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首页> 外文期刊>Journal of cellular biochemistry. >Density Gradient Centrifugation for the Isolation of Cells of Multiple Lineages
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Density Gradient Centrifugation for the Isolation of Cells of Multiple Lineages

机译：密度梯度离心分离多个谱系的细胞

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We recently developed a simple strategy for the enrichment of mesenchymal stem cells (MSCs) with the capacity for osteoblast, chondrocyte, and adipocyte differentiation. On transplantation, the progenitor-enriched fraction can regenerate bone with multiple lineages of donor origin. Although comprising multiple precursor cell types, the population is enriched >100-fold in osteoprogenitors, hence the name highly purified osteoprogenitors (HipOPs). To establish a new modified method of purifying pure MSCs, it is useful to know the expression patterns of surface markers on heterogeneous MSCs and committed cells such as osteoblasts, adipocytes, and chondrocytes. However, calcium deposition by osteoblasts is a critical obstacle in visualizing the expression patterns of surface markers. We now report a new method of separating differentiated osteoblastic HipOPs (OB-HipOPs) from calcium deposits using the Percoll density gradient centrifugation technique. After centrifuge separation, calcium deposits were observed at the bottom of the centrifuge tube, and living OB-HipOPs were harvested from the 10-70% fractions. However, there were no living cells in the 70-80% fraction. We concluded that living OB-HipOPs are separated by one 10-70% Percoll gradient. Furthermore, we analyzed the expression patterns of putative MSC markers on differentiated HipOPs. FACS analysis revealed that Sca-1, CD44, CD73, CD105, and CD106 were decreased in OB-HipOPs. In adipogenic- and chondrogenic-HipOPs, Sca-1, CD73, CD105, and CD106 were decreased. This new technique is a helpful tool to identify MSC surface markers and to clarify in more detail the differentiation stages of osteoblasts. J. Cell. Biochem. 116: 2709-2714, 2015. (c) 2015 Wiley Periodicals, Inc.
机译：我们最近开发了一种简单的策略，用于富集具有成骨细胞，软骨细胞和脂肪细胞分化能力的间充质干细胞（MSC）。移植时，富含祖细胞的级分可以使供体来源的多个谱系再生骨骼。尽管包含多种前体细胞类型，但该群体的骨祖细胞富集> 100倍，因此被称为高度纯化的骨祖细胞（HipOPs）。要建立纯化纯MSC的新改良方法，了解异质MSC和定型细胞（如成骨细胞，脂肪细胞和软骨细胞）上表面标志物的表达模式非常有用。然而，成骨细胞的钙沉积是可视化表面标志物表达模式的关键障碍。我们现在报告使用Percoll密度梯度离心技术从钙沉积物中分离分化成骨细胞HipOPs（OB-HipOPs）的新方法。离心分离后，在离心管底部观察到钙沉积，并从10-70％的馏分中收获活的OB-HipOP。但是，在70-80％的比例中没有活细胞。我们得出的结论是，活的OB-HipOP被一个10-70％的Percoll梯度分开。此外，我们分析了分化的HipOPs上假定的MSC标记的表达模式。 FACS分析表明，OB-HipOPs中的Sca-1，CD44，CD73，CD105和CD106降低。在成脂性和成软骨性HipOPs中，Sca-1，CD73，CD105和CD106降低。这项新技术是鉴定MSC表面标志物并更详细阐明成骨细胞分化阶段的有用工具。 J.细胞。生化。 116：2709-2714，2015。（c）2015 Wiley Periodicals，Inc.

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