Human bone marrow m1crovascular endothelial cells induce expansion of CD34t progenitors by direct cellular contact
We have shown that human bone marrow endothelial cells (BMEC) support proliferation and differentiation of cord blood (CB) CD34+ cells for 70 days in the absence of any added exogenous cytokines (Blood, 10/95). Binding of CD34+ cells to BMEC is divalent cation dependent and can be completely reversed with brief treatment of coculturcs with 2mM EOT A (Blood, 7/94). Based on these findings, we have designed an assay system to determine if direct cellular contact between BMEC monolayers and CD34+ cells is critical for expansion of progenitors in long-term cocullures. 8X104CD34+ cells isolated from CB were co-incubated with I06 BMEC cells either in direct cellular contact (Contact) or physically separated by a 0.4 micron microporous transweit plate (noncontact). Every len days the proliferating cells were demidepopulaled and me progenitor content was evaluated by agarose assay and the pbenotype by flow cylometry. After 20 days in coculture there was a 40 fold expansion (3.2X10 cells) of CD45+ bematopoietic cells in contact experiments. 20±3 percent (6.4X105cells)(n3) of the proliferating cells in these contact experiments were directly attached to the BMEC monolayers and were detached by brief treatment with EDTA for further analysis. Flow cytomctric analysis of the CD45+ EDTA released cells showed that 25±5% of the adherent cells (1.6X 105 cells) were CD34-fCD45+, 20±4% CD34+CD33+, and 23±6% were CD34+CD38+. Even though there was a 38 fold expansion of CD45+ cells in transwell experiments no CD34+CD45+ cells were detected. On day 20 of coculture, the total number of progenitors (CFU-GM, CPU-MIX, or BFU-E) per well, generated on the contact cultures were 2.1±0.7X 10 for the nonadherent cells and 2.50±0.25X 101 for the EDTA released cells, and 3.310.5X104 for (he transwell experiments. The number of progenitors and CD34+CD45+ cells generated from the BMEC adherent cells were maintained beyond 70 days, while the nonadherent cells as well as the transwells experiments were depleted of progenitors by day SO of coculture. No progenitors were detected on BMEC monolayers after removal of hematopoietic cells with EDTA, suggesting that EDTA treatment results in complete removal of adherent progenitors. These results suggest that direct cellular contact between CD34+ cells and BMEC monolayers results in expansion of total number of CD34+CD45+ cells, and direct cellular contact is critical for the preservation and possible expansion of progenitors in coculture experiments.