Preparation of rLN511 sBM. (A) A schema of the rLN511 sBM preparation procedure. The rLN-10 cells, in which the recombinant human LN511 molecule was overexpressed, were seeded on a cell culture insert (a), and allowed to form the BM substratum (b). After the construction of BM, rLN-10 cells were removed (c). And then mouse ES or iPS cells were seeded on the synthesized BM (sBM) substratum and cultured (d). (B) Structural and components analysis of the rLN511 sBM. (a) Transmission electron micrograph (TEM) of the extracellular matrix structure beneath rLN-10 cells that were cultured for 2 weeks on an oligo-GlcNAc-coated fibrillar collagen matrix. A lamina densa was formed beneath the cells (arrows). A number of anchoring filaments (arrow heads) connecting between the basal surface of rLN-10 cells and the lamina densa were confirmed. (b) TEM of a bared lamina densa substratum. The covering rLN-10 cells were removed by the treatment of alkali and detergent. (c) Scanning electron micrograph (SEM) of a bared lamina densa substratum. Initial matrix of collagen fibrils was observed through a small window opened at lamina densa. (d) Immunofluorescent microscopy of the transverse section in rLN10 cell culture. Laminin (d1) and type IV collagen (d2) were continuously integrated beneath rLN-10 cells. (e) Horizontal viewing of a bared ECM after the removal of rLN-10 cells. The integration of laminin (e1) and type IV (e2) collagen was observed.

ES cells cultured on rLN511 sBM substratum alone gave rise to definitive endoderm cells. (A) Quantification of endodermal cells grown on the (a) sBM or (b) M15 cells. E-cadherin-positive CXCR4-positive DE cells were quantified by flow cytometry on d8. (B) Real-time PCR analysis of the cells grown on the sBM or M15 cells. On d8 or d15, the expression levels of Pou5f1, Sox17, Foxa2 and Pdx1 were quantified, and normalized with that of β-actin. Undifferentiated ES cells were used as a control. Values represent means ± s.e. (n=3).

Differentiation of mouse ES cells into pancreatic cell lineages on the sBM substratum. (A) Schematic representation of the differentiation conditions used. Mouse ES or iPS cells seeded on the sBM substratum were cultured with serum-free medium throughout the differentiation. At d0 to d10, 20 ng/ml activin A and 50 ng/ml bFGF were supplemented in the medium, and 1 μM retinoic acid (RA) was further supplemented from d10 to d13. After d13, the glucose concentration was switched from 4500 mg/l to 1000 mg/l, and 10 mM nicotinamide and 10 nM glucagon-like peptide (GLP1) were supplemented. (B) Fluorescent images of the Pdx1 ES cell line, SK7, grown on the sBM substratum. Pdx1 expression became detectable at d10 and increased until d15. After d15, Pdx1-expressing cells formed three-dimensional clusters. On d28, strong Pdx1 expression was observed in the differentiated cell clusters. Scale bars: 100 μm. (C) Quantification of Pdx1-expressing cells. On d15, Pdx1-positive cells were quantified by flow cytometry. (D) Differentiation of the Ins1 ES cell line, ING112, on the sBM substratum. The green fluorescence of ING112 on d26 and d28 is shown. The expression of Ins1 became detectable on d26. Scale bars: 100 μm.

Gene-expression analysis of mouse ES or iPS cells differentiated on the sBM substratum. (A) Expression of mature pancreas endocrine and exocrine markers. RNA from undifferentiated ES cells (SK7, ud ES) and E13.5 fetal pancreas (FP) was used as a control (negative and positive, respectively). sBM, differentiated ES cells grown on the sBM substratum; d15 and d28, whole differentiated ES cells at the indicated day after initiation of differentiation. d28/M15, differentiated ES cells grown on M15 at d28. The amount of cDNA was normalized with the expression level of β-actin. Ins1 transcripts became detectable at d28 in ES cells cultured on sBM but not in those on M15. Pdx1, Pancreatic duodenum homeodomein 1; Ins1, Insulin1; Gcg, glucagon; Sst, somatostatin; Ptf1a, pancreas specific transcription factor 1a; Amy, amylase. (B) Expression of immature (NeuroD1, Nkx2-2, Nkx6-1, Pax4, Pax6) and mature (Isl1, Glut2, Iapp) β-cell markers. NeuroD1, neurogenic differentiation 1; Nkx2-2, NK2 transcription factor related locus 2; Nkx6-1, NK6 homeobox 1; Pax4, paired box gene 4; Pax6, paired-box gene 6; Isl1, ISL1 transcription factor; Glut2, Glucose transporter type 2; Iapp, islet amyloid polypeptide. (C) RT-PCR analysis of iPS cells grown on the sBM substratum. RNA from undifferentiated iPS cells (ud iPS) was used as a negative control. SBM, differentiated iPS cells grown on the sBM substratum; d16, d20 and d28, whole differentiated ES cells at the indicated day after initiation of differentiation.

