Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Generating human bone marrow organoids for disease modeling and drug discovery

Nature Protocols (2024)Cite this article

Subjects

Abstract

The bone marrow supports and regulates hematopoiesis, responding to physiological requirements for blood cell production over ontogeny and during pathological challenges. Interactions between hematopoietic cells and niche components are challenging to study mechanistically in the human context, but are important to delineate in order to explore the pathobiology of blood and bone marrow disorders. Organoids are proving transformative in many research settings, but an accurate human bone marrow model incorporating multiple hematopoietic and stromal elements has been lacking. This protocol describes a method to generate three-dimensional, multilineage bone marrow organoids from human induced pluripotent stem cells (hiPSCs), detailing the steps for the directed differentiation of hiPSCs using a series of cytokine cocktails and hydrogel embedding. Over 18 days of differentiation, hiPSCs yield the key lineages that are present in central myelopoietic bone marrow, organized in a well-vascularized architecture that resembles native hematopoietic tissues. This presents a robust, in vitro system that can model healthy and perturbed hematopoiesis in a scalable three-dimensional microenvironment. Bone marrow organoids also support the growth of immortalized cell lines and primary cells from healthy donors and patients with myeloid and lymphoid cancers, including cell types that are poorly viable in standard culture systems. Moreover, we discuss assays for the characterization of organoids, including interrogation of pathogenic remodeling using recombinant TGF-ß treatment, and methods for organoid engraftment with exogenous cells. This protocol can be readily adapted to specific experimental requirements, can be easily implemented by users with tissue culture experience and does not require access to specialist equipment.

Key points

  • An ex vivo three-dimensional system for modeling human bone marrow is presented. Human induced pluripotent stem cells cultured in a collagen-enriched hydrogel with stimulatory cytokines generate vascularized bone marrow organoids containing mesenchymal stromal cells, fibroblasts, endothelial and hematopoietic cells.

  • The bone marrow organoids can be engrafted with adult donor-derived cells to study the dynamics between these cells and the bone marrow niche, using downstream assays including imaging, genetic characterization and flow cytometry.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Schematic of differentiation workflow and timeline (Steps 1–67).
Fig. 2: Key steps in BMO generation (Steps 1–53).
Fig. 3: Characterization of BMOs using imaging and flow cytometry.
Fig. 4: Imaging of organoids to assess engraftment with exogenous cells and fibrosis (Steps 54–67, 68–71 and 80).

Similar content being viewed by others

Data availability

The original research relating to this protocol can be accessed in a previous publication22 and via the Github repository (https://github.com/aokhan/BMorganoidV1/). Single-cell RNA sequencing data relevant to the original publication are available at the Gene Expression Omnibus (accession GSE196684). Source data are provided with this paper.

Code availability

Code used to analyze RNA sequencing data relevant to the original publication is available at https://github.com/aokhan/BMorganoidV1/ and https://github.com/supatt-lab/SingCellaR.

References

  1. Tuveson, D. & Clevers, H. Cancer modeling meets human organoid technology. Science 364, 952–955 (2019).

    Article  CAS  PubMed  Google Scholar 

  2. Kim, J., Koo, B.-K. & Knoblich, J. A. Human organoids: model systems for human biology and medicine. Nat. Rev. Mol. Cell Biol. 21, 571–584 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Veninga, V. & Voest, E. E. Tumor organoids: opportunities and challenges to guide precision medicine. Cancer Cell 39, 1190–1201 (2021).

    Article  CAS  PubMed  Google Scholar 

  4. Li, R. et al. A pro-inflammatory stem cell niche drives myelofibrosis through a targetable galectin 1 axis. Preprint at bioRxiv https://doi.org/10.1101/2023.08.05.550630 (2023).

  5. Méndez-Ferrer, S. et al. Bone marrow niches in haematological malignancies. Nat. Rev. Cancer 20, 285–298 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Lucas, D. Structural organization of the bone marrow and its role in hematopoiesis. Curr. Opin. Hematol. 28, 36–42 (2020).

