To realize cardiac regeneration using human induced pluripotent stem cells (hiPSCs), strategies for cell preparation, tissue engineering and transplantation must be explored. Here we report a new protocol for the simultaneous induction of cardiomyocytes (CMs) and vascular cells [endothelial cells (ECs)/vascular mural cells (MCs)], and generate entirely hiPSC-engineered cardiovascular cell sheets, which showed advantageous therapeutic effects in infarcted hearts. The protocol adds to a previous differentiation protocol of CMs by using stage-specific supplementation of vascular endothelial cell growth factor for the additional induction of vascular cells. Using this cell sheet technology, we successfully generated physically integrated cardiac tissue sheets (hiPSC-CTSs). HiPSC-CTS transplantation to rat infarcted hearts significantly improved cardiac function. In addition to neovascularization, we confirmed that engrafted human cells mainly consisted of CMs in >40% of transplanted rats four weeks after transplantation. Thus, our HiPSC-CTSs show promise for cardiac regenerative therapy.

Isolation of specific cell types, including pluripotent stem cell (PSC)-derived populations, is frequently accomplished using cell surface antigens expressed by the cells of interest. However, specific antigens for many cell types have not been identified, making their isolation difficult. Here, we describe an efficient method for purifying cells based on endogenous miRNA activity. We designed synthetic mRNAs encoding a fluorescent protein tagged with sequences targeted by miRNAs expressed by the cells of interest. These miRNA switches control their translation levels by sensing miRNA activities. Several miRNA switches (miR-1-, miR-208a-, and miR-499a-5p-switches) efficiently purified cardiomyocytes differentiated from human PSCs, and switches encoding the apoptosis inducer Bim enriched for cardiomyocytes without cell sorting. This approach is generally applicable, as miR-126-, miR-122-5p-, and miR-375-switches purified endothelial cells, hepatocytes, and insulin-producing cells differentiated from hPSCs, respectively. Thus, miRNA switches can purify cell populations for which other isolation strategies are unavailable.

Although stem cell therapy is a promising strategy for cardiac restoration, the heterogeneity of transplanted cells has been hampering the precise understanding of the cellular and molecular mechanisms. Previously, we established a cardiovascular cell differentiation system from mouse pluripotent stem cells, in which cardiomyocytes (CMs), endothelial cells (ECs), and mural cells (MCs) can be systematically induced and purified. Combining this with cell sheet technology, we generated cardiac tissue sheets reassembled with defined cardiovascular populations. Here, we show the potentials and mechanisms of cardiac tissue sheet transplantation in cardiac function after myocardial infarction (MI). Transplantation of the cardiac tissue sheet to a rat MI model showed significant and sustained improvement of systolic function accompanied by neovascularization. Reduction of the infarct wall thinning and fibrotic length indicated the attenuation of left ventricular remodeling. Cell tracing with species-specific fluorescent in situ hybridization after transplantation revealed a relatively early loss of transplanted cells and an increase in endogenous neovascularization in the proximity of the graft, suggesting an indirect angiogenic effect of cardiac tissue sheets rather than direct CM contributions. We prospectively dissected the functional mechanisms with cell type-controlled sheet analyses. Sheet CMs were the main source of vascular endothelial growth factor. Transplantation of sheets lacking CMs resulted in the disappearance of neovascularization and subsequent functional improvement, indicating that the beneficial effects of the sheet were achieved by sheet CMs. ECs and MCs enhanced the sheet functions and structural integration. Supplying CMs to ischemic regions with cellular interaction could be a strategic key in future cardiac cell therapy.

Molecular mechanisms controlling arterial–venous specification have not been fully elucidated. Previously, we established an embryonic stem cell differentiation system and demonstrated that activation of cAMP signaling together with VEGF induces arterial endothelial cells (ECs) from Flk1+ vascular progenitor cells. Here, we show novel arterial specification machinery regulated by Notch and β-catenin signaling. Notch and GSK3β-mediated β-catenin signaling were activated downstream of cAMP through phosphatidylinositol-3 kinase. Forced activation of Notch and β-catenin with VEGF completely reconstituted cAMP-elicited arterial EC induction, and synergistically enhanced target gene promoter activity in vitro and arterial gene expression during in vivo angiogenesis. A protein complex with RBP-J, the intracellular domain of Notch, and β-catenin was formed on RBP-J binding sites of arterial genes in arterial, but not venous ECs. This molecular machinery for arterial specification leads to an integrated and more comprehensive understanding of vascular signaling.

