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Counting stem cells: methodological constraints

Nature Methods volume 9, pages 567–574 (2012)Cite this article

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Abstract

The number of stem cells contributing to hematopoiesis has been a matter of debate. Many studies use retroviral tagging of stem cells to measure clonal contribution. Here we argue that methodological factors can impact such clonal analyses. Whereas early studies had low resolution, leading to underestimation, recent methods may result in an overestimation of stem-cell counts. We discuss how restriction enzyme choice, PCR bias, high-throughput sequencing depth and tagging method could affect the conclusions of clonal studies.

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Figure 1: Models of clonal kinetics in hematopoiesis.
Figure 2: Reports of number of viral integration sites or hematopoietic clonality in multiple studies.
Figure 3: Strategies for identifying retroviral tags for clonal analysis.
Figure 4: Predicted PCR amplification efficiency of genomic fragments digested with different enzymes.
Figure 5: Combinatorial strategy to select optimal combination of restriction enzymes.
Figure 6: Clonal analysis in barcoded cells.
Figure 7: Clonal kinetics in mice transplanted with barcoded cells.

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Acknowledgements

We thank B. Dykstra for critical reading of the manuscript, and M. Ritsema, H. Moes, G. Mesander and R.-J. van der Lei for technical assistance. This study was supported by the Netherlands Organization for Scientific Research (VICI grant to G.d.H. and TopTalent grant to E.V.), and the Netherlands Genomics Initiative (Horizon Grant 050-71-055 to G.d.H. and the EU FP7 grant EuroSystem contract 200720).

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  1. Laboratory of Stem Cell Research, European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands

    Leonid V Bystrykh, Evgenia Verovskaya, Erik Zwart, Mathilde Broekhuis & Gerald de Haan

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  1. Leonid V Bystrykh
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  2. Evgenia Verovskaya
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Contributions

L.V.B. conceptualized the paper; L.V.B., M.B. and E.V. performed experiments; L.V.B., E.V. and E.Z. analyzed the data; L.V.B., E.V. and G.d.H. wrote the paper; and G.d.H. provided funding.

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Correspondence to Leonid V Bystrykh or Gerald de Haan.

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The authors declare no competing financial interests.

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Supplementary Tables 1,3 (PDF 326 kb)

Supplementary Table 2

Excel script for combinatorial restriction enzyme selection. (XLSX 18 kb)

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Bystrykh, L., Verovskaya, E., Zwart, E. et al. Counting stem cells: methodological constraints. Nat Methods 9, 567–574 (2012). https://doi.org/10.1038/nmeth.2043

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