Characterization of endothelial cell induced from human iPS cell with stimulation-elimination method.

<p>Immunofluorescent staining for (a) CD31 (green) and (b) VE-Cadherin (red) in human iPS cell-derived endothelial cells. Nuclei were visualized with DAPI (blue). (c-g) Relative mRNA log₀ ratio in endothelial cells at differentiation day 9 (EC (D9)) and undifferentiated human iPS cell (iPSC) compared with human umbilical vein endothelial cell (HUVEC). CD31 (c), VE-Cadherin (d), eNOS (e), CD34 (f), and CD133 (g). (h) Tube formation assay. Human iPS cell-derived endothelial cells were recultured on Matrigel Basement Membrane Matrix GFR coated dish. (i) Immunofluorescent stained of CD31 for recultured cells on Matrigel. (j,k) Acetyl-LDL incorporation assay. Endothelial cells were incubated with acetylated LDL labeled with 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindo-carbocyanine perchlorate (DiI-Ac-LDL). Bright-field (j) and fluorescent (k) images. Scale bars: 50 μm in (a), (b) and (i), 100 μm in (h), 200 μm in (j) and (k).</p

Stimulation-elimination method for efficient endothelial cells induction from human pluripotent stem cells.

<p>(a) Schematic representation of stimulation-elimination method. (b) Distinct expression pattern of VE-cadherin (VECad) and CD31 in stimulation-elimination method at differentiation day 9. (c) The yield of endothelial cells at differentiation day 9 from one human pluripotent stem cell in stimulation method or stimulation-elimination method. (d) The yield of endothelial cells at differentiation day14 from one replated endothelial cells at differentiation day 9 in two groups.</p

Time course of endothelial cell and pre-endothelial cell marker.

<p>(a) Representative expression time course of VE-cadherin (VECad) and CD31 under stimulation method (VEGF+cAMP) or vehicle without VEGF and cAMP by FACS. (b) Time course of VE-Cadherin-positive cell ratio in two groups. (c) Yield of VE-Cadherin positive endothelial cells per 1cm² in two groups. (d) Time course of total cell counts in two groups. (e) Representative expression time course of VEGF receptor 2 (VEGFR2) and VE-cadherin in stimulation method (VEGF+cAMP) or vehicle without cAMP and VEGF. Arrows: non-responder cells to VEGF and cAMP stimulation.</p

Efficient induction of endothelial cells from human pluripotent stem cells with supplementation of VEGF and cAMP.

<p>(a) Schematic representation of the stimulation method. (b) Ratio of VE-Cadherin positive cell per total cells at differentiation day 9 by flow cytometory in stimulation method group (VEGF+cAMP), VEGF administration groups (VEGF) and no administration groups (vehicle). (c) Mean yield of endothelial cells per 1cm² in three groups. (d) Distinct expression pattern of VE-cadherin in stimulation method at differentiation day 9 (left panel) and post-VE-Cadherin purification (right panel).</p

Regional wall motion evaluated by circumference strain at mid region.

<p>Regional wall motion evaluated by circumference strain at mid region.</p

Regional wall motion evaluated by circumference strain at base region.

<p>Regional wall motion evaluated by circumference strain at base region.</p

Transplantation of L-CTSs onto porcine infarct hearts.

<p>(A) Time course of the animal experiments. MI, myocardial infarction; UCG, ultrasound cardiogram (echocardiogram); LVG, left ventriculogram; Tx, treatment. (B) Surgical view during L-CTS transplantation. The first L-CTS is shown at black arrow and the second L-CTS is shown at white arrow. (C) M-mode view of the echocardiogram. Dotted yellow lines indicate the endocardial boundary of anterior or posterior wall. PreTx, before transplantation; Tx2w, 2 weeks after transplantation. (D) systolic function after transplantation. *p<0.05 vs Sham, ‡p<0.01 vs PreTx. (E) Left ventriculogram at 4 weeks after transplantation. Red: end-diastolic phase, orange: end-systolic phase. (F) Ejection fraction calculated by left ventriculogram. *p<0.05 vs Sham. PreMI, before MI induction.</p

Attenuation of LV remodeling after L-CTS transplantation.