    Article  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Jardine, L. et al. Blood and immune development in human fetal bone marrow and Down syndrome. Nature 598, 327–331 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Baryawno, N. et al. A cellular taxonomy of the bone marrow stroma in homeostasis and leukemia. Cell 177, 1915–1932.e16 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Tikhonova, A. N. et al. The bone marrow microenvironment at single-cell resolution. Nature 569, 222–228 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Baccin, C. et al. Combined single-cell and spatial transcriptomics reveal the molecular, cellular and spatial bone marrow niche organization. Nat. Cell Biol. 22, 38–48 (2020).

    Article  CAS  PubMed  Google Scholar 

  12. Kent, D., Dykstra, B. & Eaves, C. Isolation and assessment of long‐term reconstituting hematopoietic stem cells from adult mouse bone marrow. Curr. Protoc. Stem Cell Biol. 3, 2A.4.1–2A.4.23 (2007).

    Article  Google Scholar 

  13. Bradley, T. & Metcalf, D. The growth of mouse bone marrow cells in vitro. Aust. J. Exp. Biol. Med. Sci. 44, 287–300 (1966).

    Article  CAS  PubMed  Google Scholar 

  14. de L, M. et al. Cord-blood engraftment with ex vivo mesenchymal-cell coculture. N. Engl. J. Med. 367, 2305–2315 (2012).

    Article  Google Scholar 

  15. Khan, A. O. et al. Post-translational polymodification of β1-tubulin regulates motor protein localisation in platelet production and function. Haematologica 1, 243–260 (2022).

    Google Scholar 

  16. Feng, Q. et al. Scalable generation of universal platelets from human induced pluripotent stem cells. Stem Cell Rep. 3, 817–831 (2014).

    Article  CAS  Google Scholar 

  17. Ng, E. S. et al. Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta–gonad–mesonephros. Nat. Biotechnol. 34, 1168–1179 (2016).

    Article  CAS  PubMed  Google Scholar 

  18. Jing, R. et al. EZH1 repression generates mature iPSC-derived CAR T cells with enhanced antitumor activity. Cell Stem Cell 29, 1181–1196.e6 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Cao, X. et al. Differentiation and functional comparison of monocytes and macrophages from hiPSCs with peripheral blood derivatives. Stem Cell Rep. 12, 1282–1297 (2019).

    Article  CAS  Google Scholar 

  20. Ebrahimi, M. et al. Differentiation of human induced pluripotent stem cells into erythroid cells. Stem Cell Res. Ther. 11, 483 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Moreau, T. et al. Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming. Nat. Commun. 7, 11208 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Khan, A. O. et al. Human bone marrow organoids for disease modelling, discovery and validation of therapeutic targets in hematological malignancies. Cancer Discov. https://doi.org/10.1158/2159-8290.cd-22-0199 (2022).

  23. Zhao, Z. et al. Organoids. Nat. Rev. Methods Prim. 2, 94 (2022).

    Article  CAS  Google Scholar 

  24. Wimmer, R. A., Leopoldi, A., Aichinger, M., Kerjaschki, D. & Penninger, J. M. Generation of blood vessel organoids from human pluripotent stem cells. Nat. Protoc. 14, 3082–3100 (2019).

    Article  CAS  PubMed  Google Scholar 

  25. Wimmer, R. A. et al. Human blood vessel organoids as a model of diabetic vasculopathy. Nature 565, 505–510 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Popescu, D.-M. et al. Decoding human fetal liver haematopoiesis. Nature 574, 365–371 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Roy, A. et al. Transitions in lineage specification and gene regulatory networks in hematopoietic stem/progenitor cells over human development. Cell Rep. 36, 109698 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Raic, A., Naolou, T., Mohra, A., Chatterjee, C. & Lee-Thedieck, C. 3D models of the bone marrow in health and disease: yesterday, today, and tomorrow. MRS Commun. 9, 37–52 (2019).

    Article  CAS  PubMed  Google Scholar 

  29. Sharipol, A., Lesch, M. L., Soto, C. A. & Frisch, B. J. Bone marrow microenvironment-on-chip for culture of functional hematopoietic stem cells. Front. Bioeng. Biotechnol. 10, 855777 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Bessy, T., Itkin, T. & Passaro, D. Bioengineering the bone marrow vascular niche. Front. Cell Dev. Biol. 9, 645496 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Voeltzel, T. et al. A minimal standardized human bone marrow microphysiological system to assess resident cell behavior during normal and pathological processes. Biomater. Sci. 10, 485–498 (2021).

