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Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation

An Author Correction to this article was published on 09 July 2019

This article has been updated

Abstract

Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation1, which is a curative therapy for numerous diseases including immunodeficiencies and leukaemias2. Although substantial effort has been applied to identifying HSC maintenance factors through the characterization of the in vivo bone-marrow HSC microenvironment or niche3,4,5, stable ex vivo HSC expansion has previously been unattainable6,7. Here we describe the development of a defined, albumin-free culture system that supports the long-term ex vivo expansion of functional mouse HSCs. We used a systematic optimization approach, and found that high levels of thrombopoietin synergize with low levels of stem-cell factor and fibronectin to sustain HSC self-renewal. Serum albumin has long been recognized as a major source of biological contaminants in HSC cultures8; we identify polyvinyl alcohol as a functionally superior replacement for serum albumin that is compatible with good manufacturing practice. These conditions afford between 236- and 899-fold expansions of functional HSCs over 1 month, although analysis of clonally derived cultures suggests that there is considerable heterogeneity in the self-renewal capacity of HSCs ex vivo. Using this system, HSC cultures that are derived from only 50 cells robustly engraft in recipient mice without the normal requirement for toxic pre-conditioning (for example, radiation), which may be relevant for HSC transplantation in humans. These findings therefore have important implications for both basic HSC research and clinical haematology.

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Fig. 1: High levels of TPO synergize with low levels of SCF and fibronectin to enhance HSC expansion.
Fig. 2: PVA can replace serum albumin for ex vivo HSC expansion.
Fig. 3: Long-term ex vivo expansion of functional HSCs.
Fig. 4: Ex vivo expanded HSCs engraft in nonconditioned recipients.

Data availability

All graphed datasets can be found in the Supplementary Data. Additional data files will be made available by the corresponding authors upon reasonable request. A detailed protocol is available at the Protocol Exchange29.

Change history

  • 09 July 2019

    An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

References

  1. Osawa, M., Hanada, K., Hamada, H. & Nakauchi, H. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 273, 242–245 (1996).

    ADS  CAS  Article  Google Scholar 

  2. Copelan, E. A. Hematopoietic stem-cell transplantation. N. Engl. J. Med. 354, 1813–1826 (2006).

    CAS  Article  Google Scholar 

  3. Morrison, S. J. & Scadden, D. T. The bone marrow niche for haematopoietic stem cells. Nature 505, 327–334 (2014).

    ADS  CAS  Article  Google Scholar 

  4. Boulais, P. E. & Frenette, P. S. Making sense of hematopoietic stem cell niches. Blood 125, 2621–2629 (2015).

    CAS  Article  Google Scholar 

  5. Yamazaki, S. et al. Nonmyelinating Schwann cells maintain hematopoietic stem cell hibernation in the bone marrow niche. Cell 147, 1146–1158 (2011).

    CAS  Article  Google Scholar 

  6. Kumar, S. & Geiger, H. HSC niche biology and HSC expansion ex vivo. Trends Mol. Med. 23, 799–819 (2017).

    Article  Google Scholar 

  7. Eaves, C. J. Hematopoietic stem cells: concepts, definitions, and the new reality. Blood 125, 2605–2613 (2015).

    CAS  Article  Google Scholar 

  8. Ieyasu, A. et al. An all-recombinant protein-based culture system specifically identifies hematopoietic stem cell maintenance factors. Stem Cell Reports 8, 500–508 (2017).

    CAS  Article  Google Scholar 

  9. Coutu, D. L., Kokkaliaris, K. D., Kunz, L. & Schroeder, T. Three-dimensional map of nonhematopoietic bone and bone-marrow cells and molecules. Nat. Biotechnol. 35, 1202–1210 (2017).

    CAS  Article  Google Scholar 

  10. Gekas, C. & Graf, T. CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age. Blood 121, 4463–4472 (2013).

    CAS  Article  Google Scholar 

  11. Umemoto, T. et al. Integrin-αvβ3 regulates thrombopoietin-mediated maintenance of hematopoietic stem cells. Blood 119, 83–94 (2012).

