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