Haematopoietic stem cells do not asymmetrically segregate chromosomes or retain BrdU

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Abstract
In 1975, John Cairns proposed the 'immortal strand' hypothesis as a mechanism by which adult stem cells might minimize accumulation of mutations. This is achieved by selectively retaining chromosomes containing 'old' DNA as shown by the analysis of the DNA strands that incorporate radioactive label or a nucleotide analogue such as bromodeoxyuridine (BrdU). A new contribution to this ongoing debate is published in this issue. Kiel et al. show that the immortal strand model is not a general property of stem cells since it does not apply to haematopoietic stem cells. These cells cannot be identified on the basis of BrdU label retention, and do not retain older DNA during division. The 'immortal strand' hypothesis has been proposed as a mechanism by which adult stem cells minimize accumulation of mutations. This is achieved by selectively retain chromosomes containing 'old' DNA, as shown by the analysis of the DNA strands that incorporated radioactive label or a nucleotide analogue such as bromodeoxyuridine. A new important contribution to this ongoing debate shows that 'the immortal strand' model does not apply to haematopoietic stem cells and BrdU label retention should not be viewed as a fundamental characteristic of stem cells. Stem cells are proposed to segregate chromosomes asymmetrically during self-renewing divisions so that older (‘immortal’) DNA strands are retained in daughter stem cells whereas newly synthesized strands segregate to differentiating cells1,2,3,4,5,6. Stem cells are also proposed to retain DNA labels, such as 5-bromo-2-deoxyuridine (BrdU), either because they segregate chromosomes asymmetrically or because they divide slowly5,7,8,9. However, the purity of stem cells among BrdU-label-retaining cells has not been documented in any tissue, and the ‘immortal strand hypothesis’ has not been tested in a system with definitive stem cell markers. Here we tested these hypotheses in haematopoietic stem cells (HSCs), which can be highly purified using well characterized markers. We administered BrdU to newborn mice, mice treated with cyclophosphamide and granulocyte colony-stimulating factor, and normal adult mice for 4 to 10 days, followed by 70 days without BrdU. In each case, less than 6% of HSCs retained BrdU and less than 0.5% of all BrdU-retaining haematopoietic cells were HSCs, revealing that BrdU has poor specificity and poor sensitivity as an HSC marker. Sequential administration of 5-chloro-2-deoxyuridine and 5-iodo-2-deoxyuridine indicated that all HSCs segregate their chromosomes randomly. Division of individual HSCs in culture revealed no asymmetric segregation of the label. Thus, HSCs cannot be identified on the basis of BrdU-label retention and do not retain older DNA strands during division, indicating that these are not general properties of stem cells.