Differentiation and transplantation of functional pancreatic beta cells generated from induced pluripotent stem cells derived from a type 1 diabetes mouse model

Abstract

The nonobese diabetic (NOD) mouse is a classical animal model for autoimmune type 1 diabetes (T1D), closely mimicking features of human T1D. Thus, the NOD mouse presents an opportunity to test the effectiveness of induced pluripotent stem cells (iPSCs) as a therapeutic modality for T1D. Here, we demonstrate a proof of concept for cellular therapy using NOD mouse-derived iPSCs (NOD-iPSCs). We generated iPSCs from NOD mouse embryonic fibroblasts or NOD mouse pancreas-derived epithelial cells (NPEs), and applied directed differentiation protocols to differentiate the NOD-iPSCs toward functional pancreatic beta cells. Finally, we investigated whether the NPE-iPSC-derived insulin-producing cells could normalize hyperglycemia in transplanted diabetic mice. The NOD-iPSCs showed typical embryonic stem cell-like characteristics such as expression of markers for pluripotency, in vitro differentiation, teratoma formation, and generation of chimeric mice. We developed a method for stepwise differentiation of NOD-iPSCs into insulin-producing cells, and found that NPE-iPSCs differentiate more readily into insulin-producing cells. The differentiated NPE-iPSCs expressed diverse pancreatic beta cell markers and released insulin in response to glucose and KCl stimulation. Transplantation of the differentiated NPE-iPSCs into diabetic mice resulted in kidney engraftment. The engrafted cells responded to glucose by secreting insulin, thereby normalizing blood glucose levels. We propose that NOD-iPSCs will provide a useful tool for investigating genetic susceptibility to autoimmune diseases and generating a cellular interaction model of T1D, paving the way for the potential application of patient-derived iPSCs in autologous beta cell transplantation for treating diabetes.

FIG. 1.

Generation of NOD-iPSCs from a NOD mouse. (A) Quantitative real-time PCR analysis of expression of pancreatic-related genes (HNF-3 beta, PDX1, and PAX6). Relative expression levels were normalized to the average expression of the control ESC line G4-2. (B) Time schedule of NOD-iPSC generation by infection with lentiviruses encoding OCT4, SOX2, KLF4, and c-MYC. (C) Typical images of ESC-like colonies. NOD-iPSC lines were established from NMs and NPEs. Representative images of colonies after 20 passages, and detection of alkaline phosphatase activity. (D) Genotyping of NOD-iPSCs generated from NMs and NPEs. Two microsatellite markers corresponding to the Idd locus were examined in NOD-iPSCs. Note that all of the NOD-iPSCs examined exhibited the NOD genetic background and differed from that of the C57/BL6 mice. (E) RT-PCR analysis of expression of ESC marker genes in NOD-iPSC lines. Primers for OCT4, SOX2, KLF4, and c-MYC specifically detected transcripts from endogenous genes. (F) Immunostaining for the expression of pluripotency markers OCT4, and SSEA-1. Nuclei were stained with DAPI; G4-2 ESCs were used as positive controls. (G) Bisulfite genomic sequencing of the OCT4 promoter region. Open and closed circles indicate unmethylated and methylated CpGs dinucleotides, respectively. Six representative sequenced clones from NMEF, G4-2 ESC, NMEF-iPS1, and NPE-iPS1 are shown. (H) Representative fluorescence-activated cell-sorting analysis of the expression profiles of OCT4, SOX2, and SSEA-1 in NOD-iPSCs and G4-2 ESCs. Scale bars, 200 μm (Day 14 colony; B), 500 μm (Phase and AP; B), and 50 μm (OCT4 and SSEA-1; E). AP, alkaline phosphatase; ESC, embryonic stem cell; HNF 3 beta, hepatocyte nuclear factor 3 beta; KLF4, Krüppel-like factor 4; NOD, nonobese diabetic; iPSCs, induced pluripotent stem cells; NOD-iPSCs, NOD mouse-derived iPSCs; NMs, NOD mouse embryonic fibroblasts; NPEs, NOD mouse pancreas-derived epithelial cells; OCT4, octamer-binding transcription factor 4; PAX6, paired box gene 6; PDX1, pancreatic and duodenal homeobox 1; SOX2, SRY-box containing the gene 2; SSEA-1, stage-specific embryonic antigen-1; RT-PCR, reverse transcription-polymerase chain reaction. *p<0.05. Color images available online at www.liebertonline.com/scd

FIG. 2.

In vitro and in vivo differentiation of NOD-iPSCs into the 3 germ layers. (A) Phase-contrast images of embryoid bodies derived from NOD-iPSCs. (B) RT-PCR analyses of differentiation markers for the 3 germ layers (endoderm: AFP and GATA4; mesoderm: brachyury and BMP4; ectoderm: nestin and beta tubulin III). (C) Immunocytochemistry of NOD-iPSC lines (NM-iPS1 and NPE-iPS1) using markers for the 3 germ layers (endoderm: AFP; mesoderm: brachyury; ectoderm: nestin and beta tubulin III). (D) Hematoxylin/eosin staining of teratoma sections. Cells were transplanted intramuscularly into the hind leg of individual NOD/SCID mice. Hematoxylin/eosin staining of teratoma sections revealed smooth muscle (M), cartilage (C), nerve fibers (N), skin (S), gut-like epithelium (G), and respiratory epithelium (R). (E) Production of a chimeric mouse derived from NOD-iPSCs. NOD-iPSCs and control ESCs (G4-2) were injected into the space between the zona pellucida and blastomeres of host 8-cell embryos through a perforation created by a XYClone laser. Chimeric mice generated from NOD-iPSCs. Red arrows indicate that the chimerism originated from the NPE-iPSCs. Scale bars, 500 μm (A), and 100 μm (C, D). AFP, alpha-fetoprotein. Color images available online at www.liebertonline.com/scd

FIG. 3.