    CAS  Article  Google Scholar 

  12. Csaszar, E. et al. Rapid expansion of human hematopoietic stem cells by automated control of inhibitory feedback signaling. Cell Stem Cell 10, 218–229 (2012).

    CAS  Article  Google Scholar 

  13. Kawasaki, T. & Kawai, T. Toll-like receptor signaling pathways. Front. Immunol. 5, 461 (2014).

    Article  Google Scholar 

  14. Netea, M. G., Van der Graaf, C., Van der Meer, J. W. & Kullberg, B. J. Recognition of fungal pathogens by Toll-like receptors. Eur. J. Clin. Microbiol. Infect. Dis. 23, 672–676 (2004).

    CAS  Article  Google Scholar 

  15. Loures, F. V. et al. Toll-like receptor 4 signaling leads to severe fungal infection associated with enhanced proinflammatory immunity and impaired expansion of regulatory T cells. Infect. Immun. 78, 1078–1088 (2010).

    CAS  Article  Google Scholar 

  16. Wilkinson, A. C., Morita, M., Nakauchi, H. & Yamazaki, S. Branched-chain amino acid depletion conditions bone marrow for hematopoietic stem cell transplantation avoiding amino acid imbalance-associated toxicity. Exp. Hematol. 63, 12–16.e1 (2018).

    CAS  Article  Google Scholar 

  17. Taya, Y. et al. Depleting dietary valine permits nonmyeloablative mouse hematopoietic stem cell transplantation. Science 354, 1152–1155 (2016).

    ADS  CAS  Article  Google Scholar 

  18. Hernandez-Segura, A., Nehme, J. & Demaria, M. Hallmarks of cellular senescence. Trends Cell Biol. 28, 436–453 (2018).

    CAS  Article  Google Scholar 

  19. de Haan, G. & Lazare, S. S. Aging of hematopoietic stem cells. Blood 131, 479–487 (2018).

    Article  Google Scholar 

  20. Flach, J. et al. Replication stress is a potent driver of functional decline in ageing haematopoietic stem cells. Nature 512, 198–202 (2014).

    ADS  CAS  Article  Google Scholar 

  21. Kane, M. T. & Bavister, B. D. Protein-free culture medium containing polyvinylalcohol, vitamins, and amino acids supports development of eight-cell hamster embryos to hatching blastocysts. J. Exp. Zool. 247, 183–187 (1988).

    CAS  Article  Google Scholar 

  22. Wiles, M. V. & Johansson, B. M. Embryonic stem cell development in a chemically defined medium. Exp. Cell Res. 247, 241–248 (1999).

    CAS  Article  Google Scholar 

  23. Hu, Y. & Smyth, G. K. ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J. Immunol. Methods 347, 70–78 (2009).

    CAS  Article  Google Scholar 

  24. Yamamoto, R. et al. Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells. Cell 154, 1112–1126 (2013).

    CAS  Article  Google Scholar 

  25. Yamamoto, R. et al. Large-scale clonal analysis resolves aging of the mouse hematopoietic stem cell compartment. Cell Stem Cell 22, 600–607.e4 (2018).

    CAS  Article  Google Scholar 

  26. Bhattacharya, D., Rossi, D. J., Bryder, D. & Weissman, I. L. Purified hematopoietic stem cell engraftment of rare niches corrects severe lymphoid deficiencies without host conditioning. J. Exp. Med. 203, 73–85 (2006).

    CAS  Article  Google Scholar 

  27. Shimoto, M., Sugiyama, T. & Nagasawa, T. Numerous niches for hematopoietic stem cells remain empty during homeostasis. Blood 129, 2124–2131 (2017).

    CAS  Article  Google Scholar 

  28. Seita, J. et al. Lnk negatively regulates self-renewal of hematopoietic stem cells by modifying thrombopoietin-mediated signal transduction. Proc. Natl Acad. Sci. USA 104, 2349–2354 (2007).

    ADS  CAS  Article  Google Scholar 

  29. Wilkinson, A. C. et al. Ex vivo mouse hematopoietic stem cell expansion using polyvinyl alcohol. Protoc. Exch. https://doi.org/10.21203/rs.2.9270/v1 (2019).

