Additional file 1: of Late stage definitive endodermal differentiation can be defined by Daf1 expression

Daf1-positive cells are negative for Nanog expression. Mouse Nanog-iPS cells, in which GFP expression is driven by Nanog promoter [45], are differentiated into DE. Cxcr4+/E-cadherin + cells were sorted and analyzed for Daf1 and Nanog-GFP expression. Daf1-positive cells are negative for Nanog expression. (TIF 312 kb

Additional file 3: of Late stage definitive endodermal differentiation can be defined by Daf1 expression

Primer sequences used for RT-PCR analysis. Primer sequences used for detection of gene expression in Fig. 1, 2. (DOCX 15 kb

Establishment of an ELISA system for quantification of the secreted mouse Cer1 protein.

<p>(A) Schematic drawing of the ELISA system. (B) Standard curve using purified Cer1. (C) ELISA assays for the secreted Cer1 protein at differentiation days (D) 1, D3, D5, or D7. The supernatant was sampled 48 h (lower panel) after replacement with fresh media. Time-dependent expressions of Cer1 in the supernatant, detected by immunoprecipitation, are also shown (bottom panels).</p

Upregulated genes (>5-fold) classified as ‘inflammatory response genes’.

<p>Upregulated genes (>5-fold) classified as ‘inflammatory response genes’.</p

Upregulated genes (>5-fold) classified as ‘chemotaxis genes’.

<p>Upregulated genes (>5-fold) classified as ‘chemotaxis genes’.</p

Additional file 2: of Late stage definitive endodermal differentiation can be defined by Daf1 expression

Flow cytometric analyses of the cell cycle. Histograms of flow cytometric analyses of Daf1-DE and Daf1 + DE (n = 5) are shown. Cell cycle was analyzed by measuring DNA quantities using DyeCycle. (TIF 314 kb

Islet apoptosis increased with increasing glucose concentration.

<p>(A) Islets were cultured in low-glucose (100 mg/dL, left) and high-glucose (900 mg/dL, right) media for 24 hours. Lower panels show high-magnification images. (B) The caspase-3/7–positive area increased after a 24-hour exposure to various glucose concentrations from 0 to 600 mg/dL. (C) Quantitative analysis of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095451#pone-0095451-g005" target="_blank">Figure 5B:</a> media with glucose concentrations of 0 mg/dL (dotted line), 50 mg/dL (small broken line), 100 mg/dL (dot-broken line), 200 mg/dL (large broken line), 300 mg/dL (middle broken line), and 600 mg/dL (solid line) were used.</p

Graft survival improved as a result of insulin treatment.

<p>Kidneys from mice in the 50-islet TP group at (A) the time of transplantation, (B) day 1, and (C) week 18. (A) and (B) are from the same mouse. Kidneys from mice in the 50-islet TP+INS Tx group at (D) the time of transplantation, (E) day 1, and (F) week 18. (D) and (E) are from the same mouse. Lower panels show high-magnification images. Arrowheads indicate each islet, and arrows show aggregated islets. (G–L) Grafts were removed 6 hours post-transplantation and analyzed by TUNEL assay (G–I) or stained with anti-cleaved caspase-3 (J–L). Grafts of the 50-islet TP and 50-islet TP+INS Tx groups were compared (*<i>p</i><0.05, two-tailed unpaired Student’s <i>t-</i>test). (I, L) Proportions of the number of positive cells to total cells in the grafts. DAPI (blue), insulin (green), and TUNEL or anti-cleaved caspase-3 (red).</p

Beneficial Effect of Insulin Treatment on Islet Transplantation Outcomes in Akita Mice

<div><p>Islet transplantation is a promising potential therapy for patients with type 1 diabetes. The outcome of islet transplantation depends on the transplantation of a sufficient amount of β-cell mass. However, the initial loss of islets after transplantation is problematic. We hypothesized the hyperglycemic status of the recipient may negatively affect graft survival. Therefore, in the present study, we evaluated the effect of insulin treatment on islet transplantation involving a suboptimal amount of islets in Akita mice, which is a diabetes model mouse with an <i>Insulin 2</i> gene missense mutation. Fifty islets were transplanted under the left kidney capsule of the recipient mouse with or without insulin treatment. For insulin treatment, sustained-release insulin implants were implanted subcutaneously into recipient mice 2 weeks before transplantation and maintained for 4 weeks. Islet transplantation without insulin treatment did not reverse hyperglycemia. In contrast, the group that received transplants in combination with insulin treatment exhibited improved fasting blood glucose levels until 18 weeks after transplantation, even after insulin treatment was discontinued. The group that underwent islet transplantation in combination with insulin treatment had better glucose tolerance than the group that did not undergo insulin treatment. Insulin treatment improved graft survival from the acute phase (i.e., 1 day after transplantation) to the chronic phase (i.e., 18 weeks after transplantation). Islet apoptosis increased with increasing glucose concentration in the medium or blood in both the <i>in vitro</i> culture and <i>in vivo</i> transplantation experiments. Expression profile analysis of grafts indicated that genes related to immune response, chemotaxis, and inflammatory response were specifically upregulated when islets were transplanted into mice with hyperglycemia compared to those with normoglycemia. Thus, the results demonstrate that insulin treatment protects islets from the initial rapid loss that is usually observed after transplantation and positively affects the outcome of islet transplantation in Akita mice.</p></div

Establishment of an ELISA system for quantification of the secreted human Cer1 protein.

<p>(A) Time-dependent expressions of <i>CER1</i> and <i>SOX17</i> transcripts on differentiation day 2 (D2) and D6 of the human iPS cell line, 201B7 assayed by RT-PCR are shown. (B) SDS-PAGE analysis of recombinant human CER1 protein. (C) Western blot analysis of recombinant human CER1 (standard) and immunoprecipitation of the supernatant using undifferentiated iPS cells (D0) and differentiated DE (D5) medium. (D) Standard curve using recombinant human CER1.</p