Generating human bone marrow organoids for disease modeling and drug discovery

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.

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OTHERS GENERATION OF COMPLEX BONE MARROW ORGANOIDS FROM HUMAN INDUCED PLURIPOTENT STEM CELLS Article Open access 19 February 2024 BIOENGINEERED NICHES THAT RECREATE PHYSIOLOGICAL

EXTRACELLULAR MATRIX ORGANISATION TO SUPPORT LONG-TERM HAEMATOPOIETIC STEM CELLS Article Open access 10 July 2024 EXPANSION OF HUMAN MEGAKARYOCYTE-BIASED HEMATOPOIETIC STEM CELLS BY

BIOMIMETIC MICRONICHE Article Open access 18 April 2023 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

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(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 * These authors contributed equally: Antonio Rodriguez-Romera, Zoë C. Wong, Yuqi Shen. AUTHORS AND AFFILIATIONS * 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 * Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham,

UK Jasmeet S. Reyat, Julie Rayes & Abdullah O. Khan * Department of Physiology, Anatomy and Genetics, Medical Sciences Division, University of Oxford, Oxford, UK Jasmeet S. Reyat Authors

* Aude-Anais Olijnik View author publications You can also search for this author inPubMed Google Scholar * Antonio Rodriguez-Romera View author publications You can also search for this

author inPubMed Google Scholar * Zoë C. Wong View author publications You can also search for this author inPubMed Google Scholar * Yuqi Shen View author publications You can also search for

this author inPubMed Google Scholar * Jasmeet S. Reyat View author publications You can also search for this author inPubMed Google Scholar * Natalie J. Jooss View author publications You

can also search for this author inPubMed Google Scholar * Julie Rayes View author publications You can also search for this author inPubMed Google Scholar * Bethan Psaila View author

publications You can also search for this author inPubMed Google Scholar * Abdullah O. Khan View author publications You can also search for this author inPubMed 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.

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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

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and applicable law. Reprints and permissions ABOUT THIS ARTICLE 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_ 19, 2117–2146 (2024). https://doi.org/10.1038/s41596-024-00971-7 Download citation * Received: 28 February 2023 * Accepted: 22 December 2023 *

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