STROMA-CONDITIONED MEDIA IMPROVE EXPANSION OF HUMAN PRIMITIVE HEMATOPOIETIC STEM-CELLS AND PROGENITOR CELLS

It has been reported that stroma-dependent cultures support proliferat ion of hematopoietic stem cells (HSC). In order to investigate the eff ect of soluble stromal factors, we developed short-term serum-low liqu id cultures in which the effect of stroma-conditioned media (SCM) from the murine FBMD-1, and human L87/4 and L88/5 cell lines was studied o n the maintenance and expansion of various human HSC subsets in CD34-p ositive selected mobilized peripheral blood stem cells (PBSC) from aut ologous transplants of lymphoma and multiple myeloma patients. The hum an cobblestone area forming cell (CAFC) assay was employed to determin e the frequencies of both the CAFC weeks 2 to 4 as tentative indicator s of progenitor and transiently repopulating HSC, and the more primiti ve CAFC weeks 6 to 8 as indicators of long-term repopulating HSC. In 7 -day liquid cultures containing interleukin-3 (IL-3), stem cell factor (SCF) and IL-6, we recovered 3.0-fold more colony-forming cells (CFC) and 1.7- to 1.9-fold more CAFC weeks 2 and 4. The absolute number of primitive CAFC weeks 6 and 8 were only maintained (1.1- to 1.4-fold) i n these liquid cultures. This modest expansion was significantly impro ved by the addition of SCM from the FBMD-1, L87/4 or L88/5 cell lines. Output CFC numbers were 6.8-, 5.8- and 9.9-fold higher, respectively, than the input values, while absolute CAFC week 2 to 4 numbers were 4 .5-, 10.2- and 10.2-fold expanded, respectively. The addition of SCM a lso improved expansion of the more primitive CAFC week 6 to 8 stem cel l subsets by 2.2-, 4.5- and 4.9-fold, respectively. The addition of gr anulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CS F (GM-CSF), IL-1 beta, IL-11 or macrophage inflammatory protein-1 alph a to cultures containing IL-3, SCF and IL-6 could not explain the SCM effect and in all these combinations SCM addition further increased th e recovery of HSC subsets. Similarly, addition of anti-cytokine antibo dies (ie alpha-G-CSF, alpha-GM-CSF, alpha-IL-11, alpha-leukemia inhibi tory factor) to liquid cultures containing IL-3, SCF, IL-6 and SCM cou ld not neutralize the SCM effect. These data indicate that SCM signifi cantly enhances expansion of primitive HSC and progenitor cells from C D34-selected PBSC in 7-day cultures and in synergistic combination wit h multiple cytokines at optimal concentrations. As a result, SCM is a useful component of short-term liquid culture procedures for clinical expansion or manipulation of primitive HSC.