Differentiation of hPSCs on vitronectin variant-coated surfaces into hepatic lineage cells.

<p>(A) Hepatic progenitor cells were stained for AFP and HNF4A at days 10 and 15. In merged images, nuclei were counterstained with DAPI. (B) Expression of mRNAs for specific markers of undifferentiated cells (<i>POU5F1</i> and <i>NANOG</i>), hepatoblasts (<i>DLK1</i>, <i>AFP</i>, and <i>HNF4A</i>) and hepatocytes (<i>HNF4A</i>, <i>ALB</i>, <i>ASGR1</i>, and <i>TDO2</i>) was analyzed by RT-PCR. 201B6 human iPS cells were differentiated into hepatocyte-like cells, and total RNA was isolated from two independent differentiation experiments. Total RNAs from fetal and adult livers were used as controls for differentiated cells. (C and D) Cellular uptake of DiI-LDL (C) or ICG (D) by undifferentiated or differentiated (day 20) 201B6 iPS cells on R-Fc-coated surface. Scale bars indicate 100 μm. (E) Heatmap of qPCR analysis using PrimerArray assays and hierarchical clustering to compare expression levels of specific genes during hepatic differentiation. 201B6 cells were differentiated into hepatocyte-like cells on an R-Fc-coated surface (201B6 d20), and the expression levels of marker genes were compared with those of K3 human iPS cells differentiated on a Matrigel-coated surface (K3 d20). Well ID indicates 88 specific primer pairs of PrimerArray assays (PH017).</p

Differentiation of hPSCs into definitive endoderm cells on various substrates.

<p>(A) Most cells expressed the pluripotency marker POU5F1 on the hE-cad-Fc-coated surface, but almost all cells differentiated into definitive endoderm cells expressing GATA4 on vitronectin- and Matrigel-coated surfaces. (B) The vitronectin-coated surface supported differentiation of K3 cells into ALB⁺ hepatocyte-like cells at day 20 of differentiation. (C) Differentiating cells detached from the vitronectin-coated surface at day 10, although these cells expressed HNF4A. Scale bar indicates 100 μm. (D) Quantitative PCR analyses of cadherin and MMP gene expression during differentiation of human iPS cells. 201B6 cells were differentiated into hepatocyte-like cells on an R-Fc-coated surface, and total RNA was isolated at days 0, 5, 10, 15, and 20 of differentiation. Data are the means ± SD of three independent differentiation experiments. *p < 0.001, **p < 0.005 vs. day 0 (d0) sample.</p

Differentiation of hPSCs on vitronectin variant-coated surfaces into hepatoblasts.

<p>(A and B) H9 human ES cells were induced to differentiate into the hepatic lineage on R-Fc- or NC-Fc-coated surfaces. The cells were fixed and stained at day 5 (definitive endoderm stage) and day 10 (specified hepatic lineages) to detect pluripotency markers (A: POU5F1; B: NANOG) and definitive endoderm markers (A: GATA4; B: SOX17). Inserted figures showed the magnified images. (C) H9 hES and K3 hiPS cells were induced to differentiate into definitive endoderm cells on Matrigel or R-Fc, and the expression of CXCR4 was analyzed by flow cytometry at day 5. Undifferentiated cells were used as a negative control. (D) Differentiated cells were stained for HNF4A (a marker of specified cells) at day 10. Scale bars indicate 100 μm.</p

Maintenance of hPSCs on vitronectin variant-coated surfaces.

<p>(A) Immunocytochemistry of NANOG, SOX17, POU5F1, and T (Brachyury) in K3 hiPS cells cultured on recombinant human vitronectin (hVTN)-, R-Fc- or NC-Fc-coated dishes for 5 days. Nuclei were counterstained with DAPI. Scale bar indicates 100 μm. (B) Differentiation potential of hiPS cells cultured on R-Fc or NC-Fc into the ectodermal (SOX1), mesodermal (T), and endodermal (SOX17) lineages was examined. 201B6 cells were maintained on Matrigel, R-Fc or NC-Fc for 7 passages and transferred on Matrigel-coated surface to induce differentiation. (C) Characterization of teratomas formed by human iPS cells cultured on the R-Fc-coated surface. Teratomas generated from three different human iPS cell lines (253G1, 454E2, and TIG120-4f1) contained tissues derived from all three germ layers. Typical tissues are shown. Ectoderm: mature and immature central nervous systems (CNS), and optic nerve; Mesoderm: cartilage, kidney, striated muscle, and vasculature; Endoderm: intestine. Scale bar indicates 100 μm.</p

Morphology of hPSCs on vitronectin variant-coated surfaces.

<p>Phase contrast micrographs showing the morphology of H9 human ES cells (A) and K3 human iPS cells (B) cultured on surfaces coated with Matrigel, R-Fc, NC-Fc, or D6E mutants at 3 days after seeding. Scale bar indicates 100 μm. (C) Morphological observation of 201B6 and 253G1 hiPS cells that were maintained on Matrigel-, R-Fc- or NC-Fc-coated surface for 6 passages.</p

Primer pairs used for construction of hVTN variants-Fc.

<p>Underline indicates PciI and NotI recognition sites, and mutated sequence is shown in italic letters.</p

ES cells show higher proliferation and higher transfection efficiency on the E-cad-Fc-coated surface.

