Mitochondrial DNA spectra of single human CD34+ cells, T cells, B cells, and granulocytes.

Previously, we described the age-dependent accumulation of mitochondrial DNA (mtDNA) mutations, leading to a high degree of mtDNA heterogeneity among normal marrow and blood CD34+ clones and in granulocytes. We established a method for sequence analysis of single cells. We show marked, distinct mtDNA heterogeneity from corresponding aggregate sequences in isolated cells of 5 healthy adult donors-37.9% +/- 3.6% heterogeneity in circulating CD34+ cells, 36.4% +/- 14.1% in T cells, 36.0% +/- 10.7% in B cells, and 47.7% +/- 7.4% in granulocytes. Most heterogeneity was caused by poly-C tract variability; however, base substitutions were also prevalent, as follows: 14.7% +/- 5.7% in CD34+ cells, 15.2% +/- 9.0% in T cells, 15.4% +/- 6.7% in B cells, and 32.3% +/- 2.4% in granulocytes. Many poly-C tract length differences and specific point mutations seen in these same donors but assayed 2 years earlier were still present in the new CD34+ samples. Additionally, specific poly-C tract differences and point mutations were frequently shared among cells of the lymphoid and myeloid lineages. Secular stability and lineage sharing of mtDNA sequence variability suggest that mutations arise in the lymphohematopoietic stem cell compartment and that these changes may be used as a natural genetic marker to estimate the number of active stem cells.

Mitochondrial DNA sequence heterogeneity in circulating normal human CD34 cells and granulocytes.

We have reported marked mitochondrial DNA (mtDNA) sequence heterogeneity among individual CD34 clones from adult bone marrow (BM) and the age-dependent accumulation of mtDNA mutations in this mitotically active tissue. Here, we show direct evidence of clonal expansion of cells containing mtDNA mutations and that the mtDNA sequence may be easily determined by using peripheral blood (PB) as a CD34 cell source. Analysis of 594 circulating CD34 clones showed that 150 (25%) had mtDNA sequences different from the same donor's corresponding aggregate sequence. Examination of single granulocytes indicated that 103 (29%) from the same 6 individuals showed mtDNA heterogeneity, with sequences distinct from the corresponding aggregate tissue sequence and from the sequences of other single granulocytes. Circulating and BM CD34 cells showed virtually identical patterns of mtDNA heterogeneity, and the same changes were seen in progeny granulocytes as in their progenitors, indicating that blood sampling could be used in studies to determine whether mtDNA reflects an individual's cumulative or recent exposure to mutagens; as a marker of individual hematopoietic progenitors, stem cells, and their expansion; and for the detection of minimal residual disease in hematologic malignancies of CD34 cell origin.