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Polylox barcoding reveals haematopoietic stem cell fates realized in vivo
Open accessAbstract
Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites, viral barcodes, and strategies based on transposons and CRISPR-Cas9 genome editing; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system. Polylox recombination in situ reaches a practical diversity of several hundred thousand barcodes, allowing tagging of single cells. We have used this experimental system, combined with fate mapping, to assess haematopoietic stem cell (HSC) fates in vivo. Classical models of haematopoietic lineage specification assume a tree with few major branches. More recently, driven in part by the development of more efficient single-cell assays and improved transplantation efficiencies, different models have been proposed, in which unilineage priming may occur in mice and humans at the level of HSCs. We have introduced barcodes into HSC progenitors in embryonic mice, and found that the adult HSC compartment is a mosaic of embryo-derived HSC clones, some of which are unexpectedly large. Most HSC clones gave rise to multilineage or oligolineage fates, arguing against unilineage priming, and suggesting coherent usage of the potential of cells in a clone. The spreading of barcodes, both after induction in embryos and in adult mice, revealed a basic split between common myeloid-erythroid development and common lymphocyte development, supporting the long-held but contested view of a tree-like haematopoietic structure.
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TL;DR
The authors developed Polylox, an artificial Cre-loxP recombination cassette that enables endogenous DNA barcoding with a practical diversity of several hundred thousand barcodes in situ. Using Polylox to fate-map haematopoietic stem cells in mice, they found that adult HSC clones are often multilineage or oligolineage and that haematopoiesis follows a bifurcating myelo-erythroid versus common lymphoid structure.
Key findings
- Polylox recombination can generate 1,866,868 distinct codes in silico, with all codes reachable by 10 recombination events; experimentally, 52% of reads from 30,000 splenic B cells were recombined and 849 different barcodes were identified.
- Adult bone marrow HSC were a mosaic of embryo-derived clones: >95% of adult HSC had recombined barcodes after E9.5 tamoxifen induction, and 14 high-confidence single-progenitor barcodes gave adult clone sizes from 0.2% to 3.8%, including clones above 1.5%.
- Most HSC clones gave multilineage or oligolineage fates, arguing against unilineage priming; among 117 peripheral barcodes, 54 were represented in all assayed lineages and 44 in several lineages.
- Barcode concordance revealed a basic split between common myeloid-erythroid development and common lymphocyte development, with B1a as a distinct lineage within the lymphoid branch; CMP barcodes were found in both EryP and granulocytes with highly correlated frequencies, but about 1/3 of EryP and granulocyte barcodes were undetected in CMP.
Methods
- Polylox consists of ten loxP sites in alternating orientations spaced 178 bp apart, flanking nine unique DNA segments derived from the Arabidopsis thaliana AT2G21770 gene; it was targeted into the Rosa26 locus in ES cells and mice.
- In vivo barcoding used Rosa26Polylox/CreERT2 or Rosa26PolyloxTie2MCM mice, with tamoxifen induction in embryos at E9.5 or in adult mice; sorted cell populations were analyzed by PCR and single molecule real time (SMRT) sequencing across the Polylox locus.
- Barcodes were decoded by aligning the nine forward and reverse-complement segments to circular consensus reads; barcode generation probabilities (Pgen) were computed with a Markov chain model over all possible excisions and inversions.
Limitations
- The paper states that barcodes may be missed in samples, so the range of fates acquired by particular clones may sometimes be underestimated; it also states that it cannot formally conclude that individual cells within a clone underwent multilineage differentiation.
- The paper notes that the quantitative output of adult HSC is controversial, that adult sampling repeats showed lower overlap indicating overall smaller clone sizes, and that paucity of barcodes in CLP precluded robust correlation analysis.
- The Methods state that no randomization was done, no blinding was done, no animals were excluded, and no statistical methods were used to predetermine sample size.
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“The spreading of barcodes, both after induction in embryos and in adult mice, revealed a basic split between common myeloid-erythroid development and common lymphocyte development, supporting the long-held but contested view of a tree-like haematopoietic structure.”
