**1. Introduction**

Mesenchymal stem/stromal cells (MSCs), a multipotent stem/progenitor cell type, were initially described in bone marrow by Friedenstein et al. as rapid adherence to tissue culture vessels and the discrete "fibroblast" colonies approximately 50 years ago [1, 2]. Julius Cohnheim, a German-Jewish pathologist, firstly proposed that a fibroblast-like cell population for nonhematopoietic cells in bone marrow were involved in wound repair over 150 years ago [3]. In the late 1980s, Caplan firstly coined the name

"mesenchymal stem cell (MSC)" [4]. Since then, MSCs have gained much attention over the last three decades. Many laboratories focusing on MSCs have developed diverse methods to isolate and expand MSCs from a variety of tissues. While the assessment of characteristics of MSCs is necessitated in different platforms/laboratories, most researchers come to acknowledge the lack of a universally accepted criteria to define MSCs. To address this question of cell equivalence, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) proposes three minimal criteria to define MSCs [5]: property of MSC plastic adherence, the expression of specific cellular surface antigen, and capacity for trilineage mesenchymal differentiation (osteogenesis, chondrogenesis and adipogenesis).

Human MSCs from different tissues have the varied phenotypic features, the morphologic inconsistency, and heterogeneous functional behavior [6–8]. Indeed, the properties of stem cell have not been well established yet. Due to the unknown *in vivo* multipotent properties of MSCs, the issue of MSC nomenclature remains actively controversial. In 2019, ISCT MSC committee issued a position statement on nomenclature of MSCs clarifying the functional definition to emphasize the functional distinction of mesenchymal stem versus stromal cells [9].

MSCs have been considered as a promising therapeutic tool in tissue engineering and regenerative medicine. MSCs are well known to be present in almost every type of adult tissues, such as bone marrow [10–12], adipose tissue [10, 13, 14], lung [11, 15], synovial tissue [16, 17], dental pulp and periodontal ligament [18]. Notably, it has become apparent that MSCs are identified in the various human embryonic tissues, such as fetal bone marrow [19], fetal liver [20], aorta gonad-mesonephros and yolk sac [21], as well as multiple neonatal birth-associated tissues, such as placenta [10, 22, 23], amniotic and chorionic membrane [23, 24], umbilical cord tissue [10, 23–25], and umbilical cord blood [26, 27]. Therefore, different platforms/laboratories may use different type of tissue sources and methodologies for isolation and expansion of MSCs. This chapter firstly outlines protocols for standardized isolation and expansion of human bone marrow-derived MSCs (BM-MSCs), a major source of human MSCs, as well as BM-MSCs' characteristics, cryopreservation and thawing. Protocols for the preparation of MSCs derived from the other tissue types are similar to that of BM-MSCs, except tissue sample processing differentially. Human BM-MSCs are estimated at a very low frequency at approximately 0.001–0.01% of total nucleated cells [28, 29], and, therefore, human BM-MSCs are likely to be kind of difficult to isolate and harvest. This chapter will then focuses on optimal functional assays and application on the basis of our previous studies, which would be useful for researchers working with MSCs in basic research and translational and clinical applications, such as osteogenesis, chondrogenesis, adipogenesis, colony forming unit-fibroblast (CFU-F) assay, 3-D cellular co-culture, MSC homing and migration. Last but not least, the long-term culture associated alterations of MSCs' properties will be also discussed in this chapter.
