Biological characteristics and dopaminergic neural-like cell differentiation potential of human amniotic membrane-derived mesenchymal stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND:Human amniotic membrane-derived mesenchymal stem cells (AMSCs) are considered to be one kind of adult stem cells that can be easily obtained in large quantities without using an invasive method. Because of their low immunogenicity, anti-inflammatory properties, multipotency of differentiation and without ethical issue, human amniotic membrane-derived mesenchymal stem cells have been proposed as a good candidate to be used in celltherapy and regenerative medicine. However, the biological properties and the differentiation capacity of human amniotic membrane-derived mesenchymal stem cells are stil poorly characterized. OBJECTIVE:To establish a practical method for isolation and purification of human amniotic membrane-derived mesenchymal stem cells, and to study the biological characteristics and dopaminergic neural-like celldifferentiation potential of the human amniotic membrane-derived mesenchymal stem cells. METHODS:Human amniotic membrane-derived mesenchymal stem cells were disassociated and isolated from the amniotic membrane by trypsin and col agenase based enzymic digestion, and purified by percol mediated density gradient centrifugation. Expressions of surface antigens and transcription factors of the human amniotic membrane-derived mesenchymal stem cells were determined by flow cytometry and western blot assays. Based on the osteogenic and adipogenic induction, the multipotent differentiation capability of human amniotic membrane-derived mesenchymal stem cells was determined. Induction of neural celldifferentiation of human amniotic membrane-derived mesenchymal stem cells was conducted in Neurabasal conditioning medium with ATRA supplement. Neural cellassociated bio-markers were determined by immunofluoresence staining and confocal microscope. RESULTS AND CONCLUSION:In this study, we performed a practical method to isolate and purify human amniotic membrane-derived mesenchymal stem cells and amniotic epithelial cells simultaneously, with high cells yield. We demonstrated a group of constitutive expressions of neural antigens and embryonic associated transcription factor proteins (OCT-4, SOX-2 and KLF4) in fresh isolated human amniotic membrane-derived mesenchymal stem cells as wel as in human amniotic membrane-derived mesenchymal stem cells after in vitro passage, which suggested that the human amniotic membrane-derived mesenchymal stem cells not only possessed intrinsic tendency to neural celldifferentiation, but also maintained their stem cellcharacteristics after in vitro passage. We stimulated the human amniotic membrane-derived mesenchymal stem cells in the neurobasal-A and B27 based conditioning medium to induce neural celldifferentiation. The induced human amniotic membrane-derived mesenchymal stem cells displayed an up-regulation of expression in panel of neural and dopaminergic associate molecules (β-tubulin III, neuron-specific nuclear protein, tyrosine hydroxylase, glial fibril ary acidic protein, myelin basic protein and nestin) by flow cytometry and immunofluorescence staining, which demonstrated the multipotent differentiation capability and dopaminergic neuron-like differentiation potential of the human amniotic membrane-derived mesenchymal stem cells.