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Multipotent mesenchymal stem cells (MSCs) are an attractive candidate for regeneration of damaged cells, tissues, and organs. Due to limited availabilities, MSC populations must be rapidly expanded to satisfy clinical needs. However, senescence attributed to extensive in vitro expansion compromises the regenerative and therapeutic potential of MSCs. In this chapter, we describe a step-by-step protocol that aims to induce adipogenic and osteogenic differentiation of in vitro aged human MSCs and highlight noteworthy issues that may arise during the process.

Background: Adult mesenchymal stem cells (MSCs) hold great promise for regenerative medicine because of their self-renewal, multipotency, and trophic and immunosuppressive effects. Due to the rareness and high heterogeneity of freshly isolated MSCs, extensive in-vitro passage is required to expand their populations prior to clinical use; however, senescence usually accompanies and can potentially affect MSC characteristics and functionality. Therefore, a thorough characterization of the variations in phenotype and differentiation potential of in-vitro aging MSCs must be sought., Methods: Human bone marrow-derived MSCs were passaged in vitro and cultivated with either DMEM-based or αMEM-based expansion media. Cells were prepared for subculture every 10 days up to passage 8 and were analyzed for cell morphology, proliferative capacity, and surface marker expression at the end of each passage. The gene expression profile and adipogenic and osteogenic differentiation capability of MSCs at early (passage 4) and late (passage 8) passages were also evaluated., Results: In-vitro aging MSCs gradually lost the typical fibroblast-like spindle shape, leading to elevated morphological abnormality and inhomogeneity. While the DMEM-based expansion medium better facilitated MSC proliferation in the early passages, the cell population doubling rate reduced over time in both DMEM and αMEM groups. CD146 expression decreased with increasing passage number only when MSCs were cultured under the DMEM-based condition. Senescence also resulted in MSCs with genetic instability, which was further regulated by the medium recipe. Regardless of the expansion condition, MSCs at both passages 4 and 8 could differentiate into adipocyte-like cells whereas osteogenesis of aged MSCs was significantly compromised. For osteogenic induction, use of the αMEM-based expansion medium yielded longer osteogenesis and better quality., Conclusions: Human MSCs subjected to extensive in-vitro passage can undergo morphological, phenotypic, and genetic changes. These properties are also modulated by the medium composition employed to expand the cell populations. In addition, adipogenic potential may be better preserved over osteogenesis in aged MSCs, suggesting that MSCs at early passages must be used for osteogenic differentiation. The current study presents valuable information for future basic science research and clinical applications leading to the development of novel MSC-based therapeutic strategies for different diseases.

Background: Mesenchymal stem cells (MSCs) are viewed as a having significant potential for tissue engineering and regenerative medicine therapies. Clinical implementation of MSCs, however, demands that their preparation be stable and reproducible. Given that environmental and bioprocessing parameters such as substrate stiffness, seeding densities, culture medium composition, and mechanical loading can result in undirected differentiation of the MSC population, the objective of this study was to systematically investigate how hydrodynamic loading influences the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) towards the osteochondral lineages both in the presence and absence of exogenous, inductive factors., Methods: Expanded bovine MSCs were suspended in 2.5 % agarose, cast in a custom mold, and placed into either static or one of two dynamic culture environments consisting of "high" and "low" magnitude shear conditions. Constructs were supplemented with varying concentrations (0, 1, 10, 100 ng/mL) of either TGF-β3 or BMP-2 throughout cultivation with tissue samples being collected following each week of culture., Results: In the absence of exogenous supplementation, hydrodynamic loading had little effect on cell phenotype at either magnitude of stimulation. When cultures were supplemented with BMP-2 and TGF-β3, MSCs gene expression progressed towards the osteogenic and chondrogenic pathways, respectively. This progression was enhanced by the presence of hydrodynamic loading, particularly under high shear conditions, but may point the chondrogenic cultures down a hypertrophic path toward osteogenesis reminiscent of endochondral ossification if TGF-β3 supplementation is insufficient., Conclusions: Moving forward, these results suggest bioprocessing conditions which minimize exposure of chondrogenic cultures to fluid shear stress to avoid undesirable differentiation of the MSC population.

Controlled differentiation of mesenchymal stem cells (MSCs) into the chondrogenic lineage is crucial for in vitro generation of neocartilage, yet achieving it remains challenging. Traditional protocols for MSC differentiation using exogenous inductive molecules, such as transforming growth factor-β, fall short in meeting the needs of clinical applications because they yield differentiated cells that exhibit hypertrophic characteristics and subsequently facilitate endochondral bone formation. The objective of the current study was to deliver endogenous inductive factors from juvenile articular chondrocytes to bone marrow-derived MSCs to drive MSC chondrogenic differentiation through cocultivation of the two cell types in the absence of direct physical contact and exogenous stimulators. An initial chondrocyte/MSC ratio of 63:1 was identified as the appropriate proportion of the two cell populations to ensure that coculture-driven MSC-differentiated (CDMD) cells replicated the cellular morphology, behavior, and phenotype of articular chondrocytes. In a three-dimensional agarose system, CDMD cells were further shown to develop into robust neocartilage structurally and mechanically stronger than chondrocyte-laden constructs and with reduced hypertrophic potential. Although MSCs tended to lose the ability to express CD44, an important regulator in cartilage biology, during the coculture induction, CDMD cells regained this function in the three-dimensional tissue cultivation. The present work establishes a chondrocyte/MSC coculture model that serves as a template to better understand chondrocyte-driven MSC differentiation and provides insights for improved strategies to develop clinically relevant cartilage tissue replacements.