DNA barcoding-based paired daughter cell analysis reveals division preferences of hematopoietic stem cells

Fukushima, T.; Nishiyama, A.; Koide, S.; Isobe, T.; Yabushita, T.; Asada, S.; Goyama, S.; Iwama, A.; Yamazaki, S.; Tamura, T.; Kitamura, T.; Suda, T.; Tanaka, Y.

doi:10.64898/2026.07.24.739718

bioRxiv · 10.64898/2026.07.24.739718

DNA barcoding-based paired daughter cell analysis reveals division preferences of hematopoietic stem cells

Fukushima, T.·Nishiyama, A.·Koide, S.·Isobe, T.·Yabushita, T.·Asada, S.·Goyama, S.·Iwama, A.·Yamazaki, S.·Tamura, T.·Kitamura, T.·Suda, T.·Tanaka, Y.

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Abstract

Hematopoietic stem cells(HSCs) maintain their pools by stem-stem division and produce mature blood cells through stem-progenitor or progenitor-progenitor division. A paired daughter cell(PDC) assay combined with single cell transplantation is a powerful method to compare the lineage outputs of two HSC daughter cells. However, single-cell transplantation precludes large-scale analysis of daughter-cell pairs, as only one cell can be transplanted per recipient. Here, we developed a DNA barcoding-based PDC assay to overcome this limitation, enabling simultaneous analysis of 476 daughter pairs from individual HSC divisions and revealing that daughter-cell fates are coordinated and that HSC division patterns are biased toward stem-stem and progenitor-progenitor divisions rather than stem-progenitor divisions. These findings indicate that HSC fate outcomes are directed toward symmetric division outcomes. Integration of single-cell RNA sequencing with DNA barcoding revealed a continuum of HSC states--from balanced HSCs to myeloid-biased HSCs and ultimately to a low-output HSC subset--in which progressively reduced production of mature blood cells relative to stem cell expansion, a proxy for stem-stem division bias, exhibits distinct activities of transcription factors and signaling pathways. Overall, our analysis uncovers characteristic patterns of HSC division and links stem maintenance with distinct molecular features.

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Fukushima, T., Nishiyama, A., Koide, S., Isobe, T., Yabushita, T., Asada, S., Goyama, S., Iwama, A., Yamazaki, S., Tamura, T., Kitamura, T., Suda, T., Tanaka, Y.. 2026-07-24. DNA barcoding-based paired daughter cell analysis reveals division preferences of hematopoietic stem cells. https://doi.org/10.64898/2026.07.24.739718

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