Enhancing the Functionality of Human Bone Marrow-Derived Mesenchymal Stromal Cells Using Signaling Modulators.

Bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) possess an inherent capacity to support hematopoiesis in vitro. As a result, they are often used as feeder layers for the ex vivo expansion of hematopoietic stem cells (HSCs) or coinfused with HSCs to improve the success of clinical transplants. However, MSCs isolated from non-hematopoietic tissues such as adipose or dental tissue may not be as effective as BM-MSCs in supporting hematopoiesis. Additionally, BM-MSCs tend to lose their functional ability after extended in vitro culture, which is necessary for producing sufficient cell numbers for clinical use. These limitations underscore the importance of pretreating MSCs with suitable signaling modulators to boost their functionality. Reliable in vitro assays play a crucial role in finding such signaling compounds and evaluating their effects on MSCs. Conventional assays, such as long-term-culture-initiating assay (LTC-IC assay) or colony-forming-unit assay (CFU assay), chemotactic migration assays (e.g., Transwell assay), etc., primarily evaluate the functional competence of HSCs and HSPCs, but provide little insight into the hematopoiesis-supportive capacity of MSCs. This chapter describes two such assays: one is designed to assess the hematopoietic stem/progenitor cell (HSPC)-supporting capacity of primed MSCs, and the other is specifically aimed at their ability to support the growth of primitive HSCs. These assays can serve as initial screens to test the impact, whether positive or negative, synergistic or antagonistic, of known signaling compounds, bioactive peptides, growth factors, and cytokines on MSC function or to discover new entities that could boost the functionality of MSCs.