Type I diabetes (T1D) is an autoimmune disorder that leads to the destruction of pancreatic β-cells. The consequences of T1D are dysregulation of metabolism leading to cardiovascular complications, neuropathy and renal failure. Islet transplantation is the most effective way to treat T1D. Unfortunately, the shortage of cadaveric organs is a major obstacle to the effective management of T1D patients. An alternative source of insulin producing cells (IPCs) could significantly improve patient treatment. We seek to establish human induced pluripotent stem (iPS) cells as a novel source of IPCs that are patient-specific, obviating the need for immunosuppression. If successful, such a novel alternative could provide for an unlimited source of IPCs.

Here, we established a robust 3D culture protocol for differentiating a T1D patient's iPS cells into IPCs. Using a carefully optimized protocol, we drive the iPS cells through 5 stages of differentiation before they become glucose-responsive insulin producing cells. First, the iPS cells are converted into definitive endoderm (DE) cells that are >95% CXCR4⁺Sox17⁺. Subsequently, we are able to differentiate these cells into Pdx1⁺ pancreatic precursor cells (>90%) using Retinoic Acid and small molecule blockade of hepatic differentiation cues, such as Shh and BMP4. These cells are then coaxed into endocrine precursor cells using Notch signaling inhibition. Finally, they become >50% insulin⁺ IPCs with treatment of IGF-1, nicotinamide and GLP-1, which is an incretin that promotes islet proliferation and pancreatic β cell function.

The cell clusters develop into tight large spheroids that strongly express insulin as well as pancreatic β cell transcription factors such as Nkx6.1 and Nkx2.2. Additionally, these IPCs secrete insulin in vitro after high glucose stimulation. When transplanted into diabetic mice, IPCs correct hyperglycemia and form distinct organoids that are highly vascularized. Therefore, our data demonstrate that dermal fibroblasts can be reprogrammed into iPS cells, which generate functional, glucose-responsive IPCs. This strategy allows for the unlimited generation of IPCs with the potential for long-term clinical management of T1D patients.

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