Dynamic expression patterns of pancreatic lineage genes during direct pancreatic differentiation from NOD-iPSCs. (A) The flow chart of the differentiation protocol. NOD-iPSCs and G4-2 ESCs were differentiated into the definitive endoderm (step 1; 3 days), pancreatic progenitor cells (step 2; 4 days), and expanded progenitor cells (step 3; 4 days). (B) and (C) Dynamic gene expression patterns of the differentiated NOD-iPSCs were analyzed at each step of induction in 3 separate experiments by quantitative RT-PCR (qRT-PCR) analysis. (B) The expression level of OCT4, an ES cell pluripotency marker, rapidly decreases around step 1. (C) The differentiated cell samples were collected at steps 1, 2, 3, and 4 and analyzed by qRT-PCR for SOX17, PDX1, ISL-1, and Insulin. (D) Flow cytometry analysis of the differentiated NOD-iPSCs showed the presence of SOX17-positive cells after 3-day induction with activin A and wort. For each sample, gene expression was normalized to that of GAPDH. *P< 0.05. ISL-1, Islet-1.

FIG. 4.

Differentiation of NOD-iPSCs into insulin-producing cells by a stepwise direct differentiation protocol. (A) Immunocytochemical staining confirming stepwise differentiation. NOD-iPSCs and G4-2 ESCs were differentiated into definitive endoderm (Step 1; 3 days; markers, SOX17 and FOXA2); pancreatic progenitor (Step 2; 4 days; marker, PDX1); expanded progenitor (Step 3; 4 days; markers, PAX6 and ISL-1). (B) Quantitative real-time PCR (Step 1 markers: HNF-3 beta, CXCR4, GATA4, and SOX17; Step 2 markers: PDX1, SOX9, and HLXB9; and Step 3 markers: PAX6, ISL-1, NeuroD1, and Neurogenin). Gene expression was normalized to GAPDH. *P<0.05 versus G4-2 control. Scale bars, 100 μm (SOX17, FOXA2, and PDX1; A), and 50 μm (PAX6 and ISL-1; A). The insets are a higher magnification. FOXA2, forkhead box A2. Color images available online at www.liebertonline.com/scd

FIG. 5.

Differentiation of NOD-iPSCs into pancreatic insulin-producing cells. (A) Immunocytochemical staining reveals that 2 NOD-iPSC lines (NM-iPS1 and NPE-iPS1), and control ESCs (G4-2), differentiate into pancreatic beta-like cells that express PDX1, Insulin, C-peptide, Glucagon, and Somatostatin. (B) Quantitative real-time PCR analyses indicates that these 2 NOD-iPSC lines could be induced to express islet cell-specific marker genes including IAPP, Insulin, and GLUT2 at the final induction stage. Gene expression was normalized to GAPDH. *P<0.05 versus G4-2 control. (C) Analysis of insulin release from NOD-iPSC-derived insulin-producing cells. NOD-iPSC-derived populations were stimulated with 2.5 mM d-glucose, 25 mM d-glucose, 30 mM KCl, or 100 μM carbachol, and the amount of insulin released into the culture supernatant was analyzed by ELISA. NM-iPSCs, NPE iPSCs, and G4-2 ESCs showed significant differences in insulin release (*P<0.01) when stimulated with 2.5 mM versus 25 mM d-glucose, 30 mM KCl, and 100 μM carbachol. Scale bars, 50 μm (A). The insets are a higher magnification and the white arrows represent Glucagon or Somatostatin. Color images available online at www.liebertonline.com/scd

FIG. 6.

Transplantation of NPE-iPSC-derived insulin-producing cells into STZ-induced diabetic NOD/SCID mice. (A) Blood glucose levels in transplanted STZ-induced diabetic mice. NPE-iPSC-derived insulin-producing cells were transplanted into STZ-induced diabetic mice. Shown in the graph are sham-transplanted control STZ-induced mice (PBS treated group; n=3; black squares), and STZ-induced mice transplanted with NPE-iPSC-derived insulin producing cells (n=8; gray circles). (B) Circulating insulin levels in transplanted STZ-induced diabetic mice. Insulin in blood from untreated normal mice (n=3), sham-transplanted diabetic mice (n=3), and diabetic mice transplanted with NPE-iPSC-derived insulin-producing cells (n=8), after 3 weeks. *P<0.05 versus sham-transplanted control. STZ, streptozotocin.

FIG. 7.

Analysis of a grafted kidney. (A, B) H&E staining of the grafted kidney. Grafts were removed 5 weeks after transplantation and analyzed by hematoxylin/eosin staining of either the nontransplanted kidney (A) or the NPE-iPSC-grafted kidney (B). The black arrows represent the site of the kidney capsule injection. K, kidney; E, engrafted cells. (C) Expression of insulin, and C-peptide in the graft. Brown diaminobenzidine staining was positive. Sections were counterstained with hematoxylin (blue). Scale bars, 500 μm (A-B), 50 μm (C). H&E, hematoxylin/eosin. Color images available online at www.liebertonline.com/scd