Hyperglycemia Impaired Early Pancreatic Differentiation of Human Embryonic Stem Cells Through Aberration of DNA Methylation Patterns

Abstract

The prevalence of gestational diabetes ranged from 5% to 10% in different ethnics raised global concern [1, 2]. Fetal exposure to hyperglycemic intrauterine environment was correlated with an increased incidence of complications such as impaired glucose tolerance and type II diabetes later in life [3]. Although previous studies in animal models showed the involvement of alterations in DNA methylation of pancreatic genes [4, 5], the molecular mechanisms on this association in human remained largely unknown. We therefore hypothesized maternal hyperglycemia dysregulated human fetal pancreatic development through aberration in DNA methylation. In our study, a human embryonic stem cell (hESC) line, VAL3, was used as an in-vitro model for pre-implantation embryo. To simulate hyperglycemic intrauterine environment, VAL3 was cultured under hyperglycemic condition and subjected to DNA methylome profiling. Hyperglycemia resulted in 1,391 hypermethylated (annotated to 788 genes) and 1,284 hypomethylated (annotated to 924 genes) CpG sites in VAL3. Gene ontology analysis revealed that the hypermethylated genes were significantly enriched in two biological processes, embryonic morphogenesis and cell fate commitment. Interestingly, some of the genes (NKX6-2, SOX9, TBX1, EXT1) were reported to be crucial for fetal pancreas specification [6]. Targeted bisulfite sequencing validated the promoter region of NKX6-2, a gene important for pancreatic -cell specification, was significantly hypermethylated upon hyperglycemia treatment. Next, VAL3 was differentiated into early intermediates lineages (definitive endoderm: DE; pancreatic progenitor: PP) along the pancreatic pathways. It was found that hyperglycemia treated VAL3 had impaired early pancreatic differentiation. The mRNA expressions of intermediate markers (DE: SOX17, FOXA2, CXCR4 and EOMES; PP: NKX6-2) were significantly downregulated even when differentiation was conducted in normal glucose level. This is the first study to profile the global DNA methylation changes in hESCs upon hyperglycemic treatment. Our present findings also suggested transient hyperglycemia treatment of hESCs led to impairment of early pancreatic differentiation.