Figure 2. A low mitochondrial activity marks self-renewing HSCs.

(a) Experimental paradigm to assess mitochondrial activity as a discriminator of self-renewing from differentiating LT-HSCs in culture. Freshly isolated LT-HSCs were labelled with CFSE. At the end of the culture, cells that divided one time were further sorted into TMRM^low and TMRM^high phenotypes and transplanted into lethally irradiated recipient mice together with helper cells. Blood reconstitution was assessed at 4, 8 and 16 weeks. (b) CFSE-labelled LT-HSCs were cultured under expansion conditions for 2 days and progeny that underwent one division were sorted into TMRM^low and TMRM^high phenotypes, and 100 cells of each population (along with 2 million helper cells) were transplanted into lethally irradiated recipients. (c) The TMRM^low fraction of the first generation of daughter cells (that is, dividing one time) exhibited strikingly higher long-term multi-lineage blood reconstitution efficiency compared with TMRM^high cells, providing evidence for self-renewing versus differentiating HSC divisions in culture (n=10 for each condition). Assessment of blood chimerism is shown for total blood (top panel) as well as the lymphoid and myeloid lineages (bottom panels; error bar: s.e.m.; t-student, ***P<0.001). (d) BM derived from each of the TMRM^low primary recipients (from c) was injected into four secondary recipient mice after 1 year of the primary transplant. Blood chimerism (average of four secondary recipients corresponding to each primary recipient) show long-term multi-lineage reconstitution in secondary transplants (error bar: s.e.m.).