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Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding

Abstract

Disentangling cellular heterogeneity is a challenge in many fields, particularly in the stem cell and cancer biology fields. Here we demonstrate how to combine viral genetic barcoding with high-throughput sequencing to track single cells in a heterogeneous population. We use this technique to track the in vivo differentiation of unitary hematopoietic stem cells (HSCs). The results are consistent with single-cell transplantation studies but require two orders of magnitude fewer mice. In addition to its high throughput, the high sensitivity of the technique allows for a direct examination of the clonality of sparse cell populations such as HSCs. We show how these capabilities offer a clonal perspective of the HSC differentiation process. In particular, our data suggest that HSCs do not equally contribute to blood cells after irradiation-mediated transplantation, and that two distinct HSC differentiation patterns co-exist in the same recipient mouse after irradiation. This technique can be applied to any virus-accessible cell type for both in vitro and in vivo processes.

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Figure 1: Experimental workflow.
Figure 2: DNA barcode library and delivery.
Figure 3: Background noise sequences.
Figure 4: Lineage bias of HSC differentiation after irradiation.
Figure 5: Clonal correlations of hematopoietic populations.

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Acknowledgements

We thank G. Mantalas, T. Snyder and B. Passarelli for helping with the high-throughput sequencing; K. Schepers, I. Dimov, J. Seita, A. Czechowicz, M. Inlay M. Drukker and D. Sahoo for helpful discussions; P. Lovelace for FACS core management. We also thank L. Jerabek and T. Storm for laboratory management; C. Muscat and T. Naik for antibody conjugation; A. Mosley for animal supervision. This work is supported by NIH-R01-CA86065 and NIH-U01-HL099999. R.L. is supported by CIRM-TG2-01159.

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R.L. and I.L.W. designed the experiments. R.L. performed the experiments. N.F.N. and S.R.Q. set up and carried out the high-throughput sequencing. R.L. analyzed the data and wrote the manuscript. All authors edited the manuscript.

Corresponding authors

Correspondence to Rong Lu or Irving L Weissman.

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The authors declare no competing financial interests.

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Lu, R., Neff, N., Quake, S. et al. Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding. Nat Biotechnol 29, 928–933 (2011). https://doi.org/10.1038/nbt.1977

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