<div><p>(A) The proliferative activity of ES cells on a gelatin- or E-cad-Fc-coated surface was evaluated.</p> <p>EB3 cells were seeded on gelatin-coated (open square) or E-cad-Fc-coated (filled square) dishes and the cell number was counted after staining with alamar Blue reagent.</p> <p>The data indicate means±SD of experiments (n = 3). **:<i>P</i><0.01 versus gelatinized plates.</p> <p>(B) BrdU incorporation of EB3 cells under colony-forming (on gelatin) or scattering conditions (on E-cad-Fc).</p> <p>Relative BrdU incorporation value was evaluated.</p> <p>The data indicate means±SEM. §:<i>P</i><0.001.</p> <p>(C) Transfection efficiency of ES3 cells cultured on gelatin- or E-cad-Fc-coated surface.</p> <p>Relative expression of GFP was evaluated.</p> <p>The data indicate means±SEM. §:<i>P</i><0.001 versus gelatinized plates.</p></div

Effect of LIF concentration on the maintenance of undifferentiated state on ES cells.

<div><p>(A) R1 cells were cultured for 5 days in the presence of various doses of LIF (0–1,000 units/ml), on E-cad-Fc-coated plates (open bars) or gelatin-coated plates (closed bars).</p> <p>Then cells were replated onto gelatin-coated plates and three days later, the ratios of ES cell colonies with high ALP activity were estimated.</p> <p>*:<i>P</i><0.05 vs. gelatinized plate in the presence of 1,000 units/ml LIF.</p> <p>#:<i>P</i><0.05 for ES cells cultured on E-cad-Fc-coated plates versus gelatinized plate in the presence of same concentration of LIF.</p> <p>(B) The maintenance of the pluripotent efficiency of ES cells, which was cultured on an E-cad-Fc-coated surface at a low concentration of LIF (100 units/ml), was assessed by the characterisation of teratomas.</p> <p>ES (EB3) cells were maintained on E-cad-Fc-coated surface in the presence of 100 units/ml of LIF and then transplanted into mouse testis.</p> <p>H&E staining of teratomas showed the differentiation into ectoderm (epidermis: top right, bar: 100 µm), mesoderm (striated muscle cells: top left, and cartilage: top centre, bar: 100 µm, inset: 10 µm) and endoderm (ciliated columnar epithelium, possibly bronchial epithelium: bottom left, bar: 50 µm, inset: 10 µm).</p> <p>Differentiation into ectoderm was confirmed by specific staining for the neural markers GAP-43 (bottom centre) and Neurofilament-M (bottom right).</p></div

Pluripotency of ES cells on E-cad-Fc-coated surface.

<div><p>(A) R1 cells were maintained on gelatin or E-cad-Fc for 26 days, and then were cultured to form embryoid bodies.</p> <p>After 14 days culture of embryoid bodies, expression of marker genes was analyzed by RT-PCR.</p> <p>Lane 1: undifferentiated cells; lane 2: on gelatin; lane 3: on E-cad-Fc.</p> <p>(B and C) Characterization of teratomas from ES cells (EB3) cultured on an E-cad-Fc-coated surface.</p> <p>(B) H&E staining of teratomas showed the differentiation into ectoderm (epidermis), mesoderm (cartilage, and striated muscle cells) and endoderm (ciliated columnar epithelium, possibly bronchial epithelium).</p> <p>Differentiation into ectoderm was confirmed by specific staining for the neural markers βIII-tubulin, GFAP, Neurofilament-M and GAP-43 (C).</p> <p>Scale bar indicates 50 µm.</p> <p>(D) EB3 cells cultured on gelatin- or E-cad-Fc-coated dishes for 15 days were introduced into approximately 100 blastocysts of C57BL/6 (B6) mice in each group, which yielded 4 and 7 heads of chimera pups, respectively.</p> <p>Furthermore, by mating with wild-type B6 females, 2/4 chimera males from the gelatin-coated group and 3/5 chimera males from the E-cad-Fc-treated group produced offspring with ES cell-derived coat colors, suggesting comparable chimera formation and germ-line transmission abilities in E-cad-Fc-treated ES cells.</p> <p>Germ-line transmission was also verified genetically by DNA microsatellite marker analysis.</p> <p>PCR-based microsatellite marker analysis was performed on a litter mate.</p> <p>The primer sequences for D4Mit72 and D4Mit116 microsatellite markers were obtained from Mouse Microsatellite Data Base of Japan (<a href="http://shigen.lab.nig.ac.jp/mouse/mmdbj/top.jsp" target="_blank">http://shigen.lab.nig.ac.jp/mouse/mmdbj/top.jsp</a>).</p></div

Cell adhesion, morphology of ES cells on the E-cad-Fc fusion protein-immobilized surface.

<div><p>(A) ES cells (EB3) adhered to E-cad-Fc-coated dishes with equivalent efficiency as to 0.1% gelatin-coated dishes after 3 hours of incubation.</p> <p>(B) ES cells (EB3) were cultured on E-cad-Fc-coated or fibronectin-coated dishes without serum.</p> <p>EGTA (5 mM) was added to the culture medium at 3 hours after seeding (open bar).</p> <p>Detached cells were removed and remaining cells were counted using alamar Blue reagent.</p> <p>*:<i>P</i><0.05, §:<i>P</i><0.001 vs. no treated condition (closed bar).</p> <p>(C and D) Morphological observation of ES cells (EB3) on the two different matrices.</p> <p>ES cells were cultured on polystyrene surfaces coated with 0.1% (wt/vol) gelatin (C), or 10 µg/ml E-cad-Fc (D) in the presence of LIF for 2 days.</p> <p>High magnification images are shown in (C′) and (D′).</p> <p>(E) ES cells (EB3 and R1 cells) were cultured on the plates coated with gelatin or E-cad-Fc and differentiation was induced by the withdrawal of LIF.</p> <p>Morphological characteristics were observed as phase contrast images.</p> <p>Bar indicates 100 µm.</p> <p>The data indicate means±SD of 3 separate experiments.</p></div