Differentiation Of Human Pluripotent Stem Cells Into Pancreatic Progenitor Cells

Objective Diabetes mellitus is characterized by hyperglycemia due to dysfunctional insulin production or insufficient insulin utilization.Diabetes can be simply classified as type I(Insulin dependent diabetes)and type II(Non-insulin dependent diabetes).Longterm hyperglycemia will eventually lead to the loss of beta cells function and severe metabolic syndrome such as cardiovascular disease,diabetic eye disease,kidney disease,nerve damage and diabetic foot.Diabetic syndrome greatly decreases the life quality for patients and also raises a lot of economic burden for family and sociality.There are around 425 million diabetes patients in the world and about 4 million people died of diabetes and its complications in 2017.The risk of diabetes is not only a threat to human health,but also has become a global social crisis.The traditional methods treating diabetes include being on diet,burdensome daily insulin-sensitizing drugs,insulin injection and insulin pump which can only alleviate symptoms of high blood sugar,but cannot fundamentally cure diabetes.Islet transplantation combining with proper immunorepression recipe provides an effective and reliable strategy by replacing the damaged or lacked islet cells and has achieved great control of glycemia without insulin injection,but it is largely limited by cadaveric islets source.Human pluripotent stem cells,including embryonic stem cells(ESCs)and induced pluripotent stem cells(i PSCs),could form multiple cell types and tissues composing of our body.Therefore,production of functional beta cells from human ESCs or i PSCs could be a promising possibility for the curing of diabetes.How to obtain adequate,stable,functional beta cells is one of the hot topic in current reseach.BMP,WNT,SHH and other signaling pathways developmentally regulate the endodermal lineage patterning and thus study of the signaling pathways crosstalk should facilitate pancreatic differentiation.This thesis will focus on the signaling pathways regulation to improve the pancreatic progenitor cells and further obtain more beta cells.In addition,we have evaluated the supporting of chitin for ESCs and our preliminary data suggests chitin could be used in 3D culture of insulin-positive beta cells in vitro.Method Through the test of cultivation time for differentiation of pancreatic progenitor cells and RNA-sequencing analysis,we manipulated the signals during endodermal development.Flow cytometry,immunofluorescence staining and q PCR were used to measure the expression levels of pancreatic progenitor cells marker andthe other lineages marker of endoderm.q PCR was used to detect m RNA expression of WNT signaling between control group and BMP inhibition group.Immunofluorescence staining and Western blotting were used to test the protein change between control group and BMP inhibition group.q PCR and immunofluorescence staining were used to observe the beta cells differentiation from pancreatic progenitor cells.We also tested the result in i PSCs.MTT detected the survival of PANC1,L02 and ESCs cultured with the biomaterial chitin.The location of ESCs and chitin were stained by Calcein-AM/PI.The supporting of chitin for spontaneous differentiation of ESCs was determined by q PCR.Results After testing of cultivatin time and multiple signals during endoderm development,we identified the most potent chemicals,BMP inhibitors K02288 and LDN193189,which both improved the pancreas duodenum hox 1(PDX1)expression during pancreatic progenitor cells differentiation.Upon treated with the BMP inhibitor LDN193189,liver-specific facter alpha fetoprotein(AFP)decreased.Meanwhile,the intestinal key transcriptional factor caudal-related homeobox gene(CDX2)had also declined.Downstream gene expression of WNT signaling and the location of beta-catenin showed that the activity of WNT down-regulation after BMP inhibition.Coordination of WNT and BMP inhibition could improve PDX1 expression of pancreatic progenitor cells,as well as the insulin-positive beta cells generation by following differentiation.For the chitin test,we found PANC1,L02,ESCs could be supported by chitin,furthermore,ESCs could proliferate and differentiate with chitin.Conclusion Inhibition of BMP improved the generation of PDX1-positive cells during definitive endoderm cells to pancreatic progenitor cells;BMP inhibition led to suppressed WNT/beta-catenin signaling activity during pancreatic differentiation;Inhibition of BMP and WNT could promote PDX1 expression of pancreatic progenitor cells;Further differentiation of pancreatic progenitor cells could generate beta cells.Chitin could be used as material for embryonic stem cells proliferation and differentiation.