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Blood, 1 November 2005, Vol. 106, No. 9, pp. 3271-3284.
Prepublished online as a Blood First Edition Paper on July 14, 2005; DOI 10.1182/blood-2005-01-0150.
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Submitted January 12, 2005
Accepted May 21, 2005
Mitochondrial DNA spectra in single CD34+ cells, T-cells, B-cells and granulocytes
Yoji Ogasawara, Kazutaka Nakayama, Magdalena Tarnowka, J. Philip McCoy, Jr, Sachiko Kajigaya, Barbara C Levin, and Neal S Young*
National Institutes of Health, Hematology Branch, National Heart, Lung and Blood Institute, Bethesda, MD, USA
National Institutes of Health, Flow Cytometry Core Facility, National Heart, Lung and Blood Institute, Bethesda, MD, USA
National Institute of Standards and Technology, Biotechnology Division, Chemical Science and Technology Laboratory, Gaithersburg, MD, USA
* Corresponding author; email: youngns{at}mail.nih.gov.
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 as well as in granulocytes. We established a method for sequence analysis of single cells. We show marked mtDNA heterogeneity distinct from each of five healthy adult donors' corresponding aggregate sequences in isolated cells; 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 due to poly-C tract variability; however, base substitutions were also prevalent: 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 two 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 lineage. 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 utilized as a natural genetic marker to estimate the number of active stem cells.
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