Supplementary Material

JCS066886 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Nuclear staining of Pdx1-positive cells grown on sBM. Pdx1-positive cells were purified by flow cytometry and stained with anti-GFP antibody and nuclear dye, DAPI. Scale bar: 100 µm

• Supplemental Figure S2 -

  Fig. S2. Comparison of the differentiation potency of mouse ES or iPS cell line. (A) Flow-cytometry analysis of E-cadherin⁺ CXCR4⁺ DE cells. SK7, ING112 ES cells or 20D-17 iPS cells were cultured on M15 cells until day 8. The differentiation potency of the ES cell lines to DE cells is in the order: 20D-17−SK7−ING112. (B) E-cadherin⁺ CXCR4⁺ DE cells grown on M15 were recovered by flow cytometry and were subjected to RT-PCR analysis. All three ES cell lines give rise to Pdx1-expressing cells.

• Supplemental Figure S3 -

  Fig. S3. HSPG2 expression in the ES cells. RT-PCR analysis of ES cells grown on sBM substratum is shown. ES, undifferentiated ES cells; sBM, differentiated ES cells grown on sBM substratum; d8, d15 and d28, differentiated ES cells at indicated differentiation day.

• Supplemental Figure S4 -

  Fig. S4. Insulin and GFP staining of the recovered graft and the islet of Ins1-GFP transgenic mouse. In the recovered graft transplanted with ING112 ES cells, the GFP signals are detected in the nuclei and the cytoplasm, and are almost completely overlapped with insulin staining (upper panels). Similarly, in the islet of the Ins1-GFP transgenic mouse line, overlapping GFP signals and insulin staining is observed (lower panels).

• Supplemental Figure S5 -

  Fig. S5. Functional analysis of the recovered graft. (A) Measurement of insulin contents. Insulin contents are indicated along with the images. Ins1-positive or -negative grafts were recovered under fluorescent microscope. Arrowheads indicate Ins1-positive cell clusters. Insulin content was determined after extraction with acid ethanol (Yamagata et al., 2002), using an insulin enzyme-linked immunosorbent assay (ELISA) kit (Sibayagi, Gunma, Japan). (B) Measurement of insulin contents in the adult mouse islet. Five or ten normal islets, equivalent to 5000 or 10,000 β-cells, respectively, were used for the quantification. (C) Measurement of glucose-dependent insulin release in the recovered grafts. The recovered grafts with (GFP⁺) or without (GFP⁻) Ins1-positive cell clusters were minced in 1 ml HKRB buffer (129 mM NaCl, 4.7 mM KCl 1.2 mM MgCl₂, 1.2 mM KH₂PO₄, 2.5 mM NaHCO₃, 2.5 mM CaCl₂, 10 mM HEPES, pH 7.4) with 0.05% bovine serum albumin (BSA). After centrifugation, pellet was resuspended in 1 ml HKRB with 0.05% BSA and 2.2 mM glucose, and preincubated at 37°C for 60 minutes. Then, cells were incubated for 60 minutes in buffer containing 2.2 mM (low glucose, white bars) or 22 mM glucose (high glucose, black bars). Concentration of released insulin was measured by ELISA kit (Sibayagi). Ten islets (10,000 β-cells) were used as a positive control.