Induction of functional islet-like cells from human iPS cells by suspension culture

Abstract

Introduction: To complement islet transplantation for type1 diabetic patients, cell-based therapy using pluripotent stem cells such as ES cells and iPS cells is promising. Many papers have already reported the induction of pancreatic β cells from these cell types, but a suspension culture system has not usually been employed. The aim of this study is to establish a suspension culture method for inducing functional islet-like cells from human iPS cells.

Methods: We used 30 ml spinner type culture vessels for human iPS cells throughout the differentiation process. Differentiated cells were analyzed by immunostaining and C-peptide secretion. Cell transplantation experiments were performed with STZ-induced diabetic NOD/SCID mice. Blood human C-peptide and glucagon levels were measured serially in mice, and grafts were analyzed histologically.

Results: We obtained spherical pancreatic beta-like cells from human iPS cells and detected verifiable amounts of C-peptide secretion in vitro. We demonstrated reversal of hyperglycemia in diabetic model mice after transplantation of these cells, maintaining non-fasting blood glucose levels along with the human glycemic set point. We confirmed the secretion of human insulin and glucagon dependent on the blood glucose level in vivo. Immunohistological analysis revealed that grafted cells became α, β and δ cells in vivo.

Conclusions: These results suggest that differentiated cells derived from human iPS cells grown in suspension culture mature and function like pancreatic islets in vivo.

Keywords: Islet; Pancreatic β cell; iPS cells.

Fig. 1

Overview of the differentiation protocol of iPS cells in suspension culture. A: Summary of 6-stage differentiation protocol including supplements and additives (growth factors and small molecules). DE: definitive endoderm, PGT: primitive gut tube, PFG: posterior fore gut, PP: pancreatic progenitor, EP: endocrine progenitor, BETA: β cell stage. B: Photograph of the spinner type 30 ml reactor with magnetic stirrer in the incubator. C, D: Representative phase-contrast images of iPS cells-derived spheroids before (C) and after (D) differentiation process. Scale bar = 200 μm.

Fig. 2

Immunohistochemical analysis and C-peptide secretion assay with the final stage spheroids. A(a-h): Representative immunofluorescent staining of spheroids at the end of the final stage. PDX1 (a), NKX6.1 (c), C-p: human C-peptide (e), GCG: glucagon (g), DAPI (b, d, f, h). Scale bar = 200 μm. B: Glucose-stimulated C-peptide secretion assay was performed for each batch of spheroids (X-axis A, B, C). Y-axis: the concentration of human C-peptide secreted in the supernatants. The concentrations of human C-peptide secreted under 2 mM glucose conditions (blue bar) and under 20 mM glucose conditions (red bar) were measured with the human C-peptide ELISA kit and a spectrophotometer. We measured the concentration of C-peptide for each batch twice and calculated the mean ± SEM. P value was shown in parenthesis. P < 0.05 was considered significant.

Fig. 3

Transplanted cells ameliorate hyperglycemia in diabetic mice Approximately 6 × 10⁶ iPSC-derived cells were injected under the left kidney capsule of the STZ-induced NOD/SCID diabetic mice. Three different batches of spheroids were used for transplantation experiments. Batch A spheroids were then transplanted into mice #1 and #2, batch B spheroids; mice #3 and #4, batch C spheroids; mice #5, #6 and #7, respectively. PBS was injected in the same site of diabetic mice as a control (mouse DM-cont-1and DM-cont-2 in G). Non-fasting blood glucose levels were examined once or twice a week (A, D, G). Blood human C-peptide (B, E, H) and glucagon levels (C, F, I) were also measured every 2 weeks or 4 weeks for transplanted mice. Survival nephrectomies were performed on some mice (mouse #4, #5, #6, #7).

Fig. 4

Glucose tolerance test and hormone secretion A: Blood glucose levels were measured during oral glucose tolerance test (glucose load; 2.0 g/kg) at 12 weeks after transplantation (mouse #3, #4). Blood human C-peptide (B) and glucagon (C) levels were also measured before glucose load (pre), 30 min and 120 min after glucose load by the ELISA kits. D: The same OGTT procedure was performed in a DM control mouse and a non-DM control mouse. Blood mouse C-peptide (E) levels were measured 30 min and 120 min after glucose load. Glucagon (F) levels were also measured before glucose load (pre), and 30 min and 120 min after glucose load by the ELISA kits.

Fig. 5

Immunohistochemical analysis of grafts in the kidney A: Representative HE staining image of the kidney (mouse #3) resected 101 days after transplantation. Square area in A is enlarged in B. C, D: Double immunofluorescent staining of the same area as B. (C) red; human C-peptide, green; glucagon, (D) red; human C-peptide, green; somatostatin. All scale bars = 200 μm.