To further study the proliferation and multi-differentiation potentials of adipose-derived stem cells (ADSCs), the cells were isolated with improved methods and their growth curves were achieved with cck-8. Surface protein expression was analyzed by flow cytometry to characterize the cell phenotype. The multi-lineage potential of ADSCs was testified by differentiating cells with adipogenic, chondrogenic, osteogenic, and myogenic inducers. The results showed that about 5 Â 10 5 stem cells could be obtained from 400 to 600 mg adipose tissue. The ADSCs can be continuously cultured in vitro for up to 1 month without passage and they have several logarithmic growth phases during the culture period. Also, the flow cytometry analysis showed that ADSCs expressed high levels of stem cell-related antigens (CD13, CD29, CD44, CD105, and CD166), while did not express hematopoiesis-related antigens CD34 and CD45, and human leukocyte antigen HLA-DR was also negative. Moreover, stem cell-related transcription factors, Nanog, Oct-4, Sox-2, and Rex-1 were positively expressed in ADSCs. The expression of alkaline phosphatase (ALP) was detected in the early osteogenic induction and the calcified nodules were observed by von Kossa staining. Intracellular lipid droplets could be observed by Oil Red staining. Differentiated cardiomyocytes were observed by connexin43 fluorescent staining. In order to obtain more stem cells, we can subculture ADSCs every 14 days instead of the normal 5 days. ADSCs still keep strong proliferation ability, maintain their phenotypes, and have stronger multi-differentiation potential after 25 passages.

Recently, a cohort of miRNAs, including miR-31, was reported to be downregulated during osteogenic induction by miR microarray analysis. It remains unclear how changes in miR-31 expression collaborate with bone transcription factors to activate the biological pathways that regulate the differentiation of bone mesenchymal stem cells (BMSCs). Here the effects of miR-31, Runx2, and Satb2 on the osteogenic differentiation of BMSCs were investigated using mimics and inhibitors of miR-31, small interfering RNA for knockdown of Runx2 and plasmids for overexpression of Runx2. Our results showed that miR-31 expression decreased progressively in BMSC cultures during differentiation. Inhibition of miR-31 dramatically increased the alkaline phosphatase activity and mineralization in BMSC cultures. Additionally, miR-31 diminished the levels of the Satb2 protein without significantly affecting Satb2 mRNA levels, and Runx2 directly repressed miR-31 expression. Overexpression of miR-31 significantly reduced expression of the osteogenic transcription factors OPN, BSP, OSX, and OCN, but not Runx2. Furthermore, the high expression of miR-31 in BMSCs cultured in the proliferation medium repressed Satb2 protein levels, which may contribute to the maintenance of BMSCs in an undifferentiated state. In conclusion, our results suggest that a Runx2, Satb2, and miR-31 regulatory mechanism may play an important role in inducing BMSC osteogenic differentiation. The results of this study provide us with a better understanding of the molecular mechanisms that govern the BMSC fate.

Adult stem cells hold great promise to be used in tissue repair and regeneration. And research interests continuously exist in both the biology and potential therapeutic applications of adult stem cells from bone marrow. In recent years, interest has rapidly grown in the developmental plasticity and therapeutic potential of stromal cells isolated from adipose tissue, called adipose-derived stem cell(ADSC). The resources of adipose tissue is much less expensive than bone marrow, with less invasive operation and more quantities. Therefore, adipose tissue represents an abundant, practical, and appealing source of donor tissue for autologous cell replacement. However, the published data about ADSC primary culture and their growth character are quite different. Therefore, the detailed biological characteristics of ADSCs are not well understood to date. To further study the proliferation and multi-differentiation potentials of ADSC, the cells were isolated with the improved methods and their growth curves were achieved with cck-8. Surface protein expression was analyzed by flow cytometry to characterize the cell phenotype. The multilineage potential of ADSCs was testified by differentiating cells with adipogenic, chondrogenic, osteogenic and myogenic inducers. The results showed that about 5×10 5 stem cells could be obtained from 400~600 mg adipose tissue. And ADSCs can be continuously cultured in vitro for up to 1 month without passage and they have several logarithmic growth phases during the culture period. Also the flow cytometry analysis showed that ADSCs expressed high level of stem cell-related antigens (CD13, CD29, CD44, CD105 and CD166), while didn't express hematopoiesisralated antigens CD34 and CD45, and human leukocyte antigen HLA-DR was also negative. Meanwhile stem cell related transcription factors, Oct-4, Sox-2 and Rex-1, were positively expressed in ADSCs. ADSC could differentiate into adipocyte, osteocyte, chondrocyte and endodermal cardiomyocyte successfully. In order to obtain more stem cells, we subcultured ADSCs every 14 days in stead of normal 5 days. ADSCs still kept strong proliferation ability, maintain their phenotypes, and had stronger multi-differentiation potential after 25 passages. By our isolated and culture method, we can obtain more and higher quality ADSCs, hADSCs as a source of stem cells are very appealing, and could also potentially be an alternative source to BMSCs for being used in allogeneic transplants and tissue engineering.

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