Application Of Stem Cell Derived β Cells And CRISPR Technology To Model And Correct Human Diabetes Mellitus

Diabetes as a class of metabolic diseases is currently affecting hundreds of millions of people worldwide.With the continuous improvement of human living standards,the incidence of diabetes in the world is still increasing year by year.Diabetes can be divided into type 1 and type 2 diabetes mellitus(T1DM and T2DM).Different pathogenic factors ultimately induce β cell deficiency or dysfunction to cause T1DM and T2DM.Therefore,it is becoming popular to reveal how β cells regulate its intracellular molecular mechanisms to respond to external stimulatory factors(such as glucose,fatty acids,immune cytokines,etc.)in diabetes research.Because hereditary permanent neonatal onset diabetes(PNDM)is a monogenic disease and tends to be more likely affected by intrinsic factors in βcells,PNDM models have become a powerful tool to investigate the mechanisms of β cell deficiency and dysfunction in diabetes,such as impaired insulin secretion.In recent years,the in-depth study of PNDM has greatly expanded our understanding of the pathogenesis of T2DM and other complex types of diabetes.At present,pluripotent stem cells based pancreatic differentiation technology is emerging as another major boost to human diabetes research and treatment.The technology enables researchers to obtain a large number of differentiated β cells from the patient or healthy human pluripotent stem cells for disease study and even for β cell transplantation to treat diabetes.Since the human β cells can be directly studied,translational barrier of research finding stemming from difference between human and animal model is expected to be overcome.As a pathogenic gene leading to PNDM,PERK works as an important signal transduction gene in the endoplasmic reticulum stress response(ER stress)and its loss-of-function results in Wolcott-Rallison syndrome(WRS)characterized by severe diabetes,skeletal dysplasia,and mental retardation.In this study,biopsy fibroblast cells from a WRS patient were reprogrammed into pluripotent stem cells,and the sequencing result found that the patient had four bases pair deletion with GAAA in the 9th exon of PERK gene.The CRISPR/Cas9 technique was used to introduce the patient’s specific mutation into the wild-type PERK gene of embryonic stem cell line Mell.Western blot experiments showed that the deletion of GAAA causing codon frame shift led to the failure of PERK protein translation.After differentiating wild-type and mutant pluripotent stem cells into pancreatic βcells,we found that the mutation did not affect β cells formation in vitro,but the total amount of insulin in PERK mutant β cells decreased as compared with wild-type.The observed increase of the ratio of pro-insulin to insulin and decreased insulin secretion indicate that the PERK gene mutation may cause diabetes by affecting the normal insulin synthesis and secretion in β cells.To understand the effects of the mutation on ER stress response and transcriptome in β cells,ER stress assays and RNA-seq were performed on both wildtype and mutant β cells.The results demonstrated that ER stress induced XBP1 splicing increased rapidly and mutated PERK failed to deposit inhibitory phosphorylation on EIF2αupon ER stress.Consistently,transcriptome analysis found that the cellular pathways related to SRP-dependent co-translational proteins targeting to ER membrane were significantly enriched after PERK mutation.These findings provide an important clue for further elucidation of how ER stress contributes to human β cell deficiency and dysfunction.Treating diabetes by β cell transplantation has been the focus of scientists for a long period,but significant progress has not been made until recently.Our study combined stepwise pancreatic differentiation protocol and CRISPR/Cas9 genome editing technology to explore the possibility of treating monogenic diabetes,especially PNDM.Firstly,somatic cells of a PNDM patient were obtained to generate induced pluripotent stem cells.Then a single base mutation in the first codon of INS gene was identified in this patient.Both CRISPR corrected stem cells and mutant cells were differentiated into β cells.It turned out that mutant β cells failed to produce insulin and C peptides,but the corrected ones restored insulin protein expression.After transplantation of mutant and corrected human β cells into a diabetic mouse model,only corrected cells were able to rescue the diabetic phenotype in mice.The results indicated that gene repaired β cells hold promising potential for treating monogenic diabetes.In summary,by using human induced pluripotent stem cell,pancreatic differentiation and CRISPR technology,we established an in vitro cell model of PNDM caused by the patient’s specific mutation in PERK gene(also known as EIF2AK3),and analyzed the effect of PERK mutation on the function of human β cells.We also identified a new mutation in INS gene that led to PNDM,and successfully corrected the mutation and repaired the mutant β cell by CRISPR/Cas9 technology,laying the foundation for the use of in vitro differentiated β cells transplantation to treat diabetes.Our study demonstrated the feasibility and great application values of in vitro pancreatic differentiation technology for modeling human disease and conducting cell therapy for diabetes.