| ADSCs (Adipose-derived stromal cells) from the adipose tissue of the body are the adult stem cells that have been characterized extensively recent years. BMSCs are one of the adult stems cells which were earliest recognized. They have very important clinical value in cell therapy, gene therapy and the regenerative medicine field. But the patients are not easy to accept the bone marrow extraction process. In 1×106 bone marrow mononuclear cells were only 2～5 BMSCs, which made access to a large number of BMSCs had been restricted. ADSCs have many similar characteristics with BMSCs. It has abundant sources, liposuction surgery is simple, autologous transplantation without immune rejection and many other advantages increasingly highlighted their strengths and potential clinical value. Expected to replace or supplement BMSCs used in cell therapy. However, in relation to BMSCs, studies on ADSCs are in the initial phase. The biological characteristics of ADSCs are comprehensive understanding. ADSCs differentiation potential in vivo and vitro and induced to other mesoderm cells need for more researches to confirm the results.The task is to establish a suitable cell growth and expanded of hADSCs (human adipose-derived sromal cells) in vitro conditions; expounded its basic biological characteristics; identification to their adipogenic, osteogenic and especially neurogenic differentiation potential. We also have done a preliminary study for its large-scale amplification.1. Studies on the basic biological characteristics of hADSCshADSCs were obtained from raw human lipoaspirates. Briefly, raw lipoaspirates were washed extensively with PBS to remove contaminating debris and red blood cells. Washed aspirates were treated with 0.075％collagenase (typeⅡ) in PBS. hADSCs were 1～2×105/ml inoculated with 100mm dish and cultured as normal conditions. Serial subcultivation when the cells growth and accounted for 80％of the culture dish. cell growth were detection by the time change, the cumulative growth multiples and others detection of EGF and FGF on cell growth kinetics. Detection hADSCs surface marker CD29, CD45, CD105 by flow cytometry, the cell cycle, DNA content were detected by flow cytometry. There is an analysis of cell ultramicrostmcture by transmission electron microscope.EGF, HGF have an significant role on promoting cell proliferation and FGF has no significant impact, hADSCs expressed CD29, CD105 and did not express CD45. CD29 and CD105 expression were 99.5％and 92.2％respectively in p3. CD105 expression was only 57.6％in p7. Cell cycle analysis showed that exponential phase of growth cells were 88.26％in GO period, 11.7％in S period and 0.03％in G2 period. The rate of apoptosis was 0.24％. DNA content analysis show that cells were normal diploid cells.hADSCs are easily separated from the adipose tissue. Per 100ml lipoaspirates can offer 2％107～4×107 nucleated cells. The morphology of hADSCs is not significantly changed with long-time culture and cells can still maintain good proliferate ability.2. Studies on cell differentiation potency of hADSCsCurrently mesenchymal stem cells have not highly recognized specific molecular marker, there are no more separation and cultured standard methods to get mesenchymal stem cells. These experiments aim to prove that the mesenchymal stem cells have the potency to differentiate into 3 cells types included osteocyte, chondrocyte, adipocyte.(1)Osteogenic differentiationhADSCs with osteogenic medium (10-7mol/L dexame thasone, 50mg/L vitamin C, 10mmol/Lβ-glycerophosphate, 10％FBS, DMEM/F12) were Induced into osteogenic cells. To confirm osteogenesis, cells were examined by RT-PCR for the expression of osteopontin genes 7 hours later after induction. Using Von Kossa staining detected the formation of calcium nodus. It was observed that formation of mineralized nods in extracellular matrix. It was a result that the induced cells and normal osteoblasts had the same ultrastructure by TEM observation for 35 days. There were a certain amount of rough endoplasmic reticulum and Golgi apparatus in the intracytoplast, and also a large number of collagen fibers in intercellular substance.(2)Adipogenic differentiationInduction of hADSCs with AM resulted in expanded cell morphology and a time-dependent increase in intracellular Oil Red O staining; an established lipid dye. Induction of hADSCs with AM resulted in expression of the adipose-specific transcription factor peroxisome-proliferating activated receptor PPAR-γNo adipogenic-morphological change was observed when hADSCs were exposed to a control medium without adipogenic stimulation.(3)Neurogenic differentiationhADSCs were induced toward the neurogenic lineage using an established protocol. We used different methods to induce neuronal differentiation. And compare the methods difference of induction neuronal morphology and protein expression.Method 1: Change the medium A (DMEM,10％FBS) to medium C (DMEM,10％FBS,1mmol/Lβ-mercaptoethanol) when the cells growth and accounted for 80％of the culture dish in p3. Cultured 24 hours later then changed medium to medium D (DMEM,1mmol/Lβ-mercaptoethanol. Cell morphology change significantly after 2h～5h. Immunocytochemistry showed positive staining of NSE. RT-PCR results also were confirmed by the induction of cell expression NSE. But 10 hours after induction cells begin to death; in vitro longest survival time is only 3-5d.Method 2:Induction of hADSCs with our neurogenic medium (DMEM/F12,10％plasma,20ng/ml FGF, 20ng/ml GDNF ). Cell morphology change significantly after 24h. Immunocytochemical results suggest that the pre-induced cell nestin expression was increased contrast to preinduction, NSE, S-100, GAP-43, GAFP, TH were positive staining.hADSCs can quickly differentiate into neuron-like cells in neurogenic differentiation medium. Immunohistochemical staining showed that these cells express neuron-specific protein. This differentiation triggered from extracellular signal is rapid. It shows that hADSCs itself have the neuronal gene expressed sequences. ADSCs have the capacity of neuroectoderm differentiation. Expected to be a new source of cells for cell therapy of Parkinson's disease. 3. The scale expansion of hADSCsWe use New Brunswick Scientific Company production of desktop biological reactor combined Fibra-Cel carriers to culture hADSCs for a large-scale cultivation. The aim is to obtain a large number hADSCs in a short time that has the similar biological characteristics with the hADSCs in vivo, and to provide adequate cell source for cell therapy and tissue engineering. SEM result showed hADSCs almost completely attached to the carriers within 48h.The cells grew slowly in the first 3-4 days and attached to the carriers with a small amount of extracellular matrix secretion. A large number of cells secreted into extracellular matrix on the 5th day. And cells established connection each other. On the 9th day, it can be saw that cells were covered by their own secretion of extracellular matrix. After the large-scale cultivation, the biological characteristics and differentiation potency of hADSCs needed to confirm though the further studies. In a word, hADSCs can differentiate into dopaminergic neuron-like cells and are a new cell source for the treatment of neurodegeneration diseases and have a great potential for wide application.
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