In vitro construction of human amniotic mesenchymal stem cell sheet and its osteogenic differentiation / 中国组织工程研究

ABSTRACT

BACKGROUND: Human amniotic mesenchymal stem cells (hAMSCs) are a kind of adult stem cells that can be extracted from discarded placenta. Compared with other mesenchymal stem cells, hAMSCs have many advantages such as noninvasive isolation, low immunogenicity and short growth cycle, and thus hAMSCs are an important source of tissue engineering seed cells. Currently, hAMSCs have been applied in the clinical treatment of diabetes. OBJECTIVE: To explore a simple method to fabricate hAMSC sheets and to study their potential to differentiate into osteocytes. METHODS: Passage 3 hAMSCs were seeded into culture plates at a high density, and the sheet-forming medium was added to fabricate hAMSC sheet. The structural characteristics of hAMSC sheets were evaluated by histological staining and scanning electron microscopy. The sheet-forming induction medium was added to the passage 3 hAMSCs for 7 continuous days, and then replaced by the osteogenic induction medium for 14 days to construct osteogenic-induced hAMSC sheets. The potential for osteogenic differentiation of hAMSC sheets was assessed by alizarin red staining, immunohistochemical staining, alkaline phosphatase activity, RT-PCR, and western blot assay. RESULTS AND CONCLUSION: Hematoxylin-eosin staining results indicated that hAMSC sheets had a multi-layered structure with the cells stacked layer-by-layer and evenly distributed. Under the scanning electron microscope, the hAMSC sheets had a multi-layered structure, and a large amount of extracellular matrices that enveloped the fusiform cells were produced. After 14 days of osteogenic induction, orange-red precipitation was observed by alizarin red staining. Immunohistochemical staining results showed a large amount of type II collagen. Compared with non-induced hAMSC sheet, alkaline phosphatase activity was significantly increased in the osteogenic induced sheet (P < 0.01). The expression levels of type I collagen, osteocalcin, and Runt-related transcript factor 2 mRNA and protein were significantly higher in the osteogenic-induced hAMSC sheet group than the non-induced hAMSC sheet group (P < 0.05). Our findings indicate that this is a simple and economic method to construct the hAMSC sheets in the normal culture medium. Moreover, hAMSC sheets have a good osteogenic differentiation potential that has been confirmed in vitro.