Abstract
Human adipose tissue-derived stem cells (hASCs) have been subjected to extensive investigation because of their self-renewal properties and potential to restore damaged tissues. In the literature, there are several protocols for differentiating hASCs into osteoblasts, but there is no report on the control of cell viability during this process. In this study, we used osteoblasts derived from hASCs of patients undergoing abdominoplasty. The cells were observed at the beginning and end of bone matrix formation, and the expression of proteins involved in this process, including alkaline phosphatase and osteocalcin, was assessed. RANKL, Osterix, Runx2, Collagen3A1, Osteopontin and BSP expression levels were analyzed using real-time PCR, in addition to a quantitative assessment of protein levels of the markers CD45, CD105, STRO-1, and Nanog, using immunofluorescence. Rhodamine (Rho123), cytochrome-c, caspase-3, P-27, cyclin D1, and autophagy cell markers were analyzed by flow cytometry to demonstrate potential cellular activity and the absence of apoptotic and tumor cell processes before and after cell differentiation. The formation of bone matrix, along with calcium nodules, was observed after 16 days of osteoinduction. The gene expression levels of RANKL, Osterix, Runx2, Collagen3A1, Osteopontin, BSP and alkaline phosphatase activity were also elevated after 16 days of osteoinduction, whereas the level of osteocalcin was higher after 21 days of osteoinduction. Our data also showed that the cells had a high mitochondrial membrane potential and a low expression of apoptotic and tumor markers, both before and after differentiation. Cells were viable after the different phases of differentiation. This proposed methodology, using markers to evaluate cell viability, is therefore successful in assessing different phases of stem cell isolation and differentiation.
Highlights
The use of adipose tissue-derived stem cells (ASCs) has contributed to clinical and experimental research in a variety of biological systems [1]
HASCs clearly showed the presence of fat droplets (Fig 1A), which disappeared after the second and third passages (Fig 1B and 1C, respectively). Human ASCs (hASCs) exhibited a fibroblastoid morphology a few days after being isolated from the adipose tissue, and this morphology was maintained during the later passages
Cytoplasmic labeling of vimentin in hASCs revealed that this protein was present in mesodermal cells that had originated from stem cells
Summary
The use of adipose tissue-derived stem cells (ASCs) has contributed to clinical and experimental research in a variety of biological systems [1]. The hASCs are characterized by their expression of mesenchymal (CD90 and CD105) [8,9,10] and pluripotency (Nanog, Sox, and Stro-1) markers [11]. This cell type does not express the hematopoietic markers, CD45 [9, 11] CD34, and CD117 [11]. HASCs can differentiate into osteoblasts [12] when cultured in an appropriate osteogenic differentiation media [13] This property makes hASCs a useful experimental model that allows for an understanding of the behavior of osteoblasts during the different stages of osteoinduction [4]
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