Bone-marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate into a number of phenotypes, including adipocytes. Adipogenic differentiation has traditionally been performed in monolayer culture, and, while the expression of a fat-cell phenotype can be achieved, this culture method is labor and material intensive and results in only small numbers of fragile adherent cells, which are not very useful for further applications. Aggregate culture is a cell-culture technique in which cells are induced to form three-dimensional aggregates; this method has previously been used successfully, among others, to induce and study chondrogenic differentiation of MSCs. We have previously published an adaptation of the chondrogenic aggregate culture method to a 96-well plate format. Based on the success of this method, we have used the same format for the preparation of three-dimensional adipogenic cultures. The MSCs differentiate rapidly, the aggregates can be handled and processed for histologic and biochemical assays with ease, and the format offers significant savings in supplies and labor. As a differentiation assay, this method can distinguish between degrees of senescence and appears suitable for testing medium or drug formulations in a high-volume, high-throughput fashion. 1. Introduction Much of the research on adult mesenchymal stem cells (MSCs) has been done on bone-marrow-derived populations. First described by Owen and Friedenstein [1], and later more fully characterized by other groups, these cells possess, to some degree, and for a number of population doublings, the defining properties of stem cells, that is, the ability to self-renew and the potential to differentiate along one or more lineages under appropriate culture conditions [1–5]. The chondrogenic, osteogenic, and adipogenic lineages are well documented, but there are likely others [6–10]. The emerging and potentially useful properties of MSCs include their paracrine effects, which may augment the repair of damaged tissues, and their immunosuppressive abilities [11, 12]. With respect to the adipogenic lineage specifically, Mackay et al. have shown that human MSC-(hMSC-) derived adipocytes, express mRNA encoding for adipogenic transcription factors (PPARγ2, C/EBPα, and SREBP1), adipokines (adipsin, leptin, APM1, and angiotensinogen), and lipid-metabolizing agents (aP2 and LPL) by day 12 of differentiation and are thus highly analogous to subcutaneous adipocytes at this time point [13]. For most of the clinical applications envisioned, a very large number of MSCs will be
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