    Article  Google Scholar 

Download references

Acknowledgements

We thank S. Takaki, Y. Ishii, H. Hasegawa, M. Hayashi and the Stanford Human Immune Monitoring Center for technical support, and J. Bhadury for advice. This research was funded by JSPS KAKENHI Grant-in-Aid for Scientific Research (JP18H05095; JP17H05086), Japan Agency for Medical Research and Development (JP18bm0404025), CIRM (LA1_C12-06917; DISC1-10555), the NIH (R01DK116944; R01HL147124) and the Ludwig Foundation. A.C.W. was funded by Bloodwise (15050), the Leukemia and Lymphoma Society (3385-19), and the JSPS. K.M.L. was supported by the NIH Director’s Early Independence Award (DP5OD024558), Siebel Stem Cell Institute, Baxter Foundation and The Anthony DiGenova Endowed Faculty Scholar.

Reviewer information

Nature thanks Thomas Graf, Ross Levine and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Contributions

A.C.W. conceptualized the research, performed experiments, analysed data and wrote the manuscript. R.I., M.K., K.S., M.M., R.V.C. and R.Y. performed experiments and analysed data. K.M.L. conceptualized the research and edited the manuscript. M.W. and Y.N. supervised experiments and edited the manuscript. H.N. conceptualized the research, supervised experiments, analysed data and wrote the manuscript. S.Y. conceptualized the research, performed and supervised experiments, analysed data and wrote the manuscript.

Corresponding authors

Correspondence to Hiromitsu Nakauchi or Satoshi Yamazaki.

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

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Extended data figures and tables

Extended Data Fig. 1 Optimizing conditions for long-term HSC culture.

a, Schematic of the standard HSC culture assay. Bone-marrow CD34KSL HSCs of C57BL/6-CD45.1 mice were sorted (50 cells per well) into U-bottomed 96-well plate wells (b for sorting scheme). HSC growth can be observed during culture by counting or flow cytometry, with medium changes made every three days (after an initial seven days in culture). After 7–28 days, functional HSC activity was determined using competitive transplantation into irradiated C57BL/6-CD45.2 mice, against 1 × 106 bone-marrow competitor cells from C57BL/6-CD45.1/CD45.2 (F1) mice. Donor chimerism within peripheral-blood myeloid, T cell and B cell lineages was determined after 4–16 weeks, or longer. Where indicated, secondary transplantation assays were performed by transplanting 1 × 106 bone-marrow cells from primary recipients into irradiated C57BL/6-CD45.2 mice. b, FACS gating strategy for sorting CD34KSL cells (gates 1–7) and CD150+CD34KSL cells (gates 1–8) from c-KIT-enriched mouse bone marrow. Representative of at least five experiments. c, Flow cytometric histograms for cell-surface c-KIT staining of HSCs following stimulating with 100 ng ml−1 TPO and 0, 10 or 100 ng ml−1 SCF for 1, 24 and 48 h. Representative of three independent cultures. d, Mean florescence intensity of c-KIT antibody staining on HSCs cultured in 100 ng ml−1 TPO supplemented with 10 ng ml−1 or 100 ng ml−1 SCF, analysed after 1–72 h in culture, relative to cultures containing 100 ng ml−1 TPO without SCF. Mean of three independent cultures. Error bars denote s.d. e, Mean donor peripheral-blood chimerism at week 16, from 1 × 104 HSC-derived cells following a 28-day-long culture on plastic (n = 5 cell cultures), collagen 1 (n = 3 cell cultures), collagen 4 (n = 4 cell cultures), fibronectin (n = 3 cell cultures), gelatin (n = 5 cell cultures) or laminin 511 (n = 4 cell cultures) culture plates (cultured in 100 ng ml−1 TPO and 10 ng ml−1 SCF with complete medium changes). Competitive transplantation against 1 × 106 bone-marrow competitors. f, Number of live cells after culturing 50 CD34KSL HSCs for 28 days on plastic (tissue-culture-treated) plates or fibronectin-coated plates. Mean of three independent cultures. Error bars denote s.d.

