A novel single-cell proliferation assay shows that long-term culture- initiating cell (LTC-IC) maintenance over time results from the extensive proliferation of a small fraction of LTC-IC

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-1α (MIP-1α), 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 expanded 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-1α. 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-1α. 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-1α. 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-1α can induce proliferation of LTC-IC, failure to conserve a large fraction of LTC-IC results in lack of long-term expansion. Insight into the fate of individual LTC-IC should now allow us to design culture systems that increase not only proliferation but also conservation of LTC-IC, ultimately leading to long-term ex vivo stem cell expansion.