Schweitzer C M, van der Schoot C E, Dräger A M, van der Valk P, Zevenbergen A, Hooibrink B, Westra A H, Langenhuijsen M M
Department of Hematology, Free University Hospital, Amsterdam, The Netherlands.
Exp Hematol. 1995 Jan;23(1):41-8.
Bone marrow endothelial cells are likely to play an important role in the homing of hematopoietic progenitor cells. In view of analyzing the interactions between endothelial cells and hematopoietic progenitor cells, we studied several methods of isolating endothelial cells from human bone marrow, including fluorescence activated cell sorting (FACS) and separation by immunomagnetic beads. FACS sorting gave the best results as contamination with other cells did not occur. After density-gradient centrifugation of bone marrow aspirates, the mononuclear cell (MNC) fraction was depleted for T cells, B cells, and myeloid cells by immunomagnetic separation. Further enrichment of endothelial cells was achieved by FACS sorting using BNH9 or S-Endo1 monoclonal antibodies (MAbs). These MAbs, in contrast to several other endothelial-cell reactive MAbs, were found to react highly specifically with sinus endothelial cells as tested by immunohistochemistry on bone marrow tissue sections and cell culture preparations and by double-colored FACS analysis on bone marrow MNCs (BMMNC). Sorted cells, which formed 0.05% of the MNC fraction, showed strong intracytoplasmic von Willebrand factor positivity. Ultrastructural analysis revealed cells with endothelial characteristics. Cells were cultured in fibronectin-coated, 24-well culture plates in endothelial-cell culture medium or long-term bone marrow culture medium. After 1 to 3 weeks of culture, a monolayer of spindle-shaped cells developed expressing endothelial cell antigens. Cells could be kept in culture for 4 to 6 weeks. In conclusion, the method described provides highly purified preparations of human bone marrow endothelium that may permit in vitro adhesion experiments with normal and leukemic hematopoietic progenitor cells.
骨髓内皮细胞可能在造血祖细胞归巢过程中发挥重要作用。为了分析内皮细胞与造血祖细胞之间的相互作用，我们研究了几种从人骨髓中分离内皮细胞的方法，包括荧光激活细胞分选（FACS）和免疫磁珠分离法。FACS分选效果最佳，因为不会出现其他细胞污染的情况。对骨髓抽吸物进行密度梯度离心后，通过免疫磁珠分离法去除单核细胞（MNC）组分中的T细胞、B细胞和髓细胞。使用BNH9或S-Endo1单克隆抗体（MAb）通过FACS分选进一步富集内皮细胞。与其他几种内皮细胞反应性MAb相比，通过对骨髓组织切片和细胞培养制剂进行免疫组织化学检测以及对骨髓MNC（BMMNC）进行双色FACS分析发现，这些MAb与窦内皮细胞具有高度特异性反应。分选得到的细胞占MNC组分的0.05%，显示出强烈的胞质内血管性血友病因子阳性。超微结构分析显示细胞具有内皮细胞特征。将细胞接种在纤连蛋白包被的24孔培养板中，置于内皮细胞培养基或长期骨髓培养基中培养。培养1至3周后，形成一层表达内皮细胞抗原的纺锤形细胞单层。细胞可在培养中维持4至6周。总之，所描述的方法提供了高度纯化的人骨髓内皮细胞制剂，这可能允许对正常和白血病造血祖细胞进行体外黏附实验。
