Quantitative assessment of the stem cell self-renewal capacity.

: Little is known about the manner in which hematopoietic stem cells (HSCs) self-renew. To address this issue, we used a serum-free single-cell culture, followed by transplantation of cultured cells into lethally irradiated mice. CD34-negative or low, c-Kit-positive, Sca-1-positive, lineage marker-negative (CD34−KSL) cells are highly enriched for murine bone marrow HSCs. Successful long-term reconstitution with a single CD34−KSL cell enabled us to study in vitro self-renewal of HSC at clonal level. Using this clonal cell transplantation system, we examined the effect of various cytokines on CD34−KSL cells. Among the cytokines examined, stem cell factor (SCF) and thrombopoietin (TPO) were minimum cytokines to induce cell division of CD34−KSL cells most efficiently. Similarly, multilineage repopulating activity was detected in the cells derived from a significant portion of single cells after culture in the presence of TPO and SCF. However, SCF + IL-3, SCF + IL-6, or SCF + IL-11 + FL appeared to be less effective for self-renewal of HSCs. The activity of HSCs as indicated by repopulation unit (RU) remaining after culture with SCF and TPO was not so different from that of freshly isolated HSCs. However, there was a substantial loss of HSC number in these cultured cells. Taken together, this study provides definitive proof that one HSC can generate at least one HSC in vitro.