Regulation of in Vitro Glucose Levels Improve Islet Precursor Production from Human Pluripotent Stem Cells

Since its discovery as a hormone in 1922, injected insulin has been used as the primary therapy for diabetic patients for managing glucose levels. While this approach has been saved millions of lives, millions of patients struggle to regulate their blood glucose. The need for a cure for diabetes is significant. Whole pancreas and isolated islet transplants have proven successful in durable reversal of diabetes in patients with islet loss from type 1 diabetes (T1DM) and severe pancreatitis, but the supply of donor tissue falls well short of the numbers needed for patients Thus, the need for alternative cell sources for the replacement of islet cells in diabetic patients emphasizes the importance of pluripotent stem cells, as these may present the best opportunity for manufacturing islet cells most suitable for transplantation. Numerous studies have demonstrated that small-scale iPS cell cultures can be directed to differentiate into functional endocrine cells by manipulating the culture conditions to mimic the process of pancreatic development. Human iPS cell lines have been generated from skin cells of both diabetic and non-diabetic donors. Regardless of the diabetic status of the donor, both types of derived cell lines have demonstrated the ability to differentiate into insulin-producing islet cells. However, as many as 10⁹ islet precursorcells might be needed for transplant for each transplant. Historically, iPS differentiation has been performed in small-scale static cultures. However, large-scale culture strategies must be implemented to provide the higher cell yield in order to obtain enough cells for clinical applications. Therefore, before stem cell derivatives can be used for clinical purposes, procedures must be developed to generate large numbers of functional cells for pre-clinical and human trials. Our preliminary studies have demonstrated that in stirred bioreactors, 1) Insulin-producing islet cells are lost during late maturation stages, and 2) Survival and proliferation are increased in cultured beta cells when glucose levels are maintained within physiological levels. In this study, stirred suspension bioreactors were modified to allow regulation of nutrient levels, and physiological glucose level (about 100mg/dL) were maintained by continuous feeding, with feed rate adjustments based on actual glucose consumption. The use of adjusted rate perfusion feeding significantly increased precursor numbers and insulin production without altering differentiation patterns in large-scale cultures.