Yuji Shiba

Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts

Transplantation studies in mice and rats have shown that human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) can improve the function of infarcted hearts, but two critical issues related to their electrophysiological behaviour in vivo remain unresolved. First, the risk of arrhythmias following hESC-CM transplantation in injured hearts has not been determined. Second, the electromechanical integration of hESC-CMs in injured hearts has not been demonstrated, so it is unclear whether these cells improve contractile function directly through addition of new force-generating units. Here we use a guinea-pig model to show that hESC-CM grafts in injured hearts protect against arrhythmias and can contract synchronously with host muscle. Injured hearts with hESC-CM grafts show improved mechanical function and a significantly reduced incidence of both spontaneous and induced ventricular tachycardia. To assess the activity of hESC-CM grafts in vivo, we transplanted hESC-CMs expressing the genetically encoded calcium sensor, GCaMP3 (refs 4, 5). By correlating the GCaMP3 fluorescent signal with the host ECG, we found that grafts in uninjured hearts have consistent 1:1 host–graft coupling. Grafts in injured hearts are more heterogeneous and typically include both coupled and uncoupled regions. Thus, human myocardial grafts meet physiological criteria for true heart regeneration, providing support for the continued development of hESC-based cardiac therapies for both mechanical and electrical repair.

Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts

Induced pluripotent stem cells (iPSCs) constitute a potential source of autologous patient-specific cardiomyocytes for cardiac repair, providing a major benefit over other sources of cells in terms of immune rejection. However, autologous transplantation has substantial challenges related to manufacturing and regulation. Although major histocompatibility complex (MHC)-matched allogeneic transplantation is a promising alternative strategy, few immunological studies have been carried out with iPSCs. Here we describe an allogeneic transplantation model established using the cynomolgus monkey (Macaca fascicularis), the MHC structure of which is identical to that of humans. Fibroblast-derived iPSCs were generated from a MHC haplotype (HT4) homozygous animal and subsequently differentiated into cardiomyocytes (iPSC-CMs). Five HT4 heterozygous monkeys were subjected to myocardial infarction followed by direct intra-myocardial injection of iPSC-CMs. The grafted cardiomyocytes survived for 12 weeks with no evidence of immune rejection in monkeys treated with clinically relevant doses of methylprednisolone and tacrolimus, and showed electrical coupling with host cardiomyocytes as assessed by use of the fluorescent calcium indicator G-CaMP7.09. Additionally, transplantation of the iPSC-CMs improved cardiac contractile function at 4 and 12 weeks after transplantation; however, the incidence of ventricular tachycardia was transiently, but significantly, increased when compared to vehicle-treated controls. Collectively, our data demonstrate that allogeneic iPSC-CM transplantation is sufficient to regenerate the infarcted non-human primate heart; however, further research to control post-transplant arrhythmias is necessary.

Creation of human cardiac cell sheets using pluripotent stem cells

Although we previously reported the development of cell-dense thickened cardiac tissue by repeated transplantation-based vascularization of neonatal rat cardiac cell sheets, the cell sources for human cardiac cells sheets and their functions have not been fully elucidated. In this study, we developed a bioreactor to expand and induce cardiac differentiation of human induced pluripotent stem cells (hiPSCs). Bioreactor culture for 14 days produced around 8×10(7) cells/100 ml vessel and about 80% of cells were positive for cardiac troponin T. After cardiac differentiation, cardiