Manochantr Sirikul, Tantrawatpan Chairat, Kheolamai Pakpoom, U-pratya Yaowaluk, Supokawej Aungkura, Issaragrisil Surapol
Division of Cell Biology, Department of Preclinical Sciences, Faculty of Medicine, Thammasat University, Pathumthani, Thailand.
J Med Assoc Thai. 2010 Dec;93 Suppl 7:S183-91.
Mesenchymal stem cells (MSCs) derived from amnion are considered to be adult stem cells that can be easily obtained in large quantities by a less invasive method in comparison to bone marrow-derived MSCs (BM-MSCs). However; the biological properties and the differentiation capacity of amnion-derived MSCs (AM-MSCs) are still poorly characterized. The objectives of this study were to isolate, characterize and explore the potential of AM-MSCs in differentiating toward neural lineage in comparison to those of BM-MSCs. To isolate AM-MSCs, amnion was digested with trypsin-EDTA and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum. The expression profiles of several MSC markers were examined by flow cytometry. AM-MSCs from passage 3-5 were used for adipogenic, osteogenic and neural differentiation assays by culturing in appropriate induction media. The expression of several neural marker genes, including MAP-2, GFAP and beta-tubulin III in AM-MSCs was determined by quantitative real time-PCR. The expression of neural-specific markers, MAP-2 and beta-tubulin III, was subsequently confirmed by immunocytochemistry using confocal laser microscope. The results demonstrated that AM-MSCs could be easily expanded to 18-20 passages while maintaining the undifferentiated state and exhibiting MSC markers (CD73, CD90, and CD105) but do not express the hematopoietic markers (CD34 and CD45). Similar to BM-MSCs, AM-MSCs were able to differentiate to several mesodermal-lineages including adipocytes and osteoblasts. Moreover; these cells could be induced to differentiate to neuron-like cells as characterized by cell morphology and the expression of several neural markers including MAP-2, GFAP and beta-tubulin III. The present study demonstrated that AM-MSCs can be easily obtained and expanded in culture. These cells also have transdifferentiation capacity as evidenced by their neural differentiation potential. According to the results, amnion can be used as an alternative source of MSCs for stem cell therapy in neurodegenerative diseases.
与骨髓间充质干细胞（BM-MSCs）相比，羊膜来源的间充质干细胞（MSCs）被认为是成体干细胞，能够通过侵入性较小的方法轻松大量获取。然而，羊膜来源的间充质干细胞（AM-MSCs）的生物学特性和分化能力仍未得到充分表征。本研究的目的是分离、表征AM-MSCs，并与BM-MSCs相比，探索其向神经谱系分化的潜力。为了分离AM-MSCs，用胰蛋白酶-EDTA消化羊膜，并在补充有10%胎牛血清的杜尔贝科改良伊格尔培养基（DMEM）中培养。通过流式细胞术检测几种间充质干细胞标志物的表达谱。第3-5代的AM-MSCs通过在适当的诱导培养基中培养用于脂肪生成、成骨和神经分化测定。通过定量实时PCR测定AM-MSCs中几种神经标志物基因的表达，包括微管相关蛋白2（MAP-2）、胶质纤维酸性蛋白（GFAP）和β-微管蛋白III。随后使用共聚焦激光显微镜通过免疫细胞化学证实神经特异性标志物MAP-2和β-微管蛋白III的表达。结果表明，AM-MSCs能够轻松扩增至18-20代，同时保持未分化状态并表现出间充质干细胞标志物（CD73、CD90和CD105），但不表达造血标志物（CD34和CD45）。与BM-MSCs相似，AM-MSCs能够分化为几种中胚层谱系，包括脂肪细胞和成骨细胞。此外，这些细胞可以被诱导分化为神经元样细胞，其特征为细胞形态以及几种神经标志物包括MAP-2、GFAP和β-微管蛋白III的表达。本研究表明，AM-MSCs能够在培养中轻松获取和扩增。这些细胞还具有转分化能力，其神经分化潜力证明了这一点。根据结果，羊膜可作为间充质干细胞的替代来源用于神经退行性疾病的干细胞治疗。
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