Itgb1 knockdown inhibits the differentiation of Pdx1-expressing cells. (A) A schematic of the experimental procedure. Non-silencing (NS) or Itgb1 knockdown (Itgb1 KD) lentivirus-containing medium was added on d10. To remove the uninfected cells, puromycin selection was performed from d13 to d15. Cells were analyzed on d15. (B) Real-time PCR analysis of Itgb1 expression in NS or Itgb1 KD ES cells. On d15, total RNA was extracted. Itgb1 expression was normalized with that of β-actin. (C) Quantification of the Pdx1-positive cells. Significant differences were observed (*P<0.05) versus NS. Values represent means ± s.e. (n=3).

HSPGs are involved in the differentiation of Pdx1-expressing cells. (A) Real-time PCR analysis of Hspg2 expression in non-silencing control (NS) and Hspg2-knockdown (Hspg2 KD) SK7 ES cells. Hspg2 expression was normalized to that of β-actin. (B) A schematic drawing of the experimental procedure. NS or Hspg2-KD ES cells were allowed to differentiate on sBM. Pdx1-positive cells were quantified on d15. In the heparitinase treatment (+heparitinase), the sBM was pre-treated with heparitinase before seeding ES cells, and then ES cell cultures were treated daily from d10 to d15. (C) Quantification of Pdx1-positive cells. Values represent means ± s.e. (n=3). Compared with NS and Hspg2 KD and/or heparitinase-treated samples, significant differences were observed (*P<0.05). *1, NS vs Hspg2 KD; *2, NS vs NS+heparitinase; *3, Hspg2 KD vs Hspg2 KD+heparitinase; *4, NS+heparitinase vs Hspg2 KD+heparitinase.

In vivo differentiation of ING112 ES cells cultured on the sBM substratum. (A) Fluorescent image of the recovered graft. ING112 ES cells grown on sBM for 28 days were transplanted under the kidney capsules of SCID mice. Green fluorescence indicates the expression of Ins1 (arrowheads). Scale bar: 1 mm. (B) Expression of mature pancreas endocrine and exocrine markers. Before, ING112 cells grown on the sBM substratum at d28 (pre-engraftment control); after, the recovered graft. MafA, v-maf musculoaponeurotic fibrosarcoma oncogene protein A; Ppy, pancreatic polypeptide. Real-time PCR analysis of the recovered graft. (C) The expression of mature pancreas endocrine (Ins1, Ppy and Sst) and exocrine (Amy) markers were normalized to that of β-actin. RNA from E13.5 fetal pancreas (FP) was used as a positive control. (D) Immunofluorescence analysis of the recovered graft. Immunostaining of GFP (a,b,e,f; green) and Ins (a; magenta) or C-peptide (C-pep, b; magenta) revealed that GFP expression overlapped with insulin-expressing cells. The expression of mature β-cell markers, MafA (c; magenta) and Nkx6-1 (d; magenta) was observed in Ins1-positive cells (c,d; green). GFP-positive cells were surrounded by Amy-positive (e; magenta) exocrine tissues, similarly to normal pancreatic tissue. Gcg (e; yellow), Sst (f; magenta), Ppy (g; magenta) and ductal marker, DBA-positive cells (h; magenta) were also observed. Samples shown in e-h are counterstained with DAPI (blue). Scale bars: 100 μm.