Adhesion of Hematopoietic Precursors to Human Stroma: Studies Using Normal Marrow Stromal Myofibroblasts and a Stromal Cell Line Transformed by SV40

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).