DOES TRANSMEMBRANE COMMUNICATION THROUGH GAP-JUNCTIONS ENABLE STEM-CELLS TO OVERCOME STROMAL INHIBITION

When long-term bone marrow cultures are treated with Amphotericin B (A B) their haemopoietic stem cells (HSC) cease growing. This is not a to xic effect of the drug because once that is removed, HSC resume clonal growth and, given sufficient time, form as many cells as HSC in untre ated cultures. Amphotericin B-evoked inhibition of blood formation is probably mediated by transmembrane communication between HSC and strom a for the following reasons: (1) AB does not stop HSC forming colony-f orming units in culture (CFU-c) when HSC are separated from stroma by culturing them on Transwell inserts above the stroma. (2) Conditioned media (CM) from AB-containing or normal long-term cultures (LTC) does not inhibit normal marrow cells forming colonies in semi-solid culture s without stromal underlays. (3) AB itself does not stop bone marrow c ells forming colonies in semi-solid cultures nor does it stop stromal cells growing or prejudice their long-term maintenance. (4) Furthermor e, growing stromal cells with AB does not alter the number of transcri pts they form for cytokines and chemokines to any large extent, includ ing TGF-beta 1. We have extensive, though circumstantial, evidence tha t gap junctions are involved in this communication. AB only stopped th e growth of HSC when we blocked intercellular communication via gap ju nctions (GJIC) (tested by micro-injection of lucifer yellow). Lipophil ic compounds that do not affect GJIC had no effect on the growth of HS C. Looking at a series of stromal cell lines from foetal liver and neo natal bone marrow we found that extensive GJIC correlated with stromal support of the late-appearing clones formed by primitive HSC (week 35 cobblestone-area forming cells, CAFC). We propose that the proliferat ion of HSC is regulated via transmembrane communication between stroma l and HSC. Our findings support the proposal that gap junctions pray a part in this stromal-dependent regulation.