| BACKGROUND:A soft tissue defect is generally defined as a large tissue void within the subcutaneous fat layer of the skin that may alter the contour of normal tissue. From cosmetic standard point, restoration of the soft tissue aesthetic function, rather than its physical function, is one of the primary goals that will minimize patients' anxiety and psychological stress associated with disfigurement. Millions of plastic and reconstructive surgeries are performed each year to repair soft tissue defects result from traumatic injury (i.e., significant burns), tumor resections (i.e., mastectomy and carcinoma removal), and congenital defects. Strategies to repair soft tissue defects, e.g. breast reconstruction procedures, collagen injections, and the use of autologous tissue transferplantation (i.e. free fat tissue grafts and tissue flaps), include the use of implants and fillers. However, no single filler material currently can meet these various clinical needs. Excess amounts of adipose tissue are present in many individuals with obesity, obtainable through liposuction, and can be used to transplant and repair soft tissue defects. However, resorption of the transplanted autologous tissue over time may result in 40 - 60% of the graft loss. This has significantly limited clinical application of the autologous fat tissue. Therefore, tissue-engineering strategies are being investigated and represent a novel and potential approach to generating adipose tissue.The primary goal of tissue engineering is to regenerate healthy tissues or organs based on patients' need, whereby eliminating tissue or organ transplantation or mechanical devices, and their related complications. Another major challenge in organ and tissue transplantation is immunological rejection after receiving allograft tissue transplantation. Evidently, tissue-engineering strategies will not be associated with these concerns. Using engineering techniques, cells collected from a health individual can be cultured and expanded to a larger cell pool. These cells can then be seeded onto a scaffold that will support cell growth and proliferation. The cell-covered on scaffold can be implanted into a site that needs surgical repair. As the cells continue growing, the scaffold material degrades or absorbs. Eventually, a new tissue mass will develop. Tissue-engineering techniques are being investigated to develop a wide range of tissues, including bone, skin, cartilage, vascular, and adipose tissues. The development of adipose tissue-engineering strategies will be essential for revolutionizing our current practice in the restoration of tissue and repair of soft tissue defects.Tissue engineering and regenerative medicine are an important part of the contemporary medical science that has evolved in parallel with biotechnological advances. These approaches combine biomaterials, growth factors, and stem cells to repair failing organs. Material scientists can now fabricate biocompatible scaffolds with a wide range of physical parameters, combining mechanical integrity with high porosity to promote cell infiltration and angiogenesis. Likewise, biochemists can produce highly purified, bioactive cytokines in large quantity, suitable for cell culture and in vivo applications. Despite these advances, the availability of stem cells represents the most potential approach in regenerative medicine, and technically remains very challenge before it can be used in clinical practice. By definition, a stem cell is characterized by its ability to self-renew and its ability to differentiate along multiple lineage pathways. Ideally, a stem cell for regenerative medicinal applications should meet the following criteria:1. Can be found in abundant quantities (millions to billions of cells)2. Can be harvested by a minimally invasive procedure3. Can be differentiated along multiple cell lineage pathways in a regulatable and reproducible manner.4. Can be safely and effectively transplanted to either an autologous or allogeneic host5. Can be manufactured in accordance with current Good Manufacturing Practice guidelines.There are several potential sources for obtaining stem cells for tissue regeneration or repair purposes. And the most commonly used cell types are adult and embryonic-origin. Embryonic stem cells are usually obtained from destroyed embryos, that has raised ethical concerns. On the other hand, use of stem cells derived from adult tissues provides an alternative, and avoids ethical issues related to embryonic stem cells. Mesenchymal stem cells may undergo self-renewal for several generations while remaining their specific characteristics. This type of stem cells is multipotent, easily isolated and cultured, and readily expanded in the laboratory setting. All these make mesenchymal stem cells an attractive and potential source in several clinical applications, including cell-based therapies for the diseases, such as Parkinson's and Alzheimer's diseases, spinal cord injuries, burns, heart disease, and osteoarthritis, among other conditions. These adult stem cells typically include hematopoietic stem cells, neural stem cells, bone marrow stromal cells, dermal stem cells, adipose-derived stem cells and fetal cord blood stem cells .Although bone marrow is the most recognized source of mesenchymal stem cells, adipose tissue has been considered as a source of multipotent cells that have the capacity of differentiating to cells of adipogenic, chondrogenic, myogenic, and osteogenic lineages when cultured with the appropriate lineage specific stimuli. Adipose-derived stem cells (ADSCs) can be obtained from tissue harvested through liposuction [termed processed lipoaspirate cells (PLAs)], or