§ Polylox barcoding reveals haematopoietic stem cell fates realized in vivo > Abstract
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“We then focused on barcodes induced with high confidence in single embryonic HSC progenitors (Pgen < 10−4), obtaining 14 such barcodes (Fig. 4b).”
§ Polylox barcoding reveals haematopoietic stem cell fates realized in vivo > Abstract
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In the paper
“The dichotomy of myelo-erythroid and lymphoid branches was not disturbed when all barcodes were analyzed, hence it remained evident even in the presence of non-clonal barcode ‘background’ (Extended Data Fig. 7e).”
§ Polylox barcoding reveals haematopoietic stem cell fates realized in vivo > Abstract
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“Polylox recombination in situ reaches a practical diversity of several hundred thousand barcodes, allowing tagging of single cells.”
§ Polylox barcoding reveals haematopoietic stem cell fates realized in vivo > Abstract
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“we induced Polylox recombination in a Rosa26Polylox/CreERT2 mouse, and 18 hours later analyzed the acutely generated barcodes in 30,000 splenic B cells. 52% of the recovered reads were recombined, indicating recombination in about half of the cells.”
§ Polylox barcoding reveals haematopoietic stem cell fates realized in vivo > Abstract
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In the citing paper
“In contrast to the transplantation setting, native haematopoiesis has been described as polyclonal32,33, whereby several thousand clones contribute to blood formation.”
From Clonal tracing with somatic epimutations reveals dynamics of blood ageing (2025) · cites this paper as [32]
Citations
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Distinct routes of lineage development reshape the human blood hierarchy across ontogeny
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Fundamental properties of unperturbed haematopoiesis from stem cells in vivo
Busch, K., Klapproth, K., Barile, M., Flossdorf, M., Holland-Letz, T., Schlenner, S. M., Reth, M., Höfer, T., & Rodewald, H.-R. (2015) · Nature · doi:10.1038/nature14242 · Cited by 769
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Metadata
- Authors
- Pei, W., Feyerabend, T. B., Rössler, J., Wang, X., Postrach, D., Busch, K., Rode, I., Klapproth, K., Dietlein, N., Quedenau, C., Chen, W., Sauer, S., Wolf, S., Höfer, T., & Rodewald, H.-R.
- Affiliations
- German Cancer Research Center (DE)
- Max Delbrück Center (DE)
- Corresponding author
- Höfer, Thomas
- Rodewald, Hans-Reimer
- ORCID
- Venue
- Nature
- Published
- 2017-08-16
- Volume
- 548
- Issue
- 7668
- Pages
- 456-460
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- PMC
- PMC5905670
- OpenAlex
- W2747263575
- Semantic Scholar
- 34620783d9c8f0e9f5a2eba79e0a3d5e90a074c4
- Cited by
- 443
- References
- 34
- Citations by year
- 2026: 43 · 2025: 47 · 2024: 49 · 2023: 41 · 2022: 41 · 2021: 45 · 2020: 68 · 2019: 47 · 2018: 54 · 2017: 8
- FWCI
- 37.59
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- MDC Repository (Max-Delbrueck-Center for Molecular Medicine) (submittedVersion)
- Topics
- Health Sciences › Medicine › Hematology › Hematopoietic Stem Cell Transplantation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
- Keywords
- hematopoietic stem cells, Cre-loxP recombination, hematopoiesis
- MeSH terms
- Animals, Attachment Sites, Microbiological, Clone Cells, Embryo, Mammalian, Female, Hematopoietic Stem Cells, Lymphocytes, Male, Mosaicism, Recombination, Genetic, Cell Lineage, Integrases, Myeloid Cells, Erythroid Cells, Mice, DNA Barcoding, Taxonomic, Cell Tracking, Single-Cell Analysis
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- Pei, W., Feyerabend, T. B., Rössler, J., Wang, X., Postrach, D., Busch, K., Rode, I., Klapproth, K., Dietlein, N., Quedenau, C., Chen, W., Sauer, S., Wolf, S., Höfer, T., & Rodewald, H.-R. (2017). Polylox barcoding reveals haematopoietic stem cell fates realized in vivo. Nature. https://doi.org/10.1038/nature23653
