The Effect Of Allogenetic Adipose-derived Mesenchymal Stem Cells In Cell-assisted Lipotransfer

【Background】Autologous fat grafting has many advantages, such as the abundance of fat in the body, little immunological rejection, low cost for patients and simplicity of the operation. Therefore, autologous fat grafting has rapidly become an important treatment for cosmetic and reconstructive surgery. However, some complications such as fat nodules and vesicles occur after fat grafting sometimes. Specifically, the unexpected and low survival rate(approximately 20–80%) is a common and important issue. Zuk et al. firstly isolated the adipose tissue-derived mesenchymal stem cells(ADSCs) from the adipose tissue in 2001. On one hand, ADSCs generate the various proangiogenic cytokines to promote vascularization of grafts and inhibit apoptosis, such as vascular endothelial growth factor, hepatocyte growth factor, insulin growth factor. On the other hand, ADSCs can differentiate into adipocytes and vascular endothelial cells to increase long-term volume retention and decrease the incidence of complications including fat nodules and vesicles. In 2007, adipose tissue was cotransplanted with isolated ADSCs in fat grafting,that was termed cell-assisted lipotransfer(CAL). CAL has increased the survival rate of fat grafts in the long term by approximately 30 %.A sufficient amount of ADSCs with high viability are required for CAL. For this reason, fat grafting assisted by ADSCs can’t be performed efficiently in the following cases. First, many patients who require filling of the depressed temporal area, cheek and breast, are usually too thin to offer sufficient ADSCs for transplantation. Second, surgeons have the difficulty in isolating ADSCs with highly viability from older patients, as the vitality of ADSCs gradually decreases with age. Third, because the biological properties of ADSCs may change in patients with some metabolic diseases, their autologous ADSCs aren’t suitable for fat grafting. Therefore, for patients who are thin, old, or have some forms of metabolic diseases, their lack of viable ADSCs is likely to result in less satisfactory therapeutic effects in CAL. Because ADSCs have low immunogenicity and immunosuppressive properties, we hypothesize that allogeneic ADSCs from healthy donors are a reasonable alternative for replacing autologous ADSCs for these patients.【Objective】1. To find the better rat donor part of ADSCs, the isolation and culture of ADSCs were conducted from the epididymal visceral adipose tissue and inguinal subcutaneous adipose tissue of Leiws and Brown Norway(BN) rats.2. To evaluate the low immunogenicity and immunosuppressive properties in the intro experiment, the cell surface molecules relevance with immunogenicity were assessed and the splenic mononuclear cells(MNCs) model co-cultured with ADSCs was established.3.Lewis rat model of the fat graft assisted by BN ADSCs was established. The first purpose of this study was to investigate whether allogeneic ADSCs could alleviate the surgically induced injury of the fat grafts. The second purpose was to discern whether allogeneic ADSCs could promote the vascularization and long-term retention of fat grafts. The third purpose was to find whether the implanted allogeneic ADSCs could elicit the immune response.【Methods】1. The cells were isolated and cultured from the epididymal visceral and inguinal subcutaneous adipose tissue of Lewis and BN rats. To identify ADSCs, the surface molecules of ADSCs were detected by flow cytometry and the induction experiments were conducted. Moreover, the cell morphology, cell growth curve and cell cycle were observed.2. The cell surface molecules relevance with immunogenicity(CD40, CD80, CD86, MHC-I, MHC-II) were assessed by flow cytometry. In MNCs model co-cultured with ADSCs, the effects of ADSCs were assessed on the proliferation, apoptosis and cell division cycle of MNCs. Moreover, the proportion of CD4/CD25/Foxp3 Treg was detected in the co-culture system.3. Lewis rat model of the fat graft assisted by BN ADSCs was established. At 7 and 14 days, the injury degree of fat grafts was assessed by immunofluorescence staining of Perilipin A and Tdtmediated dUTP-biotin nick end labeling. The degree of vascularization was evaluated by immunohistochemical staining of CD34 and ELAISA of VEGF. At 3 months, the survival quality of grafts was assessed by haematoxylin and eosin staning, and long-term volume retention of fat grafts was observed. The proportion of CD4/CD25/Foxp3 Treg and CD4/CD8 ratio were detected for evaluating the level of immune response.【Results】1. The cytobiology difference of ADSCs wasn’t found between Lewis and BN rats, however the ADSCs isolated from the difference body parts(epididymal visceral adipose tissue and inguinal subcutaneous adipose tissue) showed the different properties in histocytology and cytobiology. The less fibrous connective tissue in epididymal visceral adipose tissue resulted into time-saving and effort-saving operation in the isolated ADSCs process. The more ADSCs were obtained because of the higher proportion ADSCs in the epididymal visceral adipose tissue. Moreover, the ADSCs from epididymal visceral adipose tissue showed the better abilities of cell attachment and cell proliferation.2. ADSCs expressed the modest level of MHC-I molecules and lack of expression of MHC-II and co-stimulatory molecules(CD40, CD80, and CD86) by flow cytometry. In the MNCs model co-cultured with ADSCs, ADSCs inhabited the cell proliferation of MNCs by regulating the cell division cycle but not promoting apoptosis. Moreover, ADSCs increased the proportion of CD4/CD25/Foxp3 Treg in the co-culture system.3. In Lewis rats model of the fat graft assisted by BN ADSCs, allogeneic ADSCs similar to syngeneic ADSCs alleviated the injury degree and apoptosis in fat graft and increased the expression levels of CD34 and VEGF at 7 and 14 days. The survival quality and the volume retention of fat grafts were improved by allogeneic ADSCs at 3 months. The proportion of CD4/CD25/Foxp3 Treg and CD4/CD8 ratio indicated no obvious immune response elicited by allogeneic ADSCs.【Conclusions】1. The more ADSCs with higher proliferation ability were isolated from the epididymal visceral adipose tissue, compared with the equal inguinal subcutaneous adipose tissue.2. The isolated allogeneic ADSCs exerted the low immunogenicity and immunosuppressive properties. They could inhabit the proliferation, division and apoptosis of MNCs. However, ADSCs could promote MNCs differentiation into CD4/CD25/ Foxp3 Treg cells.3. The Lewis rat model of autologous fat grafting was established, which was assisted by allogenetic BN ADSCs. The allogenetic ADSCs isolated BN rats decreased the injury degree and apoptotic rate of fat grafts and accelerated the vascularized process at the early stage. Moreover, allogenetic ADSCs improved the survival quality and long-term retention of fat graft. In addition, the probably immune rejection caused by allogenetic ADSCs was not detected in the fat graft model.