The ability of brown adipocytes (fat cells) to dissipate energy as heat shows great promise for the treatment of obesity and other metabolic disorders. marker uncoupling protein 1 (UCP1), as well as other general adipocyte markers. Cells within microstrands were responsive to a -adrenergic agonist with an increase in gene expression of thermogenic UCP1, indicating that these Brown-Fat-in-Micrtostrands are functional. The ability to create Brown-Fat-in-Microstrands from pluripotent stem cells opens up a new arena to understanding brown adipogenesis and its implications in obesity and metabolic disorders. [1], and fail to fully recapitulate human adipocyte development [57] and metabolic processes [58]. Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells, provide a good model system for understanding early events in development [59C61] as well as an unlimited source of white, brown, and beige adipocytes [25,62C64]. The feasibility of generating brown or white adipocytes from human pluripotent stem cells has been demonstrated with up to 85C90% differentiation efficiency through cellular programming and transplantation techniques [25,58]. However, this approach includes multiple steps and relies on transferring exogenous genes to derive adipocytes from pluripotent stem cells. For instance, human pluripotent stem cells PF-2341066 are first differentiated into mesenchymal progenitor cells through embryoid body (EB) formation, followed by replating of EBs on gelatin-coated tissue culture dishes. Then, these mesenchymal progenitor cells are replated again and transduced with a lentivirus constitutively expressing the regulator genes of white or brown adipogenesis, respectively, followed by the addition of adipogenic factors such as insulin, dexamethasone, and rosiglitazone. In order to differentiate human pluripotent stem cells into functional, classic brown adipocytes without gene transfer, a specific hematopoietic cytokine cocktail has been used [63,65]. Differentiation in this manner also includes the formation of EB-like spheres as the very first step, and replating of these spheres on gelatin-coated tissue culture plates thereafter. Taken together, data from these techniques suggest that it would be beneficial PF-2341066 to recreate a three-dimensional (3D) microenvironment for pluripotent stem cell differentiation and adipogenesis [66], including BAT formation. Additionally, although BAT transplantation has been demonstrated for decades [67], cell necrosis often occurs upon transplantation of free fat, resulting in poor formation of PF-2341066 microvascular networks and graft resorption [68,69]. Altogether, there is a great need for a 3D culture system that could recreate the PF-2341066 microenvironment for BAT differentiation from pluripotent stem cells, recapitulating BAT function during culture, and provide a new vehicle to improve the stability and engraftment efficiency during BAT transplantation. We envision that cell encapsulation in alginate hydrogel microstrands could offer an effective 3D culture solution to address the needs for BAT differentiation and transplantation. Alginate is an FDA-approved biomaterial that has been demonstrated to be safe for drug delivery, stem cell culture, tissue engineering, and cell therapy [70C73]. The long tubular structure and small diameter (200 m) of alginate hydrogel microstrands can easily overcome the diffusion limitation that challenges the use of hydrogel microbeads for cell implantation [74], which allows for more efficient signaling, nutrient and oxygen exchanges, and support for high cellularity of stem cells grown in the tubular structure [75,76]. Additionally, these microstrands are easy to be handled for delivery by injection or implantation while maintaining their structural integrity. Moreover, alginate hydrogel microstrands exhibit great potential for reconstituting intrinsic morphologies and functions of living tissues [77,78]. The current approaches to fabricate hydrogel microstrands include utilizing coaxial flow and a microfluidic chip [79], flowing through a microfabricated SU-8 filter by a variety of techniques, including capillary force [75,76], wet spinning [80], composite techniques [81]. Here, we present a new microfluidic approach for cell encapsulation in alginate hydrogel microstrands, by simply driving an alginate solution to flow consistently into a calcium solution. In this study, we create Brown-Fat-in-Microstrands by encapsulating brown preadipocytes and pluripotent stem cells in 3D alginate hydrogel microstrands, and directly differentiating them into functional brown adipocytes. Mouse embryonic stem cells (ESCs) are used as model of pluripotent stem cells to test the feasibility of Mouse monoclonal to CD152 3D brown adipogenesis in alginate microstrands. PF-2341066 Mouse WT-1 brown preadipocytes are also grown within the same.