The Role Of Autophagy In Proliferation And Differentiation Of Porcine Pancreatic Stem Cells

Diabetes is one of the most emergencies around the world.It is a chronic disease which occurs when the body cannot release enough insulin or cannot use insulin effectively.It is diagnosed by the high glucose level in the blood.The constant high glucose level could affect heart,kidney,nerves and eyes,and could cause stroke and amputation.The main feature for diabetes is the shortage of pancreatic β cells.Porcine pancreatic stem cells can be differentiated into insulin producing cells and thus be treated as a promising approach for the treatment of diabetes because of the conserved insulin structure and similar blood glucose level between man and porcine.Autophagy is a highly conserved cellular mechanism which contribute to the turnover of proteins and organelles within the cell.It is reported that autophagy is important in maintaining the structure,number and functions of pancreatic stem cells.As processes of self-renewal and differentiation need strict control of proteins and organelles within the cell,autophagy is important for the two processes because it can degradate transcription factors and enzymes within the cells efficiently.But whether autophagy functions in pancreatic stem cells is still not clear.Our study explored the role of autophagy in proliferation and differentiation of pancreatic stem cells.Firstly,we used pancreatic stem cell line which could stably express EGFP-LC3 B to monitor the change of autophagosomes within the cell,and we prove that autophagy induced by serum starvation could stimulate proliferation of pancreatic stem cells.Then we assessed the role of autophagy in differentiation of pancreatic stem cells.We found that autophagy was induced during the later differentiation process,and inhibition of autophagy in this process could reduce the inducing efficiency.Our work may laid a foundation for a better understanding of mechanisms behind proliferation and differentiation of pancreatic stem cells.1.Autophagy stimulate proliferation of porcine pancreatic stem cellsFirstly,we evaluated the autophagic activity in cells of pPSCs-EGFP-LC3 B with confocal microscope and western blotting.We found that 4 hours’ starvation could activate autophagy and chloroquine could inhibit autophagy.Then to investigate role of autophagy in proliferation of pancreatic stem cells,BrdU incorporation assay,cell cycle analysis and western blotting were examined.We found percentage of BrdU positive cells and cells in S phase increased when autophagy was activated and those starved cells had higher level of PCNA.Percentage of BrdU positive cells and cells in S phase,and high level of PCNA would decrease after adding chloroquine.Meanwhile,we found active β-catenin changed with autophagy,as active β-catenin expressed more and located more in nucleus when autophagy was activated and would decrease after the treatment of chloroquine.These results indicate that autophagy stimulated proliferation of porcine pancreatic stem cells might be regulated by canonical Wnt signaling pathway.2.Autophagy contributes to the process of differentiating of pancreatic stem cells into insulin producing cellsFirstly,we successfully differentiated porcine pancreatic stem cells into insulin producing cells with the established inducing system in our lab.The islet like clusters we got from the process were DTZ positive,had high m RNA levels of insulin,Glut2,NKX6.1 and MafA,expressed proteins of insulin and C-peptide,and more importantly could release insulin under glucose challenge.Then we assessed autophagic activity during the inducing process with western blotting and found that autophagy was activated from day 4 to day 9.Day 4 is the day that we transfer induced cells from high attachment plates into ultr-low attachment plates.When we added chloroquine during day 4-10 to inhibit autophagy,these cells could not form islet like clusters.When we added chloroquine during day 5-10,clusters can be formed but cannot response to glucose challenge and release insulin.Meanwhile,active β-catenin show a similar trend with autophagy during the process,which means higher level of active β-catenin occurred when autophagy was activated and decreased level of activeβ-catenin occurred after inhibiting of autophagy.These results indicated that the impact of autophagy on the process of differentiation might be regulated by the canonical Wnt signaling pathway.