Islet-like organoids derived from human pluripotent stem cells efficiently function in the glucose responsiveness in vitro and in vivo

Abstract

Insulin secretion is elaborately modulated in pancreatic ß cells within islets of three-dimensional (3D) structures. Using human pluripotent stem cells (hPSCs) to develop islet-like structures with insulin-producing ß cells for the treatment of diabetes is challenging. Here, we report that pancreatic islet-like clusters derived from hESCs are functionally capable of glucose-responsive insulin secretion as well as therapeutic effects. Pancreatic hormone-expressing endocrine cells (ECs) were differentiated from hESCs using a step-wise protocol. The hESC-derived ECs expressed pancreatic endocrine hormones, such as insulin, somatostatin, and pancreatic polypeptide. Notably, dissociated ECs autonomously aggregated to form islet-like, 3D structures of consistent sizes (100-150 μm in diameter). These EC clusters (ECCs) enhanced insulin secretion in response to glucose stimulus and potassium channel inhibition in vitro. Furthermore, ß cell-deficient mice transplanted with ECCs survived for more than 40 d while retaining a normal blood glucose level to some extent. The expression of pancreatic endocrine hormones was observed in tissues transplanted with ECCs. In addition, ECCs could be generated from human induced pluripotent stem cells. These results suggest that hPSC-derived, islet-like clusters may be alternative therapeutic cell sources for treating diabetes.

Figure 1. Overall scheme of hESC differentiation into definitive endoderm (DE) cells.

(a) Overall protocols for the differentiation of human PSCs into pancreatic endocrine cells. DE, definitive endoderm; PE, pancreatic endoderm; EP, endocrine progenitor; EC, endocrine cell. (b) Flow cytometric analysis of CXCR4-positive cells differentiated from hESCs. CXCR4 was used as a DE marker. (c) Transcriptional expression of the DE marker genes SOX17, GATA4 and FOXA2. Relative expression is represented as the mean ± SEM (n = 3). N.D., not detected. (d) Immunostaining of the representative DE markers SOX17, FOXA2 and GATA4 in hESC-derived DE cells. Nuclear DAPI staining is shown in blue. Scale bar, 50 μm.

Figure 2. Differentiation of hESC-derived DE cells into hormone-expressing endocrine cells (ECs).

(a) Immunostaining of the representative PE marker PDX1 in hESC-derived PE cells. Nuclear DAPI staining is shown in blue. Scale bar, 50 μm. (b) Flow cytometric analysis of PDX1-positive cells differentiated from hESCs. (c) Transcriptional expression of the PE marker genes PDX1, SOX9 and HNF1ß. Relative expression is represented as the mean ± SEM (n = 3 or 4); *p < 0.05, **p < 0.01, ***p < 0.001. (d) Immunostaining for NGN3/PDX1 and NKX2.2/PDX1 in hESC-derived EP cells. Scale bar, 50 μm. (e) Transcriptional expression of NGN3, PDX1, and NKX2.2. Relative expression is represented as the mean ± SEM (n = 3 or 4); *p < 0.05, **p < 0.01, ***p < 0.001. (f) Expression of pancreatic endocrine hormones in hESC-derived ECs. INS, insulin; C-PEP, c-peptide; SST, somatostatin; PP, pancreatic polypeptide; GCG, glucagon. (g) Expression of ß cell-associated transcriptional factors in hESC-derived ECs. (h) Expression of ß cell function-related proteins in hESC-derived ECs. PC1/3, proprotein convertase 1/3; GLUT1, glucose transporter 1. Scale bar, 50 μm. (i) Transcriptional expression of pancreatic endocrine hormone genes (INS, SST, PPY, and GCG), ß cell-associated transcriptional factor genes (PDX1, NXX2.2, NKX6.1, MAFB, and MAFA), and ß cell function-related genes (PCSK1, and SLC2A1). Relative expression is represented as the mean ± SEM (n = 3 or 4); *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3. Efficient production of islet-like organoids derived from hESCs (hESC-derived ECCs).

(a) Representative image of hESC-derived ECCs. Scale bar, 100 μm. (b) Expression of pancreatic endocrine hormones, ß cell-associated transcriptional factors, ß cell function-related proteins in hESC-derived ECCs. Scale bar, 50 μm. (c) Comparison of transcriptional levels of endocrine hormone genes (INS, SST, PPY and GCG), ß cell-associated transcriptional factor genes (PDX1, NXX2.2, NKX6.1, MAFB, and MAFA), ß cell function-related genes (PCSK1, SLC2A1, and GCK), and ß cell gap junction-related genes (CDH1, and CX36) between hESC-derived ECs and ECCs. (d) Maturation of hESC-derived, ß cell-like cells in hESC-derived ECCs. Co-expression of INS/NKX6.1 and GLUT1/PDX1 was only detected in hESC-derived ECCs, not in hESC-derived ECs. Scale bar, 50 μm.

Figure 4. In vitro functionalities of islet-like organoids derived from hESCs.

Responsiveness to 27.5 mM glucose stimulation of hESC-derived ECs and ECCs was analyzed by secretion levels of human insulin (a) and human c-peptide (b). Human insulin secreted in response to secretagogues, such as 30 mM KCl (c) and 100 μM tolbutamide (d) was measured. Secreted insulin and c-peptide levels are represented as the mean ± SEM (n = 3 or 4); *p < 0.05, **p < 0.01, ***p < 0.001. All secretion assays were performed after equilibration in 2.5 mM glucose. (e) Intracellular Ca²⁺ traces from hESC-derived ECCs. Detection of intracellular Ca²⁺ was measured using Fluo-4 AM. Responsive spots within ECCs were independently noted during the overall procedure. A representative spot is indicated by a yellow square in Supplementary Movie 2. (f) Transmission electron microscopy images of hESC-derived ECCs. Insulin-containing granules are indicated by yellow arrows. M, mitochondria. Scale bar, 500 nm.

Figure 5. Therapeutic effects of hESC-derived ECCs in ß cell-deficient mice.

(a) Regulation of blood glucose levels in STZ-treated mice after transplantation with hESC-derived ECCs. The ß cell-deficient mice were produced by treatment with STZ. Blood glucose levels were measured in mice every 3 d after 4 h of fasting. The data are represented as the mean ± SEM. (b) Expression of pancreatic endocrine hormones in tissues engrafted with hESC-derived ECCs. Tissues were obtained from mice that were euthanized 13 d after the transplantation of hESC-derived ECCs; these tissues were immunostained with antibodies against respective endocrine hormones. Nuclear DAPI staining is shown in blue. Scale bar, 50 μm. (c) Co-expression of INS/GLUT1 in engrafted hESC-derived ECCs. This result demonstrates the in vivo functionality of engrafted hESC-derived ECCs for blood glucose regulation in ß cell-deficient mice. Scale bars, 50 μm. (d) Secretion of human c-peptide in the serum of mice transplanted with hESC-derived ECCs. Serum was collected 12 d after the transplantation of hESC-derived ECCs. Measured c-peptide levels are represented as the mean ± SEM (n = 3). N.D., not detected.