Wnt/β-catenin Signaling Pathway Promotes The Differentiation Of Mouse Embryonic Stem Cells Into Insulin Producing Cells

Diabetes has become one of the most important diseases endangering human health,and with improvements to standards of living,increasing stress and the influence of genetic factors,the incidence of diabetes is increasing.One of the main factors in the development of diabetes is insufficient insulin secretion in the body.Although there are many treatment methods for this disease,these traditional treatment methods can not fundamentally cure diabetes,resulting in lifelong patient reliance on drug;the identification of new alternative treatments has therefore become one of the main goals of research on diabetes.Embryonic stem cells are derived from the inner cell mass of the preimplantation blastocyst and can undergo self-replicating proliferation(self-renewal)in vitro while maintaining the potential to develop into various types of cells from different germ layers,such as the nerves,myocardium,and liver.Embryonic stem cells are of great value for clinical and transformation applications.Although many previous studies have reported the differentiation of embryonic stem cells into insulin-secreting cells,the functions of these cells are poorly understood,the efficiency of this differentiation requires improvement,and regulatory mechanisms for this differentiation have not been reported.Previous studies have reported the addition of three different solutions,named solution 1,solution 2 and solution 3(see the experimental method section for the specific formulation),in three stages of the process by which mouse embryonic stem cells differentiate into insulin-secreting cells,allowing the acquisition of functional insulin-secreting cells.Based on this protocol,we chose to study the function of theWnt/β-catenin signaling pathway in this process,as the Wnt/β-catenin signaling pathway plays an important role in the differentiation of embryos into mesendoderm.In the presence of extracellular Wnt protein,the GSK3,Axin and CK1 kinases can not form a complex for the degradation of β-catenin,and β-catenin stably accumulated in the body and entered the nucleus to activate downstream target gene expression.Therefore,in the three stages of mouse embryonic stem cell differentiation into insulin-secreting cells,Wnt signaling was activated by the addition of a small-molecule inhibitor of the GSK3 protein,CHIR99021,at a final concentration of 3 μM in the three different solutions.In the first and third stage of the differentiation process,the expression of the insulin precursor cell marker gene Pdx1 and the insulin-encoding gene Insulin was increased.These results indicated that CHIR99021 can enhance the differentiation of mouse embryonic stem cells into islet cells after the activation of Wnt signaling.To further confirm that CHIR99021 functions by activating the Wnt/β-catenin signaling pathway,we performed two experiments.First,using a pLKO.1 lentiviral plasmid-mediated system,lentivirus targeting the β-catenin gene was packaged and used to infect mouse embryonic stem cells in stages 1 and 3.Addition of the β-catenin gene to lentivirus in solution 1 in the first stage or solution 3 in the third stage,the expression of the Pdx1 and Insulin genes reduced in the obtained cells,indicating that inhibition of β-catenin activity can impair the function of CHIR99021 in mouse embryonic stem cell differentiation.Second,to verify that upregulation of the β-catenin gene can mimic the function of CHIR99021,we constructed a β-catenin gene-inducible expression plasmid that was introduced into cells using the Tet-On inducible expression system.The addition of doxycycline at a final concentration of 2 μg/ml could induce expression of the β-catenin protein.Therefore,we added doxycycline to solutions 1 and3 in the first and third stages,respectively,of the differentiation of mouse embryonic stem cells into insulin-secreting cells.The addition of this compound upregulated the expression of Pdx1 and Insulin in insulin-secreting cells,indicating that the activation ofβ-catenin can mimic the function of CHIR99021 and thereby increase the efficiency of mouse embryonic stem cell differentiation into insulin-secreting cells.In summary,we have optimized the efficiency of mouse embryonic stem cells differentiation into insulin-secreting cells.The results of this study will help optimize the differentiation of human stem cells into islet cells and provide useful insights in obtaining more islet cells to effectively lower blood glucose levels.Finally,these results will help to find a new treatment for diabetes in the future to benefit patients.