    Article  Google Scholar 

  32. Giger, S. et al. Microarrayed human bone marrow organoids for modeling blood stem cell dynamics. APL Bioeng. 6, 036101 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Fairfield, H. et al. Development of a 3D bone marrow adipose tissue model. Bone 118, 77–88 (2019).

    Article  PubMed  Google Scholar 

  34. Glaser, D. E. et al. Organ-on-a-chip model of vascularized human bone marrow niches. Biomaterials 280, 121245 (2022).

    Article  CAS  PubMed  Google Scholar 

  35. Chou, D. B. et al. On-chip recapitulation of clinical bone-marrow toxicities and patient-specific pathophysiology. Nat. Biomed. Eng. 4, 394–406 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Zhang, S., Wan, Z. & Kamm, R. D. Vascularized organoids on a chip: strategies for engineering organoids with functional vasculature. Lab Chip 21, 473–488 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Aazmi, A. et al. Engineered vasculature for organ-on-a-chip systems. Engineering 9, 131–147 (2022).

    Article  Google Scholar 

  38. Marturano-Kruik, A. et al. Human bone perivascular niche-on-a-chip for studying metastatic colonization. Proc. Natl Acad. Sci. USA 115, 1256–1261 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Cuenca, M. V. et al. Engineered 3D vessel-on-chip using hiPSC-derived endothelial and vascular smooth muscle cells. Stem Cell Rep. 16, 2159–2168 (2021).

    Article  Google Scholar 

  40. Byambaa, B. et al. Bioprinted osteogenic and vasculogenic patterns for engineering 3D bone tissue. Adv. Healthc. Mater. 6, 1700015 (2017).

    Article  Google Scholar 

  41. Simunovic, F. & Finkenzeller, G. Vascularization strategies in bone tissue engineering. Cells 10, 1749 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kopp, H.-G., Avecilla, S. T., Hooper, A. T. & Rafii, S. The bone marrow vascular niche: home of HSC differentiation and mobilization. Physiology 20, 349–356 (2005).

    Article  CAS  PubMed  Google Scholar 

  43. Demirci, S., Leonard, A. & Tisdale, J. F. Hematopoietic stem cells from pluripotent stem cells: clinical potential, challenges, and future perspectives. Stem Cells Transl. Med 9, 1549–1557 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Sugimura, R. et al. Haematopoietic stem and progenitor cells from human pluripotent stem cells. Nature 545, 432–438 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Sakurai, M. et al. Chemically defined cytokine-free expansion of human haematopoietic stem cells. Nature 615, 127–133 (2023).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Wilkinson, A. C. et al. Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation. Nature 571, 117–121 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Homan, K. A. et al. Flow-enhanced vascularization and maturation of kidney organoids in vitro. Nat. Methods 16, 255–262 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Thamodaran, V., Rani, S. & Velayudhan, S. R. Induced pluripotent stem (iPS) cells, methods and protocols. Methods Mol. Biol. 2454, 755–773 (2021).

    Article  Google Scholar 

  50. Qi, P., Zhou, Y., Wang, D., He, Z. & Li, Z. A new collagen solution with high concentration and collagen native structure perfectly preserved. RSC Adv. 5, 87180–87186 (2015).

    Article  CAS  Google Scholar 

  51. Masselink, W. et al. Broad applicability of a streamlined ethyl cinnamate-based clearing procedure. Development 146, dev166884 (2019).

    Article  PubMed  Google Scholar 

  52. Campbell, T. B., Zhang, S. Y., Valencia, A. & Passegue, E. Bone marrow stromal cell remodeling is a common feature of diverse fibrotic myeloproliferative neoplasm models. Blood 128, 25–25 (2016).

