Cell Source Exploration Of Cell Therapy For Promoting Angiogenesis In Free Grafting

Background:Free grafting is one of the basic techniques of plastic and reconstructive surgery.After transplanted to the recipient sites, the survival of the free grafted tissue dependson their blood supply: Pristine nutrient supply mainly depends on the nutrition fromplasma around the donator. But this kind of nutrient supply is limited, it can't ensurethe long-term surviving of the grafts, only can help the grafts survive the ischemiaand anoxaemi phase. The key to the long-term survival of the grafts is theneovascularization between the grafts and the recipient site initiated 48h aftertransplantation. Insufficient neovascularization always leads to failure of the freegrafting. Therefore, to explore means of promoting revasularization after free tissuetransplantation is the need of the development of plastic and reconstructive surgery,especially in the area of free fat grafting. If the problem of blood supply aftertransplantation can be solved, the main obstacles of free fat grafting such as centralnecrosis, absorption and fibrosis can be conquered.Cell therapy is a technology that uses autologous, allogeneic or xenogeneicsomatic living cells, whose biological characteristics are altered by manipulation ornot, transplante to patients for therapeutic, diagnostic or preventive purpose. Bloodtransfusion and bone marrow transplantation or stem cell transplantation are earlysuccessful examples of cell therapy. Recently, following the development of stem cell research, cell therapy has been extended to the areas of biological or immunologicaltreatments toward tumors, diabetes, or nervous system diseases. It can also be used toassist the revascularization of transplanted tissue or infarcted cardiac muscle, or topromote wound healing. By now, bone-marrow stem cells, adipose-derived stem cellsand endothelial progenitor cells all have the proof of promoting revascularization ofthe ischemic tissue.However, to satisfy the practical use of cell therapy, the seed cells must have anabundant cell source, with a robust proliferative ability and are easy to isolate fromadult human body. Considering all these needs, Endothelial progenitor cells(EPCs)and Adipose-derived Stem Cells(ASCs) are two of the candidates that best fits. EPCsare considered to be the precursor of blood vessel endothelial cells. Which can beisolated from peripheral blood and enrich to the ischemic site, join in theneovascularization. So, they were thought to be the ideal cell source of cell therapyfor promoting revascularization of the transplanted tissue. However, peripheral bloodcontains only a minimal portion of EPCs, which display a low proliferative rateduring in vitro studies. These disadvantages restricted the use of EPCs as an ideal cellsource for cell therapy. Culture of a subpopulation of EPCs named late EPC maybe asolution to this problem.Adipose-Derived Stem Cells(ASCs) can be easily harvested from liposuctionaspirates in large amounts, it is one of the pluripotent mesenchymal stem cells, whichhas strong proliferative ability in vitro. Its ability to promote neovascularization hasbeen proved by a series of animal experiments. It has been proved that ASCs candifferentiate into endothelial cells in vivo, and can secret some angiogenic cytokinesto encourage neovascularization process.Objective: To explore how to obtain the ideal cell source, which satisfies thepractical use of cell therapy for promoting revascularization of the free grafted tissue. Methods: 1,Isolation and in vitro cell culture of EPCs from adult peripheralblood.20 samples(10～50ml each) of healthy adult peripheral blood were harvestedfrom ulnar vein with informed content. The blood was diluted by PBS, and thencarefully put onto Ficoll in centrifuge tube. After centrifuge at 450g for 25rain, themononuclear cells in the middle of the tube were harvested with careful suction.Mononuclear cells were then resuspended in M199, centrifuged and washed twice,resuspended in Medium-199 supplied with 10ng/ml human vascular endothelialgrowth factor(VEGF) and 20％Fetal calf serum(FCS), then seeded onto a six-wellplate precoated with human fibronectin at a density of (0.5-3.5)×10~6 cells/cm~2 andincubated in 37℃, 5％CO2 incubator. The medium was first changed on the 4th day,and then changed every 2-3days.2,Experimental observation of development of the biological characteristics ofadult human peripheral blood EPCs cultured in vitroCell morphology and their total amount were observed and photoed over days,flow cytometry assay were performed on day 7 and day 21 respectively for CD14,CD31,CD34,CD44,CD45,vWF,flk-1,CD133 and CD11c. expression. EPCs wereseeded on the coverslips precoated with human fibronectin. The coverslips were takenout on day 7 for vWF, CD31, CD34 immunohistochemichal assay. EPCs cultured today 21 were digested, seeded onto coverslips for vWF immunohistochemical assay.3,Isolation and cell culture of human ASCs.Human ASCs were kindly provide by Zhu ming, studying for master's degree,Department of Plastic Surgery, Nanfang Hospital, Southern Medical University. Theywere cultured in high glucose DMEM+10％FCS and passaged every 3～5days.4,coculture of human ASCs and adult peripheral blood EPCs.ASCs were digested on passage 5, resuspended in complete medium, and seeded into the EPC culture plates when EPCs were given the first medium change. After24h the medium was firstly changed and then changed as EPC culture procedures.5,Endothelial induction of ASCs.ASCs were isolated from liposuction aspirates and then divided into two parts:One were cultured in EPC culture medium. The other is cultured in common DMEMmedium as negative control. Flow cytometry assay was performed 1 month afterinductive culture for CD29, CD31 and CD45.Results: 1. There were about 1/20 peripheral blood mononuclear cells (PBMCs)attached to the culture plates. On day 4, typical endothelial cell colony-formingunits(CFU-EC) were formed. On day 7, large amount of spindle-shaped cells emerged.The flow cytometry assay demonstrated the expression of monocytic related antigensCD11c, CD14 and CD44, and weak expression of endothelial related antigens CD31,flk-1 and vWF. But hematopoietic related antigens CD133 and CD34 were notdetected. The immunohistochemical staining displays that they are positive for CD31,negative for CD34, and weakly positive for vWF.2. When cells described above were cultured continuously without passage, thespindle-shaped cells began to decrease between day 7 and day 10. Meanwhile, thecobble-stone shaped cells, which consistent with the morphology of late EPC,appeared during the same time and their total amount increased continuously. Atweek 3, the spindle-shaped cells almost disappeared, while the cobble-stone shapedcells proliferated rapidly. The flow cytometry assay demonstrated that expressions ofendothelial-related cell antigens CD31,vWF significantly increased over time, whilethe expression of monocytic related antigen CD44 decreased. Furthermore thehematopoietic related antigen CD34 still remains negative expression. Theimmunohistochemical staining shows positive stain of late EPC by vWF.3. When ASCs were cocultured with EPCs, EPCs are more elongated and proliferated better than that were cultured alone. But after 4 days of their coculture,ASCs already spread the whole plate and there is not enough rooms for theproliferation of the EPCs.4. When cultured in the EPC complete medium, expression of endothelial relatedantigen CD31 were up-regulated in ASCs. The wave shape of the stem cell relatedantigen CD29 became double hump. While the hematopoietic and blood cell relatedantigen CD45 remained negative.summary: 1. Our experiments successfully isolated EPC from adult peripheralblood, and demonstrated that they express monocytic related antigens, Meanwhiledon't express hematopoietic related antigens. Thus provided new evidence ofcharacterizing EPCs.2. Our experiments domesticly first surveyed the changes of adult peripheralblood EPCs during in vitro culture. And successfully isolated and cultured endothelialoutgrowth cells, or late EPC, which has a greater potential use in cell therapy.3. We preliminarily investigated the interaction between ASCs and EPCs,observed that ASCs can assist the differentiation and proliferation of EPCs.4. We preliminarily studied the endothelial differentiation of ASCs, detectedup-regulating of CD31 when ASCs were cultured in EPCs medium.