SELF-RENEWAL OF PRIMITIVE HUMAN HEMATOPOIETIC-CELLS (LONG-TERM-CULTURE-INITIATING CELLS) IN-VITRO AND THEIR EXPANSION IN DEFINED MEDIUM

A major goal of experimental and clinical hematology is the identifica tion of mechanisms and conditions that support the expansion of transp lantable hematopoietic stem cells, In normal marrow, such cells appear to be identical to (or represent a subset of) a population referred t o as long-term-culture-initiating cells (LTC-ICs) so-named because of their ability to produce colony-forming cell (CFC) progeny for greater than or equal to 5 weeks when cocultured with stromal fibroblasts, So me expansion of LTC-ICs in vitro has recently been described, but iden tification of the factors required and whether LTC-IC self-renewal div isions are involved have remained unresolved issues, To address these issues, we examined the maintenance and/or generation of LTC-ICs from single CD34(+) CD38(-) cells cultured for variable periods under diffe rent culture conditions, Analysis of the progeny obtained from culture s containing a feeder layer of murine fibroblasts engineered to produc e steel factor, interleukin (IL)-3, and granulocyte colony-stimulating factor showed that approximate to 20% of the input LTC-ICs (represent ing approximate to 2% of the original CD34(+) CD38(-) cells) executed self-renewal divisions within a 6-week period, Incubation of the same CD34(+) CD38(-) starting populations as single cells in a defined (ser um free) liquid medium supplemented with Fit-3 ligand,steel factor, IL -3, IL-6, granulocyte colony-stimulating factor, and nerve growth fact or resulted in the proliferation of initial cells to produce clones of from 4 to 1000 cells within 10 days, approximate to 40% of which incl uded greater than or equal to 1 LTC-IC, In contrast, in similar cultur es containing methylcellulose, input LTC-ICs appeared to persist but n ot divide, Overall the LTC-IC expansion in the liquid cultures was 30- fold in the first 10 days and 50-fold by the end of another 13 weeks, Documentation of human LTC-IC self-renewal in vitro and identification of defined conditions that permit their extensive and rapid amplifica tion should facilitate analysis of the molecular mechanisms underlying these processes and their exploitation for a variety of therapeutic a pplications.