    Article  Google Scholar 

  53. Heaton, H. et al. Souporcell: robust clustering of single-cell RNA-seq data by genotype without reference genotypes. Nat. Methods 17, 615–620 (2020).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank N. Hayder and S. Reed who helped with sample banking, the University of Birmingham TechHub Imaging Core Facilities, and the Medical Research Council (MRC) Weatherall Institute of Molecular Medicine Flow Cytometry facility and Imaging core. We thank P. Garcia, G. Murphy, V. Steeples, Y. Psaras, and C. Toepffer for the generous provision of the hiPSC lines used (BU3-10, BU8C3, KOLF2). A.O.K. is funded by a Sir Henry Wellcome fellowship (218649/Z/19/Z, 218649/A/19/Z). B.P. receives funding from a Cancer Research UK Advanced Clinician Scientist Fellowship (C67633/A29034), a British Research Council (BRC) Senior Research Fellowship, the Haematology and Stem Cells Theme of the Oxford BRC, a Kay Kendall Leukemia Fund Project Grant and unit funding from the MRC (awarded to the MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine). A CC BY or equivalent license is applied to the author accepted manuscript arising from this submission, in accordance with the funders’ open access conditions.

Author information

Author notes

  1. These authors contributed equally: Antonio Rodriguez-Romera, Zoë C. Wong, Yuqi Shen.

Authors and Affiliations

  1. MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine and National Institute of Health Research, Oxford Biomedical Research Centre, University of Oxford, Oxford, UK

    Aude-Anais Olijnik, Antonio Rodriguez-Romera, Zoë C. Wong, Yuqi Shen, Natalie J. Jooss, Bethan Psaila & Abdullah O. Khan

  2. Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK

    Jasmeet S. Reyat, Julie Rayes & Abdullah O. Khan

  3. Department of Physiology, Anatomy and Genetics, Medical Sciences Division, University of Oxford, Oxford, UK

    Jasmeet S. Reyat

Authors
  1. Aude-Anais Olijnik
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Antonio Rodriguez-Romera
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zoë C. Wong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuqi Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jasmeet S. Reyat
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Natalie J. Jooss
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Julie Rayes
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Bethan Psaila
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Abdullah O. Khan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.O.K. devised the differentiation protocol and utilization methods, performed cell culture and imaging experiments, co-authored the manuscript and sourced funding for this project. B.P. devised experiments and utilization of the organoids for disease modeling, interpreted data, co-wrote the manuscript and sourced funding for this project. A.-A.O., A.R.-R., Z.C.W. and Y.S. performed flow cytometry experiments, analyzed and interpreted data, and A.-A.O. co-wrote the protocol. J.R. and J.S.R. devised and performed sectioning and related imaging experiments. N.J.J. critically reviewed and edited the manuscript and curated and interpreted data.

Corresponding authors

Correspondence to Bethan Psaila or Abdullah O. Khan.

Ethics declarations

Competing interests

B.P.: Alethiomics (co-founder, equity, consultancy, research funding), Constellation Therapeutics (consultancy), Blueprint Medicines (advisory board), Galecto (research funding), Novartis (paid speaking engagements), GSK (advisory board). A.O.K.: Alethiomics (consultancy). The other authors have no conflicts of interest to declare that are relevant to the content of this article. A patent has been filed by A.O.K. and B.P. relating to work described in this paper (GB2202025.9 and GB221664.47 (WO/2023/156774), PCT/GB2023/050348).

Peer review

Peer review information

Nature Protocols thanks Benjamin Frisch, Veronique Maguer-Satta and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

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

Related links

Key references using this protocol

Khan, A. O. et al. Cancer Discov. 13, 364–385 (2022): https://doi.org/10.1158/2159-8290.cd-22-0199

Li, R. et al. Preprint at bioRxiv (2023): https://doi.org/10.1101/2023.08.05.550630

Supplementary Information

Supplementary Information

Supplementary Figs. 1–8.

Source data

Source Data Fig. 3

Statistical source data for the population frequencies in Fig. 3d–i.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Olijnik, AA., Rodriguez-Romera, A., Wong, Z.C. et al. Generating human bone marrow organoids for disease modeling and drug discovery. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-00971-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41596-024-00971-7

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer