Apparent mtDNA sequence heterogeneity in single human blood CD34+ cells is markedly affected by storage and transport

• mtDNA sequence heterogeneity in HSCs is markedly affected by storage and transport.
• CD34+ cells that survive stress ex vivo may be enriched in quiescent primitive HSCs.
• Primitive HSCs have fewer mtDNA mutations than committed progenitors.

Single CD34+ cells from adult human peripheral blood show mtDNA sequence heterogeneity. In this study, we compared mtDNA sequence variation in single CD34+ cells from peripheral blood (PB) mononuclear cells (MNCs) from the same donors but under different conditions of storage and transport: group I, MNCs from heparinized PB that inadvertently required six days to be transported to the testing laboratory; group II, MNCs which were isolated from PB within a day of phlebotomy and frozen prior to transportation and storage. We observed more cell death for MNCs of group I than group II. Concordantly, group I CD34+ cells had a very low potential for hematopoietic colony formation in vitro compared with group II cells. CD34+ cells of group II showed an unexpectedly higher level of mtDNA sequence heterogeneity than was present in group I cells. These observations suggest that reduced mtDNA sequence heterogeneity in single CD34+ cells of group I was likely due to elimination of cells harboring mutations. CD34+ cells that survive stress ex vivo may be more enriched in quiescent primitive hematopoietic stem cells, with fewer mtDNA mutations than are present in committed progenitors. Technically, attention is required to conditions of preparation of human blood samples for single cell mtDNA analysis.