CYCLE INITIATION AND COLONY FORMATION IN CULTURE BY MURINE MARROW-CELLS WITH LONG-TERM RECONSTITUTING POTENTIAL IN-VIVO

This investigation was directed at separating long-term reconstituting (LTR) stem cells in normal murine marrow from hematopoietic precursor s detectable in short-term assays in vitro and in vivo, and then at de termining whether purified LTR cells could themselves form colonies in culture. To do so, it was first necessary to identify culture conditi ons that would induce their growth while preserving their long-term re constituting capacity. Marrow was cultured with various cytokines in l iquid suspension for 4 days, after which the surviving LTR activity wa s quantitated in a competitive in vivo assay. Activity was preserved n ear input levels with combined murine c-kit ligand (KL), interleukin-1 (IL-1), IL-6, and IL-11. When the cultures also included tritiated or unlabeled thymidine, LTR potential was eliminated, indicating that es sentially all LTR cells were induced into cell cycle with these cytoki nes. To purify them, marrow was sorted on the basis of Ly6A expression and Rhodamine123 retention. The LyGA(hi)Rh123(lo) fraction contained 85% of total recovered LTR activity but only 1% of the recovered cells measured by multilineage colony formation in spleens or in vitro. Thi s fraction was cultured in methyl cellulose with KL. IL-1, IL-6, and I L-11 for 4 to 6 days, after which colonies were isolated and injected into mice. High levels of permanent reconstitution were achievable in sublethally irradiated W-41/W-41 mice after the injection of a single reconstituting unit, and limiting dilution analysis estimated the freq uency of multilineage LTR at 1 in 11,200 unpurified adult marrow cells . In either lethally irradiated normal or sublethally irradiated W-41/ W-41 mice, 1-year lymphomyeloid reconstitutions were obtained from 1 i n 65 to 84 colonies of 2 to 16 dispersed cells, but not from larger co lonies or those with clumped cells. The results establish that resting marrow LTR cells can be separated from almost all of the more advance d clonegenic cells that are still pluripotential, can be induced to cy cle in culture by defined cytokines with preservation of their reconst ituting potential, and can be manipulated and assayed efficiently at s ingle-cell and colony levels. (C) 1996 by The American Society of Hema tology.