Embryonic Stem Cells: Differentiation into Insulin Producing Cells and Elimination of Damaged Proteins

Sammanfattning: This thesis includes two different parts: One focusing on how to induce human embryonic stem cells (hESCs) to differentiate into insulin producing cells by following the normal pancreatic development pathway. These cells have then the potential to be an unlimited source for diabetes regenerative medicine. The second part of the thesis deals with how embryonic stem cells rid themselves of damaged protein. We have discovered that stem cells have a unique mechanism for doing this. Spontaneous differentiation of hESCs (dhESCs) under two-dimensional in vitro growth conditions resulted in differentiation of pancreatic progenitors (Pdx1+/Foxa2+) and endocrine progenitors (Pdx1+/Isl1+) but not into insulin producing cells. However, co-transplantation of dhESCs with pancreatic dorsal bud and epithelium free from mesenchyme, but not with liver or telencephalon, from mouse embryos resulted in derivation of human insulin producing cells clusters with beta cell characteristic. Comparative analysis of these cells with human adult islets demonstrated that the insulin positive cells share important features with normal beta cells, such as synthesis (proinsulin) and processing (C-peptide) of insulin and nuclear localization of key beta cell transcription factors, including Foxa2, Pdx1, Nkx6.1, and Isl1. These results suggest that both the environment of the kidney capsule and instructive signals form embryonic pancreatic bud and epithelium are required to induce dhESCs into insulin producing cells. Unexpectedly, we found that undifferentiated mouse ESCs contain high levels of both carbonyls and AGEs. The damaged proteins, identified as chaperones and proteins of the cytoskeleton are the main targets for protein oxidation in aged tissues. However, our results show that mouse ESCs rid themselves of such damage upon differentiation in vitro and in vivo. This elimination of damaged proteins coincides with a considerably elevated activity of the 20S proteasome. This clear-out of damaged proteins may be a part of a previously unknown rejuvenation process at the protein level and makes it worth considering that the offspring of mammals may initially be free of protein damage because of an early developmental damage elimination rather than by a mechanism that keeps the germ-line cells free of destructive molecules.