3D culture of sorted Flk-1⁺ cells <i>in vitro</i>.

<p>(A) Representative images of tube formation assay <i>in vitro</i> (upper). Sorted Flk-1⁺ cells derived from young and old iPS cells were cultured alone for 24 hours on Matrigel. Quantitative analysis of network projections formed on Matrigel for each experimental group (lower) (n = 3 in each group). (B) Representative images of HUVEC co-cultured with Flk-1⁺ cells (upper). Sorted Flk-1⁺ cells derived from young and old iPS cells were co-cultured with HUVEC for 24 hours on Matrigel. Flk-1⁺ cells derived from young and old iPS cells (white arrow head) were confirmed. The bar indicates 200 µm. Quantitative analysis of the number of Flk-1⁺ cells derived from young and old iPS cells into HUVEC on Matrigel (lower) (n = 3 in each group).</p

Effects of cell transplantation on blood flow recovery in the ischemic hindlimb.

<p>(A) Representative LDBF images. A low perfusion signal (dark blue) was observed in the ischemic left hindlimb of control mice (PBS), whereas high perfusion signals (white to red) were detected in the ischemic left hindlimb of mice transplanted with Flk-1⁺ cells derived from young and old mice (2×10⁵ cells) on postoperative days 3, 7 and 14. (B) Quantitative analysis of the ischemic to non-ischemic limb LDBF ratio on pre- (Day-1) and postoperative days 0, 3, 7 and 14 (Control: n = 8, Young: n = 4, Old: n = 4). *p<0.05 for mice injected with Flk1⁺ cells (2×10⁵) vs. control mice. (C) Capillary density analysis. Capillary density was determined at day 21 after surgery. Collected ischemic hindlimb muscle was stained with VE-cadherin. Capillary density was calculated as below. The number of VE-cadherin positive cells per field was divided by the number of muscle fibers per field (n = 5 in each group). (D) VEGF, HGF and IGF synthesis in ischemic tissue determined by real-time PCR at day 7 after surgery following transplantation of Flk-1⁺ cells or PBS. VEGF, HGF or IGF mRNA levels were expressed relative to GAPDH mRNA levels (n = 5 in each group). N.S. = no significant difference between groups.</p

Senescence assay <i>in vitro</i>.

<p>(A) Undifferentiated and differentiated iPS cells were stained with a senescence detection kit to detect senescence associated-β-galactosidase (SA-β-Gal) around the nuclear area. (B) Quantitative analysis of the number of SA-β-Gal positive cells in undifferentiated and differentiated iPS cells. Expression of (C) SIRT and senescence associated genes such as (D) ARF and (E) p21 in Flk-1⁺ cells from young and old murine iPS cells determined by real-time PCR. SIRT, ARF and p21 mRNA levels were expressed relative to GAPDH mRNA levels (n = 3 in each group).</p

Differentiation into mature vascular cells <i>in vitro</i>.

<p>Sorted Flk-1⁺ cells derived from young and old iPS cells successfully differentiated into (A) mature endothelial cells (VE-cadherin positive) and (B) smooth muscle cells (α-SMA positive) 5 to 7 days after re-culture <i>in vitro</i>. Total nuclei were identified by DAPI counterstaining (blue). (C) Representative images of FACS analysis in differentiated cells (upper). FACS analysis was performed 5 to 7 days after re-plating of sorted Flk-1⁺ cells derived from young and old iPS cells on type IV collagen-coated dishes. Quantitative analysis of α-SMA, VE-cadherin and Ki-67 positive cells in differentiated cells (n = 5 in each group) (lower).</p

Tracking Flk-1⁺ cells during the chronic phase <i>in vivo</i>.

<p>(A) PKH26 labeled Flk-1⁺ cells from young iPS cells (red) and EGFP labeled Flk-1⁺ cells from old iPS cells (green) in ischemic muscle on postoperative day 21. Double fluorescence staining of VE-cadherin and labeled Flk-1⁺ cells in ischemic muscle. Co-localization is indicated by yellow in the merged images (magnification, ×200; bar indicates 200 µm). Total nuclei was identified by DAPI counterstaining (blue). (B) Quantitative analysis of the number of implanted Flk-1⁺ cells from young and old murine iPS cells in the chronic phase (n = 4 in each group).</p

Reproducible myogenic differentiation with the optimized protocol.

<p>(<b>a</b>) A schematic of our muscle differentiation protocol beginning with MyoD-hiPSCs. (<b>b</b>) Immunohistochemistry of differentiated MyoD-hiPSCs for MHC (red). Scale bar = 100 µm. (<b>c</b>) Percentage of MHC positive cells per total cells following MyoD-induced differentiation of 6 MyoD-hiPSC clones. (n = 3 for each clone). Data are listed as mean ±S.D.</p

Modeling Miyoshi Myopathy (MM) by patient derived-hiPSCs.

