Haematopoietic stem cells do not asymmetrically segregate chromosomes or retain BrdU

Abstract

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 cells. 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 slowly. 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.

Figure 1. Contrasting predictions regarding stem cell labelling on the basis of the immortal strand model versus random chromosome segregation

a, According to the immortal strand model, stem cells divide asymmetrically under steady-state conditions and BrdU is incorporated into newly synthesized DNA strands that are asymmetrically segregated into differentiating daughter cells with each round of division, such that stem cells retain only the unlabelled older DNA strands. b, In contrast, if chromosomes segregate randomly, then BrdU-labelled chromosomes will be stochastically lost over multiple rounds of divisions. c, In the immortal strand model, if stem cells divide symmetrically then BrdU can be incorporated into DNA strands that become the ‘older’ strands once stem cells resume asymmetric division. Under these circumstances, the BrdU⁺ older strands would be retained indefinitely in stem cells. d, In contrast, if chromosome segregation is random then BrdU⁺ chromosomes are stochastically lost over time after BrdU is discontinued.

Figure 2. Six per cent of HSCs stochastically enter the cell cycle each day

a, HSCs can be isolated by flow cytometry as CD150⁺CD48⁻CD41⁻lineage⁻Sca-1⁺c-kit⁺ cells; these represent only 0.0066 ± 0.0003% (0.007% × 94%) of bone marrow cells but contain all detectable HSC activity and are very highly enriched for HSCs (nearly 50% of single cells give long-term multilineage reconstitution in irradiated mice17). b, BrdU incorporation into HSCs (arrowheads) is evaluated by immunofluorescence after sorting HSCs onto microscope slides (DAPI is a nuclear stain). c, d, The percentage of BrdU⁺ bone marrow cells (c) and CD150⁺CD48⁻CD41⁻lineage⁻Sca-1⁺c-kit⁺ HSCs (d) after various periods of BrdU administration (3–4 independent experiments with 3–4 mice per experiment and 200–400 bone marrow cells or 100–400 HSCs counted, respectively, per mouse). Standard deviations are shown for means that are based on at least three independent experiments. e, The percentage of HSCs that enter the cell cycle each day (6%) can be derived by plotting the negative logarithm of the percentage of HSCs that were BrdU⁻over time.

Figure 3. Few HSCs retain BrdU, and most BrdU-retaining bone marrow cells are not HSCs

a, A model is shown that predicts the fraction of HSCs that retain BrdU over time after administering BrdU for 10 days, depending on whether chromosomes segregate asymmetrically or randomly and on the threshold of BrdU that can be detected by immunofluorescence (0.5n, 0.25n, 0.125n or 0.0625n labelled DNA). b, CD150⁺CD48⁻CD41⁻lineage⁻Sca-1⁺ c-kit⁺ HSCs were sorted onto a microscope slide after 10 days BrdU administration and 70 days chase (without BrdU). Arrowheads identify BrdU⁺ cells. c, Shown is the frequency of BrdU⁺ bone marrow cells and HSCs after 10 days of BrdU administration and 40, 70 or 120 days of chase. Standard deviations are shown for means that are based on at least three independent experiments. d, Shown is the frequency of BrdU⁺ bone marrow cells and HSCs after 10 days BrdU administration to neonatal mice followed by 70 or 120 days of chase, or after 4 days BrdU administration to cyclophosphamide/G-CSF-mobilized mice followed by 70 days of chase. All data are based on 3–5 independent experiments with 3 mice per experiment and 400–700 bone marrow cells or 300–400 HSCs counted per mouse.

Figure 4. HSCs segregate chromosomes randomly in vivo and in vitro

a, By the immortal strand model, stem cells sequentially exposed to CldU (for 10 days) and then IdU (for 10 days) would not incorporate both labels, with the exception of rare cells in the S/G2/M phase of their first division after switching from CldU into IdU (expected frequency <3%). b, In contrast, if chromosome segregation is random then CldU⁺ stem cells would have the same probability of incorporating IdU as unlabelled cells (expected frequency of CldU⁺IdU⁺ HSCs is ~25%). c, CldU was administered to mice for 10 days followed by IdU for 10–11 days, and CD150⁺CD48⁻CD41⁻lineage⁻Sca-1⁺ c-kit⁺ HSCs were stained. Examples of HSCs that incorporated neither label, only CldU, only IdU or both labels are shown (mean ± sd; data are based on two independent experiments with 2 or 3 mice per experiment and 100–250HSCs per mouse).HSCs fromBrdU-treated mice divided once (d)or twice (e) in culture to form daughter cells. f, The progeny of these HSCs were either all BrdU⁺ or all BrdU⁻. We detected no clones in which label was asymmetrically segregated to a subset of daughter cells.