An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells

Abstract

The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have developed an abbreviated five-stage protocol (25–30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP, SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL, and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion, targeting selected signaling pathways for 25–30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs, small molecules or genes that may have potential to influence beta-cell function.

GABA promotes β‐cell proliferation, but does not overcome impaired glucose homeostasis associated with diet‐induced obesity

“…The protocols for quantitative and digital droplet PCR (ddPCR) have been previously described (28,29). The comparative cycle threshold (2 2DDCt ) method was applied to calculate the relative expression of target gene in each sample, relative to control in the quantitative PCR.…”

Single-Cell RNA-seq Reveals a Subpopulation of Cells Underlying β Cell Expansion in the Postnatal Islets

“…These data imply that while promoting cell proliferation, the Notch pathway also helps keep endocrine cells at an immature state during development. Interestingly, γ-secretase inhibitors, by blocking the Notch pathway, have been recently introduced into several protocols designed for in vitro generation of functional β cells from human pluripotent stem cell, applied at the last stages of the in vitro culture to promote β cell maturation (Pagliuca et al 2014;Rezania et al 2014;Massumi et al 2016).…”

Glucose‐Responsiveness of Pancreatic β‐Like (GRP β‐L) Cells Generated from Human Pluripotent Stem Cells

“…At the end of stage 4, cells express NGN3 and NKX6.1 genes. These cells persist expression of PDX1 and indicate high expression levels of FOXA2, HNF4, GATA4, ISL1 , and NeuroD1 markers as well (Massumi et al., ; Rajaei et al., ). In the last stage, about 50% of the cells express C‐Peptide and few express Somatostatin.…”

“…The glucose‐stimulated insulin secretion assay as static and dynamic demonstrated the responsiveness of the GRPβ‐L cells to the glucose concentration by increasing the insulin secretion. We showed that the secretion of the insulin is regulated by an increase in the intracellular Calcium (Massumi et al., )…”

“…As previously described by Russ et al., we similarly observed that inhibition of TGF‐beta (ALK4, 5, and 7) and BMP signaling resulted in a high number of NGN3 + /NKX6.1 + endocrine progenitors (Russ et al., ). Further differentiation of the endocrine progenitors with the combination of small molecules, including ALK5 inhibitor II, thyroid hormone (T3), Notch, and receptor AXL inhibitor, led to generation of Insulin + /NKX6.1 + /MAFA + cells in a significant proportion of the differentiated cell population (Massumi et al., ; Rezania et al., ). Moreover, in vitro analyses of the generated GRPβ‐L cells through the protocol explained here, revealed that the GRPβ‐L cells have key features of human mature β cells, and most notably, they could sense the changes in the ambient glucose concentrations and secret insulin accordingly (Massumi et al., ).…”