Electron microscopic imaging of day 30 late-stage iCMCs demonstrates an increased maturity and ultra-structural organization.

<p>(A) Control HSF shows no myofibril or sarcomere. (B) hf-iPSC shows large nucleus and the cytoplasm devoid of any myofibrils and sarcomeres. (C<b>)</b> The day 7 iCMC shows disorganized sarcomeres on disorientated myofilaments. (D) The day 14 iCMC shows less organized sarcomeres and irregularly spaced myofibrils. (E) The day 30 iCMCs show a high density of organized and aligned myofibrils. (F) The higher magnification image reveals clear aligned Z-disks and organized bands in H-zone. Black arrows denoted the sarcomeres. N, nucleus; C, cytoplasm.</p

Generation of Functional Cardiomyocytes from Efficiently Generated Human iPSCs and a Novel Method of Measuring Contractility

<div><p>Human induced pluripotent stem cells (iPSCs) derived cardiomyocytes (iCMCs) would provide an unlimited cell source for regenerative medicine and drug discoveries. The objective of our study is to generate functional cardiomyocytes from human iPSCs and to develop a novel method of measuring contractility of CMCs. In a series of experiments, adult human skin fibroblasts (HSF) and human umbilical vein endothelial cells (HUVECs) were treated with a combination of pluripotent gene DNA and mRNA under specific conditions. The iPSC colonies were identified and differentiated into various cell lineages, including CMCs. The contractile activity of CMCs was measured by a novel method of frame-by-frame cross correlation (particle image velocimetry-PIV) analysis. Our treatment regimen transformed 4% of HSFs into iPSC colonies at passage 0, a significantly improved efficiency compared with use of either DNA or mRNA alone. The iPSCs were capable of differentiating both <i>in vitro</i> and <i>in vivo</i> into endodermal, ectodermal and mesodermal cells, including CMCs with >88% of cells being positive for troponin T (CTT) and Gata4 by flow cytometry. We report a highly efficient combination of DNA and mRNA to generate iPSCs and functional iCMCs from adult human cells. We also report a novel approach to measure contractility of iCMCs.</p></div

Differentiation of hf-iPSCs and he-iPSCs into neuronal cells.

<p>(A, B) hf-iPSCs and he-iPSCs were cultured under neuronal differentiation culture conditions for 21 days show significantly higher expression neuronal mRNA transcripts of Olig2 and MAP2 than the control cells. Fold expression was calculated as the ratio of he-iPSCs expression-to-parent control cells expression. Each bar represents the mean ± SEM of three replicated experiments. *p<0.05, **p<0.01. (C, D) The immunofluorescence staining of hf-iPSC and he-iPSC-derived neuronal cultures expressed neuron specific marker PGP9.5 (red) and astrocytes specific marker GFAP (green). (E<b>)</b> The qRT-PCR data show that the hf-iPSC-derived endothelial cells express the mRNA transcripts of CD31 and VE-Cadherin. (F1<b>)</b> The VE-Cadherin mRNA expression was further supported by the immunofluorescence analysis of VE-Cadherin protein expression. (F2<b>)</b> The tube formation assay showed that hf-iPSCs were capable of differentiating into endothelial cells under specific culture conditions. (G<b>)</b> The qRT-PCR data show that the endothelial cells derived from he-iPSCs express gene transcripts of CD31 and VE-Cadherin. Data are expressed as mean ± SEM, n = 3, *p<0.05. (H1<b>)</b> The VE-Cadherin mRNA expression was further supported by the immunofluorescence analysis of VE-Cadherin protein expression. (H2<b>)</b> The he-iPSCs were cultured under endothelial differentiation medium forms tubes and capillaries.</p

Characterization of iPSCs into endoderm and validation of pluripotency by teratoma assay.

<p>The hf-iPSCs and he-iPSCs were cultured under conditions conducive for hepatocyte differentiation for 25 days. Then the cells were harvested for various analyses. (A<b>)</b> The qRT-PCR data show that hepatocytes derived from hf-iPSCs significantly expressed mRNA transcripts of APOA1 and AFP. (B<b>)</b> The mRNA expression was further supported by the immunofluorescence staining of AFP protein expression. (C<b>)</b> Under hepatocyte culture, the qRT-PCR data from he-iPSC derived hepatocyte culture cells showed increased expression of hepatocyte mRNA gene transcripts of APOA1 and AFP. Each bar represents the mean ± SEM of three replicated experiments. *p<0.05, **p<0.01. (D<b>)</b> The mRNA expression of AFP was further corroborated by immunofluorescence staining AFP protein in he-iPSC derived hepatocyte culture. (E<b>)</b> The hf-iPSCs also demonstrated and differentiated into all three germ layers. H & E staining shows all three germ layers in the teratoma derived from SCID mouse. (E1<b>)</b> Neural epithelium-Ectoderm, (E2<b>)</b> Glandular cells–Endoderm, (E3<b>)</b> Cartilages–Mesoderm.</p

Differentiation of iCMCs.

<p>(A) The qRT-PCR data show that the day 14 hf-iCMCs and he-iCMCs significantly expressed CMC gene transcripts. Each bar represents the mean ± SEM of three replicated experiments, **p<0.01. (B<b>)</b> Quantification of day 14 hf-iCMCs by flow cytometry. (C) Quantification of day 14 he-iCMCs by flow cytometry. (D<b>)</b> Immunofluorescence staining of hf-iCMCs (red-Gata4, green-α-SA and blue-DAPI) on day 14, (E<b>):</b> Immunofluorescence staining of hf-iCMCs (red-CTT, green-α-SA and blue-DAPI) on day 30. Representative images are from three repeated experiments.</p

Late-stage iCMCs show better cardiac function measured by PIV method.

