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.

The developmental potency of mouse embryonic stem (ES) cells, which is the ability to contribute to a whole embryo is known to deteriorate during long-term cell culture. Previously we have shown that ES cells oscillate between Zscan4- and Zscan4+ states, and the transient activation of Zscan4 is required for the maintenance of telomeres and genome stability of ES cells. Here we show that increasing the frequency of Zscan4 activation in mouse ES cells restores and maintains their developmental potency in long-term cell culture. Injection of a single ES cell with such increased potency into a tetraploid blastocyst gives rise to an entire embryo with a higher success rate. These results not only provide a means to rejuvenate ES cells by manipulating Zscan4 expression, but also indicate the active roles of Zscan4 in the long-term maintenance of ES cell potency.

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...

SummaryA network of transcription factors (TFs) determines cell identity, but identity can be altered by overexpressing a combination of TFs. However, choosing and verifying combinations of TFs for specific cell differentiation have been daunting due to the large number of possible combinations of ∼2,000 TFs. Here, we report the identification of individual TFs for lineage-specific cell differentiation based on the correlation matrix of global gene expression profiles. The overexpression of identified TFs—Myod1, Mef2c, Esx1, Foxa1, Hnf4a, Gata2, Gata3, Myc, Elf5, Irf2, Elf1, Sfpi1, Ets1, Smad7, Nr2f1, Sox11, Dmrt1, Sox9, Foxg1, Sox2, or Ascl1—can direct efficient, specific, and rapid differentiation into myocytes, hepatocytes, blood cells, and neurons. Furthermore, transfection of synthetic mRNAs of TFs generates their appropriate target cells. These results demonstrate both the utility of this approach to identify potent TFs for cell differentiation, and the unanticipated capacity of single TFs directly guides differentiation to specific lineage fates.