Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3

Abstract

Insulin-expressing cells that have been differentiated from human pluripotent stem cells in vitro lack the glucose responsiveness characteristic of mature beta cells. Beta-cell maturation in mice was studied to find genetic markers that enable screens for factors that induce bona fide beta cells in vitro. We find that functional beta-cell maturation is marked by an increase in the glucose threshold for insulin secretion and by expression of the gene urocortin 3.

Figure 1. β-cell maturation is defined by a decrease in GSIS sensitivity to low glucose levels and by the expression of Ucn3

(A) Three independent sets of 50 islets each, from P1 or P15 mice, were sequentially perfused with basal (0.5mM, gray), low (2.8mM, blue) or high (16.7mM, red) glucose in a dynamic GSIS assay. Arrows indicate the time points at which solutions were changed. P1 islets display complete first and second phases of GSIS in response to low glucose, whereas P15 islets do not secrete insulin at this glucose concentration. (B) Triplicates of 10 islets from P1 to adult were assayed for GSIS using low glucose (2.8mM) and high glucose (16.7mM). Two age groups can be distinguished according to their stimulation index (fold change in GSIS). ***, P<5×10⁻⁵. (C) Three independent sets of ten islets each from P1, P9 or P21 were assayed for GSIS using low glucose (2.8mM, blue), high glucose (16.7mM, red), 20mM arginine (gray) or 30mM KCl (green). The difference in the amount of insulin secreted between mature and immature islets is specific to glucose. *, P<0.05; **, P<0.001; NS, not significant). (D) Blood glucose and (E) insulin levels in immature (P1, blue) and mature (P14, red) mouse pups. Insulin levels in the immature pups are higher than in the mature ones, although their blood glucose levels are lower. (F) Electron micrograph of insulin vesicles in β-cells at various ages. Scale bars = 2μm. (G) Quantification of the number of insulin vesicles vs. β-cell area of the data shown in F. (H) A scheme representing the microarray approach. Genes differentially expressed in both mature age groups compared to both immature age groups (I and II) are chosen as candidate markers. (I) Representative scattered plot from the microarray. Note high similarity (R²) in gene expression between the mature (P10) and immature (P1) samples. (J) The expression levels of most β-cell markers are unchanged during GSIS maturation. Scatter plots of global gene expression from microarrays on FACS-sorted immature (P1) and mature (P10) β-cells. Red lines mark a 2-fold difference in expression and, with the exception of MafB, gene expression is not significantly different between these stages. (K) The expression of Ucn3 mRNA at various ages as detected in the microarray. (L) Immunostaining of Ucn3 (green) and insulin (red) on pancreata from E18.5 and adult mice. Nuclei are stained with DAPI (blue). Scale bars = 50μm (M) Ucn3 is undetectable in E18.5 embryo. Ucn3 is detected at high levels and co-localizes with adult β-cells.

Figure 2. Ucn3 expression gradually increases during the course of mouse β-cell maturation in vivo and is expressed in HESC-derived β-like cells after differentiation and maturation in vivo, but not after differentiation in vitro.

(A–C) Immunostaining of Ucn3 (green) and insulin (red) on pancreata from P1, P6 and P22 mice. (D–F) Enlargement insets shown in A–C, respectively. Nuclei are stained with DAPI (blue). Scale bars = 50μm. (A, D) Ucn3 in not detected at P1 even in large islets. (B, E) At P6, some large islets express Ucn3, but small aggregates do not express the peptide (arrows). (D, F) At P22, Ucn3 is highly expressed in all islets. (G–I) Intra-cellular FACS analysis of insulin and Ucn3 at E18.5, P6 and P13. Numbers in upper quadrants represent the percentage of insulin only (left) or insulin and Ucn3 co-expressing cells (right) of all insulin-expressing cells (two upper quadrants), calculated as average±sem of three independent biological repeats (three separate litters) for each age group. (J) An outline of the experimental approach on HESCs differentiation. HESCs (ES, red) marked by Oct4 were differentiated in vitro into definitive endoderm (DE, yellow) marked by Sox17 and subsequently to pancreatic progenitors (PP, green), marked by the expression of Pdx1 and NKX6.1. The cells were transplanted into SCID-beige mice to complete maturation in vivo. (K, L) Immunostaining for Ucn3 (green) and insulin (red) on the in vitro differentiated cells shown at two magnifications (K, low magnification; L, high magnification). In vivo differentiated (transplanted) cells are shown in (M). Nuclei are stained with DAPI (blue). Scale bars = 50μm. Ucn3 is expressed in the in vivo matured cells, but not in in vitro differentiated insulin-positive β-like cells.