Deschaseaux F, Remy-Martin J P, Keating A, Hervé P, Charbord P
a Laboratory for the Study of Hematopoiesis, Transfusion Center , Besançon , France.
c Institut d'Etude et de Transfert de Gènes , Besançon , France.
Hematology. 1998;3(5):401-17. doi: 10.1080/10245332.1998.11746415.
Adhesion of hematopoietic precursors to the marrow microenvironment appears to be a prerequisite for proliferation and differentiation of hematopoietic cells. In this report, we have studied the adhesion of CFU-GM from marrow CD34+ precursors to human marrow myofibroblasts and to an human stromal cell line, L2Ori-, transformed by a vector comprising the whole of the SV40 viral sequence except for the origin of replication. This Stro-l(+) cell line presents characteristics similar to those of vascular smooth muscle cells, since (i) few cells were α-SM actin(+)while all cells were vimentin(+) but desmine(-) and a metavinculin band was consistently detected, (ii) all cells contained lysosomes filled with glycoproteins recognized by the monoclonal antibody 1B10, (iii) we detected EDa(+) EDb(-) pericellular fibronectin and intracellular β1 and β laminins and (iv) the cytokine expression pattern was similar to that of cells from colony-derived cell lines. Transformation was confirmed by abnormal and irregular growth (hallmarked by crises with rather slow growth between crises), and the presence of some very large cells with several nuclei. Although presenting an usual stromal phenotype, this cell line could not sustain hematopoiesis from marrow CD34+ cells in coculture due to a complete inability of adhesion of CD34+ cells (0% of adherent CFU-GM vs. 20% on normal stromal myofibroblasts). The lack of adhesion was explained by abnormal expression of adhesion molecules and molecules involved in the organization of extracellular matrix: (1) at the membrane level: the lack of VCAM-1 and significant differences in the distribution of CD44 and integrins α1, α3, α4 and β as compared to normal stroma; (2) at the level of focal adhesions: the predominance of the 200 kD fragment of talin (as opposed to that of 230 kD in normal stroma), and a significantly decreased expression of vinculin and α-actinin; (3) at the level of microfilaments: the decrease in polymerized actin and a large decrease of α-SM actin synthesis; and (4) at the level of extracellular matrix: very few fibronectin fibres. These data show that transformation can strongly and negatively affect the function of hematopoiesis maintenance by disrupting intercellular and extracellular matrix adhesion mechanisms of hematopoietic cells to the stroma, in particular by affecting the fibronexus. Our data suggest the need for extreme caution when using SV40 transformed cell lines and instead, make the case for the use of other means of immortalization (such as thermosensitive T, other transforming sequences, introduction of inducible promoters).
造血前体细胞与骨髓微环境的黏附似乎是造血细胞增殖和分化的前提条件。在本报告中，我们研究了骨髓CD34+前体细胞来源的CFU-GM与人骨髓肌成纤维细胞以及一种人基质细胞系L2Ori-的黏附情况。L2Ori-细胞系由一个包含除复制起点外的整个SV40病毒序列的载体转化而来。这种Stro-1(+)细胞系具有与血管平滑肌细胞相似的特征，因为：(i) 少数细胞α-SM肌动蛋白阳性，而所有细胞波形蛋白阳性但结蛋白阴性，且始终检测到一条间皮肌动蛋白带；(ii) 所有细胞都含有充满被单克隆抗体1B10识别的糖蛋白的溶酶体；(iii) 我们检测到细胞外EDA(+) EDB(-)纤连蛋白以及细胞内β1和β层粘连蛋白；(iv) 细胞因子表达模式与集落衍生细胞系的细胞相似。转化通过异常和不规则生长（以生长危机为特征，危机之间生长相当缓慢）以及存在一些具有多个细胞核的非常大的细胞得以证实。尽管呈现出常见的基质表型，但由于CD34+细胞完全无法黏附（黏附的CFU-GM为0%，而在正常基质肌成纤维细胞上为20%），该细胞系在共培养中无法维持骨髓CD34+细胞的造血。黏附缺失是由黏附分子和参与细胞外基质组织的分子的异常表达所解释的：(1) 在膜水平：缺乏VCAM-1，并且与正常基质相比，CD44以及整合素α1、α3、α4和β的分布存在显著差异；(2) 在黏着斑水平：以200 kD的踝蛋白片段为主（与正常基质中的230 kD片段相反），并且纽蛋白和α-辅肌动蛋白的表达显著降低；(3) 在微丝水平：聚合肌动蛋白减少，α-SM肌动蛋白合成大幅下降；(4) 在细胞外基质水平：纤连蛋白纤维极少。这些数据表明，转化可通过破坏造血细胞与基质之间的细胞间和细胞外基质黏附机制，特别是通过影响纤维连接桥，对造血维持功能产生强烈的负面影响。我们的数据表明，在使用SV40转化细胞系时需要格外谨慎，相反，支持使用其他永生化方法（如温度敏感T、其他转化序列、引入诱导型启动子）。
