Separation And Amplication Of Human Adipose Derived Stromal Cells And Induced Into Adipocytes

Modern Tissue engineering is a new discipline developed rapidly in recent years .Its influence is not merely of some soft tissue defects in plastic surgery and other treatment of common diseases. With the potential of stem cell differentiation further study is bound to human health plagued some major diseases, such as Alzheimer's disease, Cancer, diabetes treatment propose new ways different from traditional.Stem cells are a population possessing self-renewal capacity, long-term viability, and multilineage potential. The multilineage potential of embryonic stem cells and adult stem cells from the bone marrow has been characterized extensively. Although embryonic stem cell potential is enormous, many ethical and political issues accompany their use. Therefore, adult stem cells from the bone marrow stroma (mesenchymal stem cells, MSCs) have been proposed as an alternative source. Adipose tissue, like bone marrow, is derived from the mesenchyme and contains a supportive stroma that is easily isolated. Based on this, adipose tissue may represent a source of stem cells that could have far-reaching effects on several fields.Much of the work conducted on adult stem cells has focused on bone marrow stromalcells (BMSCs) found within the bone marrow stroma. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Preliminary studies have recently identified a putative stem cell population within the adipose stromal compartment. These cells can be induced into adipocytes.This study established the method of the saparation and amplification in vitro of human adipose derived stromal cells (ADSCs) and to investigate its multi-lineags differentiation potential. ADSCs were isolated and amplificated by collagenase digestion from lipoaspirate to detect the proliferation kinetics of ADSCs though cell time course and cumulative population doubling. ADSCs were induced into adiposcytes by different factors to assess the committed differentiations.1. Separation and amplication of ADSCsADSCs were obtained from aspirates.The aspirates were washed extensively with sterile phosphate-buffered saline (PBS) to remove contaminating debris and red blood cells. Washed aspirates were treated with 0.075% collagenase (typeⅡ) in PBS for 30 min at 37°C with gentle agitation. The cellular pellet was resuspended in DMEM/10% FBS and filtered through a 200-μm mesh filter to remove debris. The filtrate was centrifuged as detailed above and plated onto conventional tissue culture plates in control medium.2. Cell cycle and growth dynamics detectionDetection of cell cycle: Cells in the G1 phase were 90.07%; cells in the G2 phase were 0.03%; cells in the S phase were 9.00%; G2/G1 was 1.84.Detection of growth dynamics: Cells after incubation period of 1-2 days to enter a period of rapid growth. The peak was the 7th day. The number of ADSCs was amplified to the original (6357.36±335.58)times from p0 to p8. The numbers of every generation maintain stability and no downward trend. It illustrated that ADSCs can still maintain good ability to increasing after long-time generation.3. ADSCs undergo adipogenic differentiationAdipogenic (AM): DMEM /FBS (10%) with 0.5 mM isobutyl-methylxanthine (IBMX), 1μM dexamethasone, and 10μM insulin. Induction of ADSCs with AM resulted in an expanded cell morphologyand a time-dependent increase in intracellular Oil Red O staining, an established lipid dye.ADSCs were induced for 10 days. Total cellular RNA was isolated and reverses transcribed using conventional protocols. PCR amplification was performed. All primer sequences were determined using established GenBank sequences. Induction of ADSCs with AM resulted in expression of the adipose-specific transcription factor peroxisome-proliferating activated receptor-γ(PPAR-γ). The results showed that ADSCs can differertiate into adipocytes.4. Cell surface markerImmunofluorescence: ADSCs were fixed for 10 min in 4% polyoxymethylene. The fixed cells were washed in 0.01% PBS and incubated for 1 h in anti-CD44 monoclonal antibody. Then cells were washed in 0.01% PBS and incubated for 30 min in CY3-conjugated fluorescent antibody. Observed with fluorescence microscope.ADSCs were stained with red fluorescence.Flow Cytometry: ADSCs were cultured in control medium 72 h before analysis. Flow cytometry with a FACscan argon laser cytometer was performed according to a previous study. Briefly, cells were harvested in 0.25% trypsin/EDTA and fixed for 20 min in ice-cold 2% formaldehyde. The fixed cells were washed in flow cytometry buffer and incubated for 30 min in flow cytometry buffer containing fluorescein isothiocyanate-conjugated monoclonal antibodies and the following CD antigens: 29, 45, and 105. ADSCs were stained with a phycoerythrin-conjugated nonspecific IgG to assess background fluorescence. ADSCs expressed CD29 and CD105. In contrast, noexpression of the hematopoietic lineage markers CD45.In short, ADSCs were a type of adult stem cell which were found in adipose tissue recent years. ADSCs which like BMSCs can differentiate into adipocytes, chondrocytes, osteoblasts, and myoblasts in vitro. But the studies on ADSCs are now in the initial stage.Its biological characteristics are not entirely clear. With the biological characteristics and mechanism of differentiation deeply studies, ADSCs will lay the foundation for cell replacement therapy, gene therapy and organ transplant.