IntroductionVascular endothelial growth factor (VEGF) signaling is a key regulator of vascular development during embryogenesis as well as neovascularization in the adult. [1][2][3] Intensity of VEGF signaling is strictly controlled during vascular development through ligandreceptor interaction. 4,5 Flk1 (also designated as VEGF receptor-2) is tyrosine-phosphorylated much more efficiently than Flt1 (VEGF receptor-1) upon VEGF binding and is thought to be the major receptor in endothelial cells (ECs) for VEGF-induced responses. [6][7][8] Whereas Flk1-null mice die at embryonic day 8.5 (E8.5) to E9.5 with no organized blood vessels, 9 Flt1-null mice die at midgestation with vascular overgrowth and disorganization. 10,11 Flt1 tyrosine kinase-deficient homozygous mice, in which VEGF can bind to the cell-surface domain of Flt1 but cannot conduct kinase signaling, developed normal vessels and survived, 12 indicating that VEGF signal intensity on Flk1 is regulated by absorption of VEGF to the higher affinity receptor, Flt1. VEGF-A heterozygotes die early in gestation due to failure in vascular system formation. 13 On the other hand, 2-to 3-fold overexpression of VEGF-A from its endogenous locus results in aberrant heart development and lethality at E12.5 to E14, 14 indicating that strictly balanced VEGF function is important in normal embryogenesis.Neuropilin-1 (NRP1) is a type 1 membrane protein, which is expressed in particular classes of developing neurons 15,16 and functions as a receptor for the class 3 semaphorins mediating semaphorin-elicited inhibitory axon guidance signals to neurons. 17,18 NRP1 is also expressed in ECs of blood vessels and endocardial cells of the heart. 15,16,19 NRP1, together with Flk1, forms a specific receptor for VEGF 165 , an isoform of VEGF, and the Flk1-VEGF 165 -NRP1 complex potently enhances Flk1 signaling. 20 Coexpression of NRP1 with Flk1 in cultured ECs enhanced VEGF 165 binding to Flk1 and VEGF-elicited mitogenic and chemotactic activities. 20 Overexpression of NRP1 in mouse embryos resulted in an excess production of blood vessels and malformed hearts. 15 NRP1-null mice die midway through gestation at E10.5 to E12.5 and exhibit defects in the heart, vasculature, and nervous system. 16 These findings indicate that NRP1 plays an important role in regulating vascular development, and Flk1/NRP1 system would be important for controlling VEGF signal intensity. However, the regulatory mechanisms of Flk1/NRP1 expression in vascular development are not fully elucidated.In the early embryo and in differentiating embryonic stem (ES) cells, Flk1 expression marks a common progenitor for both blood and endothelium. [21][22][23][24] To elucidate the mechanisms underlying vascular development, we have developed a novel ES cell differentiation system that exhibits early vascular development using Flk1 ϩ cells as common progenitors for vascular cells. 25 ES cell-derived Flk1 ϩ cells can differentiate into both ECs and mural cells (MCs: vascular smooth muscle cells and pericytes) and form mature...

The efficiency of pluripotent stem cell differentiation is highly variable, often resulting in heterogeneous populations that contain undifferentiated cells. Here we developed a sensitive, target-specific, and general method for removing undesired cells before transplantation. MicroRNA-302a-5p (miR-302a) is highly and specifically expressed in human pluripotent stem cells and gradually decreases to basal levels during differentiation. We synthesized a new RNA tool, miR-switch, as a live-cell reporter mRNA for miR-302a activity that can specifically detect human induced pluripotent stem cells (hiPSCs) down to a spiked level of 0.05% of hiPSCs in a heterogeneous population and can prevent teratoma formation in an in vivo tumorigenicity assay. Automated and selective hiPSC-elimination was achieved by controlling puromycin resistance using the miR-302a switch. Our system uniquely provides sensitive detection of pluripotent stem cells and partially differentiated cells. In addition to its ability to eliminate undifferentiated cells, miR-302a switch also holds great potential in investigating the dynamics of differentiation and/or reprograming of live-cells based on intracellular information.

Opioids are effective analgesics for the management of moderate to severe cancer pain. Here we show that κ opioid receptor (KOR) agonists act as anti-angiogenic factors in tumors. Treatment with KOR agonists, U50,488H and TRK820, significantly inhibited human umbilical vein endothelial cell (HUVEC) migration and tube formation by suppressing VEGFR2 expression. In contrast, treatment with a μ opioid receptor agonist, DAMGO, or a δ opioid receptor agonist, SNC80, did not prevent angiogenesis in HUVECs. Lewis lung carcinoma (LLC) or B16 melanoma grafted in KOR knockout mice showed increased proliferation and remarkably enhanced tumor angiogenesis compared with those in wild type mice. On the other hand, repeated intraperitoneal injection of TRK820 (0.1–10 μg/kg, b.i.d.) significantly inhibited tumor growth by suppressing tumor angiogenesis. These findings indicate that KOR agonists play an important role in tumor angiogenesis and this knowledge could lead to a novel strategy for cancer therapy.

Poor engraftment of cells after transplantation to the heart is a common and unresolved problem in the cardiac cell therapies. We previously generated cardiovascular cell sheets entirely from pluripotent stem cells with cardiomyocytes, endothelial cells and vascular mural cells. Though sheet transplantation showed a better engraftment and improved cardiac function after myocardial infarction, stacking limitation (up to 3 sheets) by hypoxia hampered larger structure formation and long-term survival of the grafts. Here we report an efficient method to overcome the stacking limitation. Insertion of gelatin hydrogel microspheres (GHMs) between each cardiovascular cell sheet broke the viable limitation via appropriate spacing and fluid impregnation with GHMs. Fifteen sheets with GHMs (15-GHM construct; >1 mm thickness) were stacked within several hours and viable after 1 week in vitro. Transplantation of 5-GHM constructs (≈2 × 106 of total cells) to a rat myocardial infarction model showed rapid and sustained functional improvements. The grafts were efficiently engrafted as multiple layered cardiovascular cells accompanied by functional capillary networks. Large engrafted cardiac tissues (0.8 mm thickness with 40 cell layers) successfully survived 3 months after TX. We developed an efficient method to generate thicker viable tissue structures and achieve long-term survival of the cell graft to the heart.