<p>(A) Differentiated and beating cardiomyocytes visualized by high frame rate (1/10 sec) video microscopy as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134093#pone.0134093.s005" target="_blank">S1 Movie</a>. (B) Velocity field snapshot of the area marked by a white rectangle in panel A. Red lines represent PIV-calculated displacements, their end point is marked by black dots. Locations without red lines were stationary during the 0.1 sec long time interval. (C) The beat pattern (PIV-derived displacements, measured relative to a stationary reference state and averaged over the entire field of view) indicates that CMC contractility is periodic with a steady waveform. (D) Time development of a tissue culture area that initially consisted three aggregates. Microscopic fields (D1, D3, D5, and the video microscopy is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134093#pone.0134093.s006" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134093#pone.0134093.s007" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134093#pone.0134093.s008" target="_blank">S4</a> Movies, respectively) and characteristic beat patterns (D2, D4, D6) are shown for three consecutive days after the onset of beating at day 7. Red, green and blue curves correspond to the areas marked as 1, 2 and 3 in the microscopic fields, respectively. During the time course of three days, the aperiodic and asynchronous beat patterns consolidate into a periodic and synchronous one. (E) Average profiles of contractile peaks are shown for beat patterns characterizing area 3 in panel D from day 8 [blue], day 9 [green] and day 10 [red]. Blue, green and red colors indicate progressively older cultures. As cardiomyocytes mature, the contractile periods become shorter. (F) Average period lengths obtained from 8 different cultures at various days in vitro. As cultures mature, the beating frequency tends to increase up to 1.4 Hz, at 37 days after the onset of beating. Representative images are from three repeated experiments.</p

Characterization of pluripotency in hf-iPSCs and he-iPSCs.

<p>Quantitative real-time PCR array-based expression pattern of 86 pluripotent genes. (A, B) Among the 86 genes, 44 genes in hf-iPSCs and 49 genes in he-iPSCs were significantly up regulated in he-iPSCs at passage 3 (P3), which are represented in red color. (C, D) The selected up regulated pluripotent genes from qRT-PCR array that showed more than 100 fold mRNA expression in hf-iPSCs and he-iPSCs. Each bar represents the mean ± SEM of three replicated experiments. Fold expression was calculated as the ratio of hf-iPSCs expression-to-parent control cells expression. The hf-iPSCs and he-iPSCs at P3 cells were further analyzed by immunofluorescence staining. (E) The immunofluorescence microscopic image shows the hf-iPSCs were stained positive for the Oct4 and Nanog protein expression. (F) Similarly, the he-iPSCs were also positive for Oct4 and Nanog protein expression at P3. (G) The Western analysis showed that the endogenous Oct4, Sox2 and Nanog genes are getting activated and expressing higher levels of proteins when compared to control parent cells. Histone 3 serves as a protein loading control. Representative images are from three repeated experiments.</p

Schematic representation of iPSC derivation protocol and the generation of iPSCs from HSF and HUVECs.

<p>(A) On day 0, the parent cells, HSF and HVECs cells were treated separately with Stemcircle, a plasmid containing Oct4, Nanog, Sox2 and Lin28 for 24 hours and subsequently, a cocktail of mRNAs containing Oct4, Sox2, Klf4, cMyc, Lin28 for up to day 12. The colonies were allowed to expand in between day 13 and 16. On day 17, the fully expanded colonies were picked and grown. HSF-Human skin fibroblast, HUVECs-Human umbilical vein endothelial cells, D-day. (B1) Phage contrast microscopic image of iPSC-derived from HSF (hf-iPSCs). (B2) Live staining of Tra1-60+ cells from hf-iPSCs. (C1) Phage contrast microscopic image of iPSC-derived from HUVECs (he-iPSCs). (C2) Live staining of Tra1-60+ cells from he-iPSCs. (D) Transfection efficiency of iPSCs generated from HSF by flow cytometry analysis at passage 0 (P0). The data show 4% of cells were positive for either Oct4 or Sox2. (E) Quantification of Tra1-60+ cells by flow cytometry at P9. The data show 73.2% of cells are Tra1-60+. Data shown are representative of three independent experiments, **p<0.01. (F, G) Alkaline phosphatase staining for hf-iPSCs and he-iPSCs.</p

Echocardiographic assessment of LV function.

<p>Representative two-dimensional (A, C) and M-mode (B, D) images from vehicle-treated (A, B), and atorvastatin-treated (C, D) rats 4 wk after coronary occlusion. The infarct wall is delineated by arrowheads. Compared with the vehicle-treated heart, the atorvastatin-treated heart exhibited a smaller LV cavity, a thicker infarct wall, and improved motion of the infarct wall. Contractile activity in the infarct area is present in the atorvastatin-treated heart and virtually absent in the vehicle-treated heart. Panels (E–J) demonstrate that treatment with atorvastatin improved echocardiographic measurements of LV systolic function at 4 wk after myocardial infarction. Data are mean ± SEM. n = 8–12 rats per group. *, <i>P</i><0.05 versus vehicle-treated rats at 4 wk. BSL, baseline.</p

Hemodynamic assessment of LV function at 4 wk after atorvastatin treatment.

<p>Representative pressure-volume loops from a vehicle-treated (A) and an atorvastatin-treated (B) rat recorded during preload manipulation by a brief period of inferior vena cava occlusion. Panels C–F illustrate the quantitative analysis of hemodynamic variables including dP/dt (C), LV end-diastolic pressure (D), end-systolic elastance (E), and preload recruitable stroke work (F). Data are mean ± SEM. n = 8–12 rats per group. *, <i>P</i><0.05 versus vehicle-treated rats at 4 wk. Abbreviations: Ees, end-systolic elastance; LVEDP, left ventricular end-diastolic pressure; PRSW, preload recruitable stroke work.</p