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Mesenchymal stromal cells (MSC) are a rare, heterogeneous and multipotent population that can be isolated from several tissues. MSC were originally discovered in the bone marrow and studied for their capacity to maintain hematopoietic cells. We will describe here methods to isolate, culture, and bank MSC from human bone marrow. Then, characterization protocols by flow cytometry, clonogenic assays and doubling time evaluation will be developed. Finally, in vitro MSC culture and differentiation into osteoblasts, adipocytes, and chondrocytes will be explained. Thus, this chapter will detail all bases to work on MSC with consensus and clear methods and protocols.

Hematopoietic stem cells (HSCs) are responsible for replenishing immune cells and reside in bone marrow (BM) niches, which provide all cellular and molecular components required for their lifelong maintenance and differentiation. Although HSCs have been extensively analyzed and characterized, their ex vivo expansion, which constitutes a promising approach for therapeutic development in regenerative medicine, remains challenging. Here, we describe an original in vitro system allowing to quantify by flow cytometry the differentiation of mouse HSCs into lineage-primed multipotent hematopoietic progenitors (MPPs) in a cytokine-supplemented feeder-free medium.

B cell development is a stepwise process encompassing several B cell precursor stages that can be phenotypically distinguished, and that is taking place in the bone marrow in adults. Interestingly, within the bone marrow B cell precursors coexist with the most differentiated actors of this lineage, the plasma cells. In this chapter, we describe a method to recover cells from the bone marrow and a flow cytometric approach to identify each subpopulation of the B cell lineage that resides within the bone marrow compartment. This protocol focuses on membrane markers to discriminate all the B cell subpopulations in order to preserve cell integrity during experimentation and for further analyses.

Mesenchymal stem/stromal cells (MSCs) are multipotent adult cells that are present in several tissues including the bone marrow (BM), in which they can differentiate in a variety of cell types such as osteoblasts, chondrocytes and adipocytes. The isolation of MSCs has been carried out by many studies that aim to control their differentiation into cartilaginous and bone cells in vitro in order to use this technology in the repair of damaged tissues. Here we describe the minimum requirements and an efficient method for isolation, expansion of mouse bone-derived multipotent mesenchymal stromal cells and their differentiation into osteoblasts, responsible for the bone matrix synthesis and mineralization.