Polisetti Naresh, Chaitanya V G, Babu Phanithi Prakash, Vemuganti Geeta K
Stem Cell Biology Laboratory, L.V. Prasad Eye Institute, Banjara Hills, Hyderabad, India.
Neurol India. 2010 Mar-Apr;58(2):201-8. doi: 10.4103/0028-3886.63789.
Bone marrow mesenchymal cells have been identified as a source of pluripotent stem cells with varying degrees of plasticity in humans. However, there are a few reports on rat-derived cells, which could be good models for the research purpose. We describe here a simple method of establishing the rat bone marrow stromal cells by the principle of adhesion and document their phenotype along with their differentiation potential to other lineages.
Rat bone marrow stromal cells were isolated by three methods: direct plastic adherence, ficoll hypaque separation and a combination of both. The stromal cells obtained by these methods were characterized by fluorescent activating cell sorting (FACS) for established hematopoietic and non-hematopoietic markers. The cells obtained by combination method (combination of ficoll density gradient centrifugation and plastic adherence) were cultured and serially passaged. Transcriptional confirmation was done by reverse transcription polymerase chain reaction (RT-PCR) for vimentin and collagen type 1 alpha 1. Attempts were made to differentiate the marrow stromal cells into adipocytes, osteocytes and neuronal like cells.
Bone marrow samples from 10 rats yielded 4-5 million bone marrow mononuclear cells /ml per femur. Of the three methods tested, a combination method yielded good growth of spindle cells. The cells obtained by combined method showed high percentage of positivity for vimentin, fibronectin and CD90 and negative for hematopoietic markers. Further, RT-PCR confirmed vimentin and collagen type - 1 alpha 1 expression. Oil red O staining and Alizarin red staining confirmed adipocytic and osteogenic differentiation. On immunocytochemical analysis, the cells expressed nestin, beta-tubulin III, neurofilament and synaptophysin.
Adequate quantities of rat marrow stromal cell cultures can be established by a simple method based on adhesion properties. Their phenotypic characteristics and plasticity support the evidence that they are mesenchymal stem cells with a distinct tendency for neural lineage.
骨髓间充质细胞已被确定为人类多能干细胞的一个来源，具有不同程度的可塑性。然而，关于大鼠来源细胞的报道较少，而大鼠来源细胞可能是很好的研究模型。我们在此描述一种基于贴壁原理建立大鼠骨髓基质细胞的简单方法，并记录其表型以及向其他谱系分化的潜能。
通过三种方法分离大鼠骨髓基质细胞：直接塑料贴壁法、菲可-泛影葡胺分离法以及两者结合的方法。通过荧光激活细胞分选（FACS）对这些方法获得的基质细胞进行已确立的造血和非造血标志物的表征。对通过结合法（菲可密度梯度离心和塑料贴壁相结合）获得的细胞进行培养并连续传代。通过逆转录聚合酶链反应（RT-PCR）对波形蛋白和Ⅰ型胶原α1进行转录确认。尝试将骨髓基质细胞分化为脂肪细胞、骨细胞和神经元样细胞。
来自10只大鼠的骨髓样本每根股骨产生400 - 500万个骨髓单个核细胞/毫升。在所测试的三种方法中，结合法产生了良好的梭形细胞生长。通过结合法获得的细胞对波形蛋白、纤连蛋白和CD90呈高阳性百分比，对造血标志物呈阴性。此外，RT-PCR证实了波形蛋白和Ⅰ型胶原α1的表达。油红O染色和茜素红染色证实了脂肪生成和成骨分化。免疫细胞化学分析显示，这些细胞表达巢蛋白、β-微管蛋白Ⅲ、神经丝和突触素。
基于黏附特性的简单方法可建立足够数量的大鼠骨髓基质细胞培养物。它们的表型特征和可塑性支持了它们是具有明显神经谱系倾向的间充质干细胞这一证据。
