We have previously shown that when adult marrow CD34+/HLA-DR- cells are cultured for 5 or 8 weeks in the presence of stroma-conditioned media with interleukin-3 (IL-3) and macrophage inflammatory protein-1 alpha (MIP-1 alpha), long-term culture-initiating cells (LTC-IC) are maintained but not expanded. However, if the same cultures are evaluated after 2 weeks, we show that LTC-IC expand 5.5- +/- 0.2-fold. Because expansion of LTC-IC is likely the result of a balance between proliferation and loss of LTC-IC, we hypothesized that, although LTC-IC proliferate in these cultures, loss of a fraction of LTC-IC underlies the lack of long-term expansion. To evaluate the fate of LTC-IC (proliferation, conservation, or loss), we performed PKH-26 labeling assays and developed a single LTC-IC proliferation assay. For PKH-26 labeling assays, CD34+/HLA-DR- cells were incubated with the membrane intercallating dye, PKH-26, before culture for 14 days in stroma-noncontact cultures + IL-3 + MIP-1 alpha. Progeny was reselected by fluorescence-activated cell sorting based on their PKH-26 fluorescence intensity. These studies showed that LTC-IC proliferate because 80% of LTC-IC at week 2 had 0.5 to 1 log lower fluorescence intensity than did freshly labeled CD34+/HLA-DR- cells. To further determine the fate of LTC-IC, we also developed a single LTC-IC proliferation assay. A population of CD34+/CD33- cells, highly enriched in LTC-IC, was sorted singly in stroma-conditioned media+IL-3 + MIP-1 alpha. After 5 weeks, the content of each well was divided equally over 8 secondary stroma-containing wells and cultured for 8 weeks to determine the capacity of the single-cell progeny to initiate 1 or more secondary stromal cultures. Progeny of single-sorted cells were able to initiate up to 8 secondary long-term cultures, demonstrating that LTC-IC proliferate in stroma-conditioned media+IL-3 + MIP-1 alpha. However, more than 65% of single-sorted LTC-IC were not conserved because their progeny could no longer initiate secondary long-term cultures. This finding indicates that, although stromal factors and IL-3 + MIP-1 alpha can induce proliferation of LTC-IC, failure to conserve a large fraction of LTC-IC results in lack of long-term expansion.(ABSTRACT TRUNCATED AT 400 WORDS)