Source Data

Extended Data Fig. 2 Identification of PVA-based HSC culture conditions.

a, Fold change in MFI from cytokine immunoassays performed on HSA-based HSC cultures between day 8 and day 13. Medium changes performed at day 7 and day 10. Mean of four independent cultures with fold change relative to unconditioned medium. Error bars denote s.d. b, Mean expansion of 50 CD34+KSL haematopoietic progenitor cells at day 7, in 100 ng ml−1 TPO and 10 ng ml−1 SCF with or without addition of 0.3 ng ml−1 to 10 ng ml−1 mouse IL-6 (n = 4 cell cultures). c, Heat map displaying the MFI fold change from cytokine immunoassays using conditioned medium from HSC cultures at day 14. CD34KSL HSCs were isolated from C57BL/6 wild-type (WT), Tlr2 knockout (TLR2-KO) or Tlr4 knockout (TLR4-KO) mice, and cultured in HSA-based cultures. Dexamethasone (+Dex) at 50 nM was added, where indicated. Mean of four independent cultures with fold change relative to unconditioned medium. d, Concentration of IL-6 observed in HSA-based cultures at day 14 of wild-type HSCs (n = 8 cell cultures), Tlr2 knockout HSCs (n = 8 cell cultures), Tlr4 knockout HSCs (n = 6 cell cultures), or wild-type HSCs + dexamethasone (n = 8 cell cultures). Error bars denote s.d. e, Mean donor peripheral-blood chimerism at week 12, from HSCs cultured for 7 days, in fresh medium (n = 7 mice) or in medium composed of 50% medium collected from a 12-day-long HSC culture and 50% fresh medium (termed ‘conditioned media’, n = 7 mice). Competitive transplantation against 1 × 106 bone-marrow competitors. f, Example flow cytometry plots displaying c-KIT and SCA1 expression on the Lin progeny (left), and CD150 and CD48 expression in the KSL population (right) after a PVA-based HSC culture for seven days. Representative of four independent cultures. g, Concentration of various cytokines in conditioned medium at day 14, from HSA- or PVA-based CD34KSL HSC cultures. Mean of eight independent cultures. Error bars denote s.d. Statistical significance was calculated using t-tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. h, Relative expression of p16Ink4a, p19Arf and Trp53 in KSL cells collected from cultures at day 14 (HSA-based cultures with half-medium changes, HSA-based cultures with complete medium changes and PVA-based cultures with complete medium changes), relative to expression in freshly isolated KSL cells. Mean of three independent cultures, with gene expression normalized to Gapdh expression. Error bars denote s.d. i, Number of phospho-γ-histone 2A.X (H2A.X) nuclear foci in KSL cells at day 28, from HSA-based or PVA-based HSC cultures. Irradiated cells were included as a positive control. Forty-nine cells quantified per condition. j, Relative expression of p16Ink4a, p19Arf and Trp53 in KSL cells collected from cultures at day 14 (left): HSA-based cultures, PVA-based cultures and PVA-based cultures supplemented with 1 ng ml−1 lipopolysaccharide. Mean of technical quadruplets, with gene expression normalized to Gapdh expression. The concentration of IL-6 observed in these culture conditions is shown on the right. Mean of four independent cultures. Error bars denote s.d. k, Twenty-eight-day-long expansion of 50 CD150+CD34KSL HSCs in medium containing 87% hydrolysed PVA or >99% hydrolysed PVA. Ten thousand cells at day 28 represent ~1 HSCeq for 87% PVA and ~5 HSCeq for 99% PVA. Mean of three independent cultures. Error bars denote s.d. l, Seven-day-long expansion of 50 human cord-blood CD34+ cells in HSA- or PVA-based cultures supplemented with 10 ng ml−1 human SCF and 100 ng ml−1 human TPO. Mean of three independent cultures. Error bars denote s.d.

