Bone Marrow Environment Methods and Protocols

This volume brings together classical and cutting-edge protocols on the spatio-temporal study of the cellular subsets constituting the bone and the marrow in both mouse and human. Chapters details methods on bone marrow (BM) ecosystem, to label, sort, analyse, and culture specific cell subsets as well as techniques allowing the evaluation of the function of some of the cellular elements of the BM. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Bone Marrow Environment: Methods and Protocols aims to help new investigators to pursue the characterization of the BM microenvironment in the coming years.

Part I: Cell Culture and In Vitro Functionnal Assays

1. Culture, Expansion, and Differentiation of Human Bone Marrow Stromal Cells

Valeria Bisio, Marion Espéli, Karl Balabanian and Adrienne Anginot

2. Differentiation and Phenotyping of Murine Osteoclasts from Bone Marrow Progenitors, Monocytes, and Dendritic Cells

Julia Halper, Maria-Bernadette Madel, and Claudine Blin-Wakkach

3. Culture, Expansion, and Differentiation of Mouse Bone-derived Mesenchymal Stromal Cells

4. Feeder-Free Differentiation Assay for Mouse Hematopoietic Stem and Progenitor Cells

Vincent Rondeau, Marion Espéli and Karl Balabanian

5. In Vitro Analysis of Energy Metabolism in Bone-marrow Mesenchymal Stromal Cells

Jérôme Bourgeais and Olivier Hérault

Part II : Flow Cytometry-Based Analysis  of the Bone Marrow Cellular Compartments

6. Flow Cytometry Analysis of Mouse Hematopoietic Stem and Multipotent Progenitor Cells

Julien M.P. Grenier, Marjorie C. Delahaye, Stéphane J.C. Mancini, and Michel Aurrand-Lions

Yuki Matsushita, Wanida Ono, and Noriaki Ono

8. Immunophenotyping of the Medullary B cell Compartment in Mouse Models

Amélie Bonaud , Karl Balabanian, and Marion Espéli

9. Metabolic Analysis of Mouse Haematopoietic Stem and Progenitor Cells

Liang He, Amina Aouida, Amira Mehtar, Houda Haouas, and Fawzia Louache

10. Multicolor Immunofluorescence Staining Of Paraffin-Embedded Human Bone Marrow Sections

Francesca M. Panvini, Simone Pacini, Stefano Mazzoni, and Simón Méndez-Ferrer

11. 3D microscopy of Murine Bone Marrow Hematopoietic Tissues

YeVin Mun and César Nombela-Arrieta

12. Ex Vivo Whole-Mount Imaging of Leukocyte Migration to the Bone Marrow

Stephan Holtkamp and Christoph Scheiermann

13. Intrafemoral Delivery of Hematopoietic Progenitors

Maximilien Evrard, Immanuel Kwok, and Lai Guan Ng

14. Imaging of Bone Marrow Plasma Cells and of their Niches

Carolin Ulbricht, Raluca A. Niesner, and Anja E. Hauser

15. Intravital Imaging of Bone Marrow Microenvironment in the Mouse Calvaria and Tibia

Changming Shih, Leonard Tan, Jackson LiangYao Li, Yingrou Tan, Hui Cheng, and Lai Guan Ng

16. Intravital Imaging of Bone Marrow Niches

Myriam L. R. Haltalli and Cristina Lo Celso

Part IV : In Vivo and Ex Vivo Modelling of the Bone Marrow Ecosystem

17. Modeling Human Fetal Hematopoiesis in Humanized Mice

Seydou Keita, Bruno Canque, and Kutaiba Alhaj Hussen

18. Engraftment of Human Hematopoietic Cells in Biomaterials Implanted In Immunodeficient Mouse Models

Syed A Mian and Dominique Bonnet

19. 3D Engineering of Human Hematopoietic Niches in Perfusion Bioreactor

Steven J. Dupard and Paul E. Bourgine

20. Manufacturing a Bone Marrow-on-a-Chip Using Maskless Photolithography

Benoit Souquet, Matthieu Opitz, Benoit Vianay, Stéphane Brunet, and Manuel Théry

Part V: Sequencing-Based Analysis of Cell Heterogeneity

21. In Vivo Tracking of Hematopoietic Stem and Progenitor Cell Ontogeny by Cellular Barcoding

Tamar Tak, Almut S. Eisele, and Leila Perié

22. Single Cell Analysis of Haematopoietic Stem Cells