University of Lincoln, Brayford Pool, Lincoln LN6 7TS, UK.
Cell Biol Int. 2012 Jul;36(7):595-600. doi: 10.1042/CBI20110336.
HUCB (human umbilical cord blood) has been frequently used in clinical allogeneic HSC (haemopoietic stem cell) transplant. However, HUCB is poorly recognized as a rich source of MSC (mesenchymal stem cell). The aim of this study has been to establish a new method for isolating large number of MSC from HUCB to recognize it as a good source of MSC. HUCB samples were collected from women following their elective caesarean section. The new method (Clot Spot method) was carried out by explanting HUCB samples in mesencult complete medium and maintained in 37°C, in a 5% CO2 and air incubator. MSC presence was established by quantitative and qualitative immunophenotyping of cells and using FITC attached to MSC phenotypic markers (CD29, CD73, CD44 and CD105). Haematopoietic antibodies (CD34 and CD45) were used as negative control. MSC differentiation was examined in neurogenic and adipogenic media. Immunocytochemistry was carried out for the embryonic markers: SOX2 (sex determining region Y-box 2), OLIG-4 (oligodendrocyte-4) and FABP-4 (fatty acid binding protein-4). The new method was compared with the conventional Rosset Sep method. MSC cultures using the Clot Spot method showed 3-fold increase in proliferation rate compared with conventional method. Also, the cells showed high expression of MSC markers CD29, CD73, CD44 and CD105, but lacked the expression of specific HSC markers (CD34 and CD45). The isolated MSC showed some differentiation by expressing the neurogenic (SOX2 and Olig4) and adipogenic (FABP-4) markers respectively. In conclusion, HUCB is a good source of MSC using this new technique.
人脐带血（HUCB）已广泛应用于临床同种异体造血干细胞（HSC）移植。然而，HUCB 作为间充质干细胞（MSC）的丰富来源尚未得到充分认识。本研究旨在建立一种从 HUCB 中分离大量 MSC 的新方法，以将其视为 MSC 的良好来源。从行择期剖宫产的妇女中采集 HUCB 样本。通过将 HUCB 样本植入间充质完全培养基中并在 37°C、5%CO2 和空气孵育箱中孵育，来实施新方法（凝块斑法）。通过对细胞进行定量和定性免疫表型分析，并使用与 MSC 表型标志物（CD29、CD73、CD44 和 CD105）结合的 FITC，来确定 MSC 的存在。将造血抗体（CD34 和 CD45）作为阴性对照。在神经发生和脂肪生成培养基中检查 MSC 分化。通过免疫细胞化学对胚胎标志物：性别决定区 Y 框 2（SOX2）、少突胶质细胞-4（OLIG-4）和脂肪酸结合蛋白-4（FABP-4）进行检测。将新方法与传统的 Rosset Sep 方法进行比较。与传统方法相比，使用凝块斑法的 MSC 培养物增殖率提高了 3 倍。此外，细胞高表达 MSC 标志物 CD29、CD73、CD44 和 CD105，但缺乏特定 HSC 标志物（CD34 和 CD45）的表达。分离的 MSC 通过表达神经发生（SOX2 和 Olig4）和脂肪生成（FABP-4）标志物分别显示出一些分化。总之，使用这项新技术，HUCB 是 MSC 的良好来源。