Source Data

Extended Data Fig. 3 Characterization of long-term PVA-based HSC cultures.

a, Mean donor peripheral-blood chimerism at weeks 4–16, from 28-day-long PVA-based (CD150+CD34KSL) HSC cultures using 100 ng ml−1 TPO and 10 ng ml−1 SCF in fibronectin-coated wells with complete medium changes. Indicated cell numbers transplanted against 2 × 105 bone-marrow cells. Data from two independent transplantation experiments. b, Multilineage donor peripheral-blood chimerism at week 16, for each individual mouse in a. c, Expression of p16Ink4a, p19Arf and Trp53 in 28-day-long PVA-cultured KSL cells, relative to expression in freshly isolated KSL cells. Mean of three independent cultures, with gene expression normalized to Gapdh expression. Error bars denote s.d. d, Sanger sequencing trace of Trp53 cDNA amplified from KSL cells collected from 28-day-long PVA-based HSC cultures (n = 1 cell culture). e, Representative images of β-galactosidase activity staining of freshly isolated KSL, KSL isolated from 28-day-long PVA-based cultures and bulk 28-day-long PVA-based cultures. Representative of two biological replicates. f, Percentage of β-galactosidase-positive cells in conditions described in e. Mean of technical triplicates (50–100 cells counted per replicate). Error bars denote s.d. g, Karyotype of CD45.1+ bone-marrow repopulating progeny of expanded functional HSCs at day 28, in PVA-based media at 16 weeks after transplantation. All chromosomes that were analysed were normal in 25 out of 25 cells (performed by Nihon Gene Research Laboratories). h, Frequency of CD11a+, CD34+, CD48+, CD135+, CD201+ and ESAM+ cells within the phenotypic KSL population during ex vivo HSC culture (derived from 50 CD150+CD34KSL cells). Mean of four independent cultures. Error bars denote s.d. i, Composition of the Lin+ compartment of HSC cultures at day 28. The lineage antibody cocktail used in this study comprised CD4, CD8, CD45R, TER119, LY-6G/LY-6C and CD127. A non-overlapping FCER1+ cell population was also identified within the culture, and is quantitated relative to the Lin+ population. Mean of four independent cultures. Error bars denote s.d. j, Mean donor peripheral-blood chimerism at weeks 4–16, from 1 × 105 cells from a 57-day PVA-based HSC culture using fibronectin-coated plates and supplemented with 100 ng ml−1 TPO and 10 ng ml−1 SCF (n = 5 mice). Competitive transplantation against 1 × 106 bone-marrow competitors.

Source Data

Extended Data Fig. 4 Characterization of clonally derived HSC expansion cultures.

a, Mean number of live cells, KSL cells and CD150+KSL cells derived from single CD150+CD34KSL HSCs after 28 days of culture (n = 48 single cell cultures) (left), and mean number of live cells from bulk (50 and 500) CD150+CD34KSL HSC cultures after 28 days of culture (n = 4 cell cultures) (right). b, Proportion of phenotypic cell types that constitute cultures at day 28 derived from single CD150+CD34KSL HSCs. Only cultures with >10,000 cells were analysed (39 wells of 84 wells analysed). c, Donor peripheral-blood chimerism at week 16, from 28-day expanded single CD150+CD34KSL HSC cultures, transplanted into lethally irradiated recipients against 2 × 105 bone-marrow cells. Each column represents an individual mouse. d, Donor peripheral-blood chimerism at weeks 4–12, from one fifth of a 28-day-long culture derived from a single CD150+CD34KSL HSC, as described in Fig. 3f, g. Each column represents an individual mouse. Representative data for three independent single HSC cultures (out of ten transplanted).

Source Data

Extended Data Fig. 5 Nonconditioned transplantation into immunodeficient recipients.

a, Schematic of nonconditioned allogeneic transplantation. One hundred CD150+CD34KSL cells from C57BL/6-CD45.2 mice were expanded for 28 days before being transplanted into nonconditioned immunodeficient NOD/SCID recipient mice. b, Donor peripheral-blood chimerism at week 4, from 100 fresh HSCs (n = 5 mice) or a 28-day-long HSC culture derived from 100 HSCs (n = 5 mice), transplanted as described in a. Each column represents an individual mouse. c, Example flow cytometry plots displaying T cell (CD4 and CD8) and B cell (CD45R, also known as B220) peripheral-blood lineages within nonconditioned NOD/SCID mice at 16 weeks after transplantation (representative of 5 mice), as described in a.

Source Data

Extended Data Table 1 List of antibodies

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Wilkinson, A.C., Ishida, R., Kikuchi, M. et al. Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation. Nature 571, 117–121 (2019). https://doi.org/10.1038/s41586-019-1244-x

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