MACROPHAGE INFLAMMATORY PROTEIN 1-ALPHA, INTERLEUKIN-3 AND DIFFUSIBLEMARROW STROMAL FACTORS MAINTAIN HUMAN HEMATOPOIETIC STEM-CELLS FOR ATLEAST 8 WEEKS IN-VITRO

Factors that induce proliferation of the human hematopoietic stem cell are ill-defined. Primitive hematopoietic progenitors can be maintaine d and differentiate in stroma-dependent, long-term bone marrow culture s (LTBMC), originally described by Dexter et al. (Dexter, T. M., L. H. Coutinho, E. Spooncer, C. M. Heyworth, C. P. Daniel, R. Schiro, J. Ch ang, and T. D. Alien. 1990. Molecular Control of Haemopoiesis). Howeve r, 70-80% of primitive progenitors capable of reinitiating secondary s tromal cultures (LTBMC-initiating cells [ICI]) are lost over a period of 5 wk in such cultures. We have recently described a novel ''stroma- noncontact'' culture system, in which hematopoietic progenitors are se parated from the stromal layer by a 0.4-mu m microporous filter membra ne. Primitive progenitors in such cultures can not only differentiate into committed progenitors, but are also maintained to a greater exten t than in ''Dexter'' cultures. However, still only 50% of the original ly seeded LTBMC-IC are recovered at week 5. Since maintenance of primi tive progenitors may depend not only on growth-promoting factors but a lso on factors that inhibit differentiation and/or proliferation, we e valuated the effect of macrophage inflammatory protein 1 alpha (MIP-1 alpha) or ''stem cell inhibitor''' in combination with the growth-indu cing factor interleukin 3 (IL-3) on the recovery of LTBMC-IC from stro ma-noncontact cultures. We demonstrate that addition of MIP-1 alpha al one to stroma-noncontact cultures does not change the number of LTBMC- IC present after 8 wk, indicating that this factor may not directly in hibit or stimulate proliferation of primitive progenitors. Addition of the growth stimulatory cytokine, IL-3, alone results in exhaustion of LTBMC-IC after 8 wk of culture, possibly as a result of their termina l differentiation. However, LTBMC-IC can be maintained for at least 8 wk when grown in stroma-noncontact cultures supplemented with both MIP -1 alpha plus IL-3. This effect depends on soluble (ill-defined) strom al factors, and results from a direct interaction of these cytokines w ith the progenitor population or its progeny, but not the stroma.