-6 between phosphate buffered saline (PBS)-infused mice (n=16) and human adipose tissue derived stem cell (ASC)-infused mice (n=16) after skin allograft transfu-sion. Each of four mice in each group were measured at day 1, 2, 7, and 20 posttransplantation, respectively. Overall, ASC-in-fused mice showed statisticalliy significant decrease in serum IL-6 levels than PBS-infused mice did throughout all posttransplantation periods. a P＜0.005.

• 

  Fig. 3. Representative photographs which demonstrate a mouse skin allograft specimen containing 4’,6-diamidino-2-phenyl-indole (DAPI)-labeled human adipose-derived stem cells. (A) After obtaining the skin graft at 7-posttransplantation day, (B) the frozen section of the tissue was observed under a fluo-rescent microscope (×100). The staining with DAPI released cy-an fluorescence.

• 

  Fig. 4. mRNA expressions of proinflammatory (A) interferon (IFN)-γ , (B) interleukin (IL)-10, and (C) tumor necrosis factor (TNF)-α genes in the skin allografts at day 7 posttransplantation manifested by real-time polymerase chain reaction. The mRNA expressions in phosphate buffered saline (PBS) (n=8) and adipose tissue-derived stem cell (ASC) (n=8) were expressed as fold changes in relation to the control group (n=8). The expressions of IFN-γ, IL-2, and TNF-α gene mRNA more decreased in ASC-infused mice than they did in PBS-in-fused mice, though the difference did not reach statistical significance.

• 

  Fig. 5. Mixed lymphocyte reaction at day 30 posttransplantation. Data are expressed as fold changes related to the phosphate buffered saline group. The alloreactivity between res-ponder cells (from BALB/c mice of corresponding groups) and stimulating cells (from C57BL/6 mice) was measured using CCK assay. Adipose tissue derived stem cell (ASC) group (n=8) showed significantly reduced alloreactivity compared with PBS group (n=8) (1.00±0.16 vs. 0.70±0.16; P=0.002). a P=0.002.

• 

  Fig. 6. Representative microphotographs comparing skin allograft specimens between phosphate buffered saline (PBS)-infused mice and adipose tissue-derived stem cell (ASC)-infused mice after skin allograft transplantation. (A) At day 7 posttransplantation, the inflammatory reactions, which had been prominent in PBS-infusion group on day 7 posttransplantation, attenuated after ASC infusion on HE stain (Aa, Ab). Vasoactive endothelial growth factor (VEGF) was minimally or not expressed in PBS group, but its expression was enhanced in ASC-infused group (Ac, Ad). (B) At day 20 posttransplantation, most of full-thickness skin grafts treated with PBS were detached from the recipient’s tissue (Ba). However, the considerable number of grafts treated with ASC was persisted with limited inflammatory cell infiltration (Bb). And, VEGF was lesser expressed on day 20 posttransplantation than it did on day 7 posttransplantation in ASC-infused group (Bc, Bd).

• 

  Fig. 7. Kaplan-Meier allograft survival curves for mice which had been intravenously administrated phosphate buffered saline (PBS) and adipose tissue-derived stem cells (ASC; 1x106). Each group included 10 mice. ASC infusion markedly increased skin allograft survival in comparison with PBS infusion (P＜0.001).

• 

  Fig. 8. Representative photographs of skin allografts showing skin allograft acceptance. Two adipose tissue-derived stem cells (ASC)-infused mice (A, B) showed necrosis-free skin graft suggestive of graft acceptance at day 20 posttransplantation, showing the role of ASC inducing immunological tolerance.

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