through abdominoplasty procedures. These cells have also been identified as mesenchymal cells because they are derived from adipose tissue which is, in turn, derived from mesenchyme, like bone marrow. ADSCs have been shown to be very similar to marrow-derived stem cells in morphology and phenotype. In addition to their common multipotency, several CD antigen (or cell surface) markers on the surface of marrow stem cells have also been found on the surface of ADSCs. A wide availability, easy and safe to harvest have made ADSCs a great candidate for its clinical applications in tissue-engineering. However, genetic variation of the donors and possible contamination of ADSCs by endothelial, smooth muscle, and pericyte cell populations limit the clinical application of ADSCs. Therefore, the key issue it is critically important to purify ADSCs and identify their specific surface antigens. Although several possible cell surface antigens of ADSCs have been reported, their specificity remains to be determined. Ring cloning has been used to select clones derived from a single ADSC cell. Using lineage specific differentiation media, these clones can be used to induce adipogenesis, osteogenesis, chondrogenesis, and neurogenesis. It indicated that it is possible to purify ADSCs by clone forming.In the present study, we established a colony unit of ADSCs, investigated the cell surface antigens of these cells, and assessed the potential of adipogenic differentiation of different ADSC clones to further determine whether adipogenesis inducement impacts the specificity of these cell surface antigens, additional assays were also performed before and after three passages of adipogenesis of the ADSCs . The present study developed a novel approach to purify the ADSCs and further characterized the cell surface antigens of these cells that will promote clinical application of ADSCs in plastic and cosmetic surgeries.OBJECTIVES1. To develop a colony unit of human adipose-derived stem cells(ADSCs), and investigate the cell-surface antigens.2. To determine adipogenic differentiation potential of different clones by purifying ADSCs isolated from fatty tissues, and assessing the relation of highly adipogenic potential to cell-surface antigens.3. To compare the changes of cell-surface antigens before and after in vitro adipogenic differentiation, find the high- adipogenic potential related cell-surface antigensMETHODSADSCs were isolated and cultivated from the resection of subcutaneous fat potions in healthy donors who under dermatoplasty. Fatty tissue were digested by collagenase, and the cells were isolated and seeded in primary culture. ADSCs were cultured for two passages , then subjected to limit dilution assays to form colony unit. Flow cytometry was used to identify the expression of cell-surface antigens in the clones obtained from above experiments. The antibodies to CD29,CD44,CD34,CD54,CD106, and ABCG2 were used to determine ADSCs specific antigens. Each clone was induced for adipogenesis, and determined by Oil Red O stain. Adipogenic, chondrogenic and osteogenic lineage differentiations of the 4th generation of ADSCs was assessed by Oil O Red, Alcian Blue, and Alizarin Red staining, respectively. RESULTSThere was a large amount of ADSCs in the fatty tissue. 10 clones were obtained by colony-forming unit (CFU) assays, and after cultured for nine passages, the amount of the cells reached 2×10~6. The results of the cell-surface antigens investigation is: all of the clones were shown to be highly positive for CD29(92.9±7.4%) and CD44(94.6±6. 8%), while ABCG2 was expressed relatively low (2.5±1. 4%). The expression of CD34,CD54, and CD106 varies in different clones. Of each clone, the potential of adipogenic differentiation had a significant difference from l%～60%. Additionally, the expression of CD34, CD54, and CD106 in cells undergoing adipogenic differentiation for 7 days showed a diverse change contrast to those in cells before adipogenic differentiation. Data was analyzed by paired samples T test, and the result is no significant statistic difference. Two weeks after adipogenic differentiation of ADSCs, a significant fraction of the cells are characterized by multiple, intracellular lipid-filled droplets as indicated by Oil Red O staining, a typical function of matured fatty cells. In contrast, two weeks after chondrogenic and osteogenic induction, the cells were became positively stained by Alcian Blue and Alizarin Red, a typical function of matured chondrocyte and osteoblast cells.CONCLUSIONADSCs, harvested from adipose tissue by collagenase, is a mix population of multilineage stem cells. Clonal analysis could be used to successfully obtain purified ADSCs. Growth dynamics and morphology of the cells did not change with passages. There are differences and commonness of the expression of surface antigens among the clones. The potential of adipogenic differentiation of each clone also had variety. The difference in expression of cell surface antigens may attribute to the variation in potential of differentiation. Among those antigens identified, CD34, CD54, and CD106 showed markedly difference in each clone, maybe more associated with adipogenic differentiation clones. Additionally, the maintained expression of CD34, CD54, and CD106 after adipogenesis, while it reduced with passages, indicated that these markers may be considered as the high- adipogenic potential related cell-surface antigens. Our results suggest that the ADSCs, isolated from adipose tissue by collagenase can be the purified and certain cell surface antigens may be used to enhance ADSCs potential of adipogenicity.
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