MarShie: a clearing protocol for 3D analysis of single cells throughout the bone marrow at subcellular resolution

Till Fabian Mertens, Alina Tabea Liebheit, Johanna Ehl, Ralf Köhler, Asylkhan Rakhymzhan, Andrew Woehler, Lukas Katthän, Gernot Ebel, Wjatscheslaw Liublin, Ana Kasapi, Antigoni Triantafyllopoulou, Tim Julius Schulz, Raluca Aura Niesner and Anja Erika Hauser ()
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Till Fabian Mertens: Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin
Alina Tabea Liebheit: Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin
Johanna Ehl: Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin
Ralf Köhler: Deutsches Rheuma-Forschungszentrum (DRFZ), a Leibniz Institute
Asylkhan Rakhymzhan: Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin
Andrew Woehler: Max Delbrück Center for Molecular Medicine
Lukas Katthän: Miltenyi Biotec B.V. and Co. Bertha-von-Suttner-Straße 5
Gernot Ebel: Miltenyi Biotec B.V. and Co. Bertha-von-Suttner-Straße 5
Wjatscheslaw Liublin: Deutsches Rheuma-Forschungszentrum (DRFZ), a Leibniz Institute
Ana Kasapi: Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin
Antigoni Triantafyllopoulou: Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin
Tim Julius Schulz: German Institute of Human Nutrition (DIfE) Potsdam-Rehbruecke
Raluca Aura Niesner: Deutsches Rheuma-Forschungszentrum (DRFZ), a Leibniz Institute
Anja Erika Hauser: Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin

Nature Communications, 2024, vol. 15, issue 1, 1-19

Abstract: Abstract Analyzing immune cell interactions in the bone marrow is vital for understanding hematopoiesis and bone homeostasis. Three-dimensional analysis of the complete, intact bone marrow within the cortex of whole long bones remains a challenge, especially at subcellular resolution. We present a method that stabilizes the marrow and provides subcellular resolution of fluorescent signals throughout the murine femur, enabling identification and spatial characterization of hematopoietic and stromal cell subsets. By combining a pre-processing algorithm for stripe artifact removal with a machine-learning approach, we demonstrate reliable cell segmentation down to the deepest bone marrow regions. This reveals age-related changes in the marrow. It highlights the interaction between CX3CR1+ cells and the vascular system in homeostasis, in contrast to other myeloid cell types, and reveals their spatial characteristics after injury. The broad applicability of this method will contribute to a better understanding of bone marrow biology.

Date: 2024
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DOI: 10.1038/s41467-024-45827-6

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