Approach to get mineralized tissue using pulp cells

本研究旨在通过简单的方法从人牙髓组织中获得多能干细胞。为了得到mils，我们根据细胞表面抗原的指示分离细胞。本研究采用荧光激活细胞分选仪（Fluorescence Activated Cell Sorter，FACS）和磁细胞分选仪（Magnetic Cell Sorting，MACS），进行了以下实验：（1）无菌收集人牙髓组织，使其生长，继续生长，进行流式细胞仪分析。我们使用CD 73、74、106、146、166、271和STRO-1抗原。结果显示，除CD 166外，其余6种抗原表达水平均较低，但CD 166染色细胞中有20%的细胞呈阳性表达。(2)我们将这些细胞与上述抗原（除CD 73外）偶联，并将它们应用于MACS系统。我们继续培养抗原阳性细胞，并将其分化为成骨细胞。结果显示，茜素红染色水平在CD 74和106阳性细胞中低于在CD 74和106阴性细胞中。与CD 146和STRO-1阴性细胞相比，CD 146和STRO-1阳性细胞显示相同的茜素红染色水平。CD 166和271阳性细胞茜素红染色较CD 166和271阴性细胞明显增强。这些CD 166和271阳性细胞显示出碱性磷酸酶的高活性。CD 166和271阳性细胞的茜素红染色明显高于未选择的牙髓细胞，尤其是CD 166阳性细胞，表明利用MACS系统从人牙髓中分选高能力的成骨细胞是一种有效的方法。

The purpose of this study was to get pluripotent cells from human pulp tissues by simple technique. To get the mils, we separated cells by the indication for cell surface antigens. Using Fluorescence Activated Cell Sorter (FACS) and Magnetic Cell Sorting (MACS) systems, we have done the following experiments(1) We collected human pulp tissues aseptically, allow for them by outgrowth, continue to growth, then analysis them by FACS. We used CD73, 74, 106, 146, 166, 271, and STRO-1 antigen. As the results, the expression level of six antigen except CD166 were quite low, however positive expression were detected by 20% of CD166 stained cells.(2) We conjugated the cells to the above antigen except CD73, and apply them to MACS system. We continued to growth the antigen positive cells, and differentiated them to osteoblasts. As the results, Alizarin red staining levels were lower in CD74 and 106 positive cells than in CD74 and 106 negative cells. CD146 and STRO-1 positive cells show same Alizarin red staining levels compared with CD146 and STRO-1 negative cells. CD166 and 271 positive cells show obvious strong Alizarin red staining compared with CD166 and 271 negative cells. These CD166 and 271 positive cells show high activity of Alkaline phosphates. Alizarin red staining of CD166 and 271 positive cells were obviously higher than non selected pulp cells, especially in CD166 positive cells.Taken together from these results show that MACS system utilized for CD166 is a useful method to sort out high capacity of osteogenic cells from human pulp.