Engineered human brown adipocyte microtissues improved glucose and insulin homeostasis in high fat diet-induced obese and diabetic mice

A large population of people is affected by obesity (OB) and its associated type 2 diabetes mellitus(T2DM). There are currently no safe and long-lasting anti-OB/T2DM therapies. Clinical data and preclinical transplantation studies show that transplanting metabolically active brown adipose tissue (BAT) is a promising approach to prevent and treat OB and its associated metabolic and cardiovascular diseases. However, most transplantation studies used mouse BAT, and it is uncertain whether the therapeutic effect would be applied to human BAT since human and mouse BATs have distinct differences. Here, we report the fabrication of three-dimensional (3D) human brown adipose microtissues, their survival and safety, and their capability to improve glucose and insulin homeostasis and manage body weight gain in high-fat diet (HFD)-induced OB and diabetic mice. Methods3D BA microtissues were fabricated and transplanted into the kidney capsule of Rag1-/- mice. HFD was initiated to induce OB 18 days after transplantation. A low dose of streptozotocin (STZ) was administrated after three months HFD to induce diabetes. The body weight, fat and lean mass, plasma glucose level, glucose tolerance and insulin sensitivity were recorded regularly. In addition, the levels of human and mouse adipokines in the serum were measured, and various tissues were harvested for histological and immunostaining analyses. ResultsWe showed that 3D culture promoted BA differentiation and uncoupling protein-1 (UCP-1) protein expression, and the microtissue size significantly influenced the differentiation efficiency and UCP-1 protein level. The optimal microtissue diameter was about 100 {micro}m. Engineered 3D BA microtissues survived for the long term with angiogenesis and innervation, alleviated body weight and fat gain, and significantly improved glucose tolerance and insulin sensitivity. They protected the endogenous BAT from whitening and reduced mouse white adipose tissue (WAT) hypertrophy and liver steatosis. In addition, the microtissues secreted soluble factors and modulated the expression of mouse adipokines. We also showed that scaling up the microtissue production could be achieved using the 3D suspension culture or a 3D thermoreversible hydrogel matrix. Further, these microtissues can be preserved at room temperature for 24 hours or be cryopreserved for the long term without significantly sacrificing cell viability. ConclusionOur study showed that 3D BA microtissues could be fabricated at large scales, cryopreserved for the long term, and delivered via injection. BAs in the microtissues had higher purity, and higher UCP-1 protein expression than BAs prepared via 2D culture. In addition, 3D BA microtissues had good in vivo survival and tissue integration, and had no uncontrolled tissue overgrowth. Furthermore, they showed good efficacy in preventing OB and T2DM with a very low dosage compared to literature studies. Thus, our results show engineered 3D BA microtissues are promising anti-OB/T2DM therapeutics. They have considerable advantages over dissociated BAs or BAPs for future clinical applications in terms of product scalability, storage, purity, quality, and in vivo safety, dosage, survival, integration, and efficacy.