CRISPR-Cas9 Gene Editing Restores Beta-Cell Differentiation and Function in Patient-Specific iPSCs

Abstract

Genetic knockout studies in mice concluded that the differentiation of endocrine lineages in the pancreas and intestine requires the transcription factor neurogenin3 (NGN3). However, case reports of patients with NGN3 mutations show that most patients do not develop diabetes until later in childhood, suggesting that NGN3 may not be required for human beta cell differentiation in vivo. We have identified a patient with a previously undescribed NGN3 loss-of-function mutation and generated induced pluripotent stem cells from patient fibroblasts to analyze the role of NGN3 in human beta cell differentiation. We used established protocols to differentiate patient-specific iPSCs (PS-iPSCs) into pancreatic progenitor cells and beta-like cells in parallel with the H1 hESC line. The PS- iPSCs displayed significantly lower pancreatic progenitor cell differentiation with only 10.7% of the double positive PDX1+/NKX6.1+ cell population present, whereas the control H1s hESCs produce 60% double positive cells. After further differentiation into beta-like cells, H1 cells produced multiple endocrine cell types, while the PS-iPSC line was not able to make significant numbers of endocrine cells. To assess if the loss of NGN3 was the primary cause for the loss of pancreatic progenitor cells and beta-like cell differentiation, we used CRISPR-cas9 gene editing to correct the patient’s mutation in the PS-iPSCs (cPS-iPSCs). Correcting the patient’s mutation restored the PDX1+/NKX6.1+ cell population in the pancreatic progenitor stage. Beta-like cells differentiated from cPS- iPSCs were capable of glucose responsive insulin secretion at the end of the differentiation protocol. In conclusion, our data suggests that NGN3 may have a previously unidentified role important for the differentiation of pancreatic progenitor cells and that there may be a relationship between NKX6.1 and NGN3 that is critical for pancreatic development. Disclosure K. Millette: None. K.R. Vogt: None. A. Salas: None. P. Pitukcheewanont: None. J. Austin: None. M. Martin: None. S. Georgia: None.