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Long-term ex vivo expansion of mouse hematopoietic stem cells


Utilizing multipotent and self-renewing capabilities, hematopoietic stem cells (HSCs) can maintain hematopoiesis throughout life. However, the mechanism behind such remarkable abilities remains undiscovered, at least in part because of the paucity of HSCs and the modest ex vivo expansion of HSCs in media that contain poorly defined albumin supplements such as bovine serum albumin. Here, we describe a simple platform for the expansion of functional mouse HSCs ex vivo for >1 month under fully defined albumin-free conditions. The culture system affords 236- to 899-fold expansion over the course of a month and is also amenable to clonal analysis of HSC heterogeneity. The large numbers of expanded HSCs enable HSC transplantation into nonconditioned recipients, which is otherwise not routinely feasible because of the large numbers of HSCs required. This protocol therefore provides a powerful approach with which to interrogate HSC self-renewal and lineage commitment and, more broadly, to study and characterize the hematopoietic and immune systems.

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Fig. 1: Schematic overview of the HSC expansion culture protocol.
Fig. 2: Representative gating scheme for FACS purification of bone marrow HSCs.
Fig. 3: Representative images of mouse HSC cultures.
Fig. 4: Representative gating scheme for flow cytometric analysis of HSC cultures.
Fig. 5: Example flow cytometric analysis of HSC cultures.
Fig. 6: Representative gating scheme for flow cytometric peripheral blood analysis.
Fig. 7: Representative gating scheme for flow cytometric bone marrow analysis.

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All raw data are available upon reasonable request.


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We thank the IMSUT FACS Core Laboratory for FACS support and M. Odai for filming. This research was funded by a JSPS KAKENHI Grant-in-Aid for Scientific Research (JP18H05095; JP17H05086), the Japan Agency for Medical Research and Development (JP18bm0404025), CIRM (LA1_C12-06917; DISC1-10555), the NIH (R01DK116944; R01HL147124; R21AG061487) and the Ludwig Foundation. A.C.W. was funded by Bloodwise (15050), the Leukemia and Lymphoma Society (3385-19), and the JSPS.

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Authors and Affiliations



A.C.W. designed and validated the protocol, and wrote and edited the manuscript. R.I. designed and validated the protocol, and edited the manuscript. H.N. supervised the research and edited the manuscript. S.Y. designed and validated the protocol, supervised the research, and edited the manuscript.

Corresponding authors

Correspondence to Hiromitsu Nakauchi or Satoshi Yamazaki.

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Competing interests

H.N. is a co-founder and shareholder of ReproCELL. Inc.

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Peer review information Nature Protocols thanks Nina Cabezas-Wallscheid, James Che and David Kent for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Wilkinson, A. C. et al. Nature 571, 117–121 (2019):

Supplementary information

Video 1

Example of media change performed in a 96-well plate format

Reporting Summary

Video 2

Example of media change performed in a 24-well plate format

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Wilkinson, A.C., Ishida, R., Nakauchi, H. et al. Long-term ex vivo expansion of mouse hematopoietic stem cells. Nat Protoc 15, 628–648 (2020).

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