<p>(<b>a</b>) Morphology of patient derived MM-hiPSC clones, expanded following G418 selection for Tet-MyoD1 vector transposition. Scale bar = 200 µm. (<b>b</b>) RT-PCR analysis of endogenous pluripotent stem cell markers in MyoD-MM hiPSCs. (<b>c</b>) Efficient myogenic differentiation of MyoD-MM hiPSCs according to the protocol defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061540#pone-0061540-g003" target="_blank">Figure 3a</a>. MHC positive (left), or Myogenin positive (right) cells were observed dominantly. Scale bars = 100 µm. (<b>d</b>) DYSFERLIN expression of the myofibers from MyoD-MM hiPSCs (lane 1, 2), rescued MyoD-MM hiPSCs which expressed full-length <i>DYSF</i> cDNA driven by EF1α promoter (lane 3, 4), and control non-diseased MyoD-hiPSCs (lane 5) confirmed by western blotting. ACTB = β-actin. (<b>e</b>) Entry of FM1-43 green fluorescent dye into differentiated myofibers from MyoD-MM #5 (left), rescued MyoD-MM #5 with <i>DYSF</i> expression (middle), or control MyoD-hiPSC clone B7 #9 (right), before (0 s) and 20 s after (20 s) two photon laser-induced damage of the sarcolemmal membrane (arrow). Scale bars = 20 µm. (<b>f</b>) Summary time course data of accumulation of FM1-43 dye in laser-damaged myofibers derived from B7 #9 (black circles), MyoD-MM hiPSCs (red or blue triangles) and rescued MyoD-hiPSCs with DYSFERLIN expression (red or blue circles). n = 5 for each clone. Data are listed as mean ±S.E.</p

Generation of MyoD-hiPSCs.

<p>(<b>a</b>) Construction of the Tet-inducible <i>MYOD1</i> expressing <i>piggyBac</i> vector (Tet-MyoD1 vector). (<b>b</b>) A scheme of generation of MyoD-hiPSCs. Human iPSCs were transfected the Tet-MyoD1 vectors with transposase by lipofection. To select transfected cells, G418 were added for 5 days in the hiPSC culture media at 2 days after transfection. (<b>c</b>) MyoD-hiPSCs after 24 h in culture with or without Dox administration. Scale bar = 20 µm. (<b>d</b>) Upper lanes show dox-added MyoD-hiPSCs at d1. Lower lanes show immunodetection of MHC in Dox-induced MyoD-hiPSCs at d7. A lower-left panel shows the cells differentiated in maintenance medium from d1 to d7. A lower-right panel shows the cells differentiated in αMEM containing 5% KSR from d1 to d7. Scale bars = 200 µm. (<b>e</b>) Percentage of MHC positive cells per total cells following MyoD-induced differentiation. **<i>p</i><0.01.</p

Functional assay for differentiated MyoD-hiPSCs.

<p>(<b>a, b</b>) Electron microscopy of differentiated MyoD-hiPSCs (<b>a</b>) and differentiated human myoblast Hu5/E18 cells (<b>b</b>). Red arrows indicate myofibrils. Black arrowheads indicate future Z lines. Black arrows indicate myosin fibers. Scale bars = 500 nm. (<b>c</b>) Serial photographs of differentiated MyoD-hiPSCs co-cultured with C2C12 cells. A hiPSC-derived mCherry+ cell (white arrow) fused with a mouse-derived GFP+ cell (white arrowhead) resulting in a yellow cell (red arrow). Time increments between images = TIME. Scale bar = 100 µm. (<b>d</b>) Immunohistochemistry of MyoD-hiPSCs co-cultured with C2C12 cells. White arrows indicate human nuclei in a GFP+ murine myofiber. Scale bar = 100 µm.</p

Characterization of myofiber derived from MyoD-hiPSCs.

<p>(<b>a</b>) Time course gene expression profile for undifferentiated and myogenic markers in B7 #9 MyoD-hiPSC clone with (gray bars) or without (black bars) Dox administration (n = 3). Data are listed as mean±S.D. The data were standardized by β-actin using teratoma. The data on d0 = 1 in undifferentiated markers, such as <i>OCT3/4</i>, <i>SOX2</i> and <i>NANOG</i>. The data on d7 = 1 in other analyses. *: <i>p</i><0.05, **: <i>p</i><0.01, respectively, between Dox(–) and Dox(+). (<b>b</b>) Intracellular localization of mitochondria in both undifferentiated and differentiated MyoD-hiPSCs. Scale bar = 20 µm. (<b>c</b>) Immunohistochemistry of differentiated MyoD-hiPSCs for mature myogenic markers, such as CK-M, creatine kinase muscle isoform, Skeletal muscle Actin, and DYSTROPHIN. Scale bar = 20 µm. (<b>d</b>) Heat map of global mRNA expression comparing undifferentiated hiPSC (sample 1) and differentiated myogenic cells (samples 2-5). (<b>e</b>) Myogenic gene profile and unsupervised clustering based on markers associated with myofibers for undifferentiated hiPSCs and differentiated myogenic cells. Red color indicates up-regulated genes and blue color indicates down-regulated genes in (<b>d</b>) and (<b>e</b>).</p