<p>(A) Representative Masson’s trichrome staining at 4 weeks after transplantation. RV, right ventricle (removed). (B) The ratio of fibrotic area to the whole heart area. ***p<0.001. (C) Representative PAS staining of border region at 4 weeks after transplantation. Scale bar = 20 μm. (D) Cardiomyocyte diameter. ***p<0.001. (E) Representative vWF staining 4 weeks after L-CTS transplantation (brown). Scale bar = 20 μm. (F) Capillary density at border region. ***p<0.001. (G) Representative Ku80 staining (brown). Scale bar = 20 μm.</p

Fabrication of Mouse Embryonic Stem Cell-Derived Layered Cardiac Cell Sheets Using a Bioreactor Culture System

<div><p>Bioengineered functional cardiac tissue is expected to contribute to the repair of injured heart tissue. We previously developed cardiac cell sheets using mouse embryonic stem (mES) cell-derived cardiomyocytes, a system to generate an appropriate number of cardiomyocytes derived from ES cells and the underlying mechanisms remain elusive. In the present study, we established a cultivation system with suitable conditions for expansion and cardiac differentiation of mES cells by embryoid body formation using a three-dimensional bioreactor. Daily conventional medium exchanges failed to prevent lactate accumulation and pH decreases in the medium, which led to insufficient cell expansion and cardiac differentiation. Conversely, a continuous perfusion system maintained the lactate concentration and pH stability as well as increased the cell number by up to 300-fold of the seeding cell number and promoted cardiac differentiation after 10 days of differentiation. After a further 8 days of cultivation together with a purification step, around 1×10⁸ cardiomyocytes were collected in a 1-L bioreactor culture, and additional treatment with noggin and granulocyte colony stimulating factor increased the number of cardiomyocytes to around 5.5×10⁸. Co-culture of mES cell-derived cardiomyocytes with an appropriate number of primary cultured fibroblasts on temperature-responsive culture dishes enabled the formation of cardiac cell sheets and created layered-dense cardiac tissue. These findings suggest that this bioreactor system with appropriate medium might be capable of preparing cardiomyocytes for cell sheet-based cardiac tissue.</p></div

Cardiac differentiation of ES cells in the bioreactor.

<p>(A) Real-time PCR analysis (<i>n</i> = 3). Solid lines indicate the results of continuous medium exchange, and dotted lines indicate the results of intermittent medium exchange. *<i>p</i><0.05. (B) Representative dot plots of flow cytometric analyses of GFP(+) cells in EBs at day 10. The graph shows the percentage of GFP(+) cells at day 10 (<i>n</i> = 6). (C) GFP(+) cells were collected using a fluorescence-activated cell sorter and then seeded onto gelatin-coated dishes. GFP(+) cells expressed sarcomeric α-actinin (upper), myosin heavy chain (MHC, middle) and cardiac troponin T (lower) in a fine striated pattern. Nuclei were counterstained with DAPI. Scale bars, 20 µm. (D, E) Comparison of the numbers of cardiomyocytes among culture conditions using R1 ES cells. (D) The number of remaining cells 8 days after starting culture with G418 (<i>n</i> = 3). Data are means ± s.d. (E) At 8 days after starting culture with G418 in the bioreactor with or without regulation, the cells were dissociated and seeded onto 1% gelatin-coated 24-well plates. At 24 h after seeding, the cells were fixed and immunostained for sarcomeric-α actinin. The percentage of sarcomeric α-actinin-positive cells among the remaining cells was calculated and is shown in the graph (<i>n</i> = 3). Data are means ± s.d. Right, representative images. Nuclei were stained with DAPI. Bars, 200 µm. (F) Comparison of cardiomyocyte engraftment between medium exchange systems. Purified cardiomyocytes after treatment with G418 were co-cultured with fibroblasts at the ratio of 8∶2. At day 2, cells were fixed and immunostained with cTnT. The percentage of cTnT-positive cells was calculated and is shown in the graph (<i>n</i> = 3). Data are means ± s.d. n.s., not significant. Right, representative images. Nuclei were stained with Hoechst33258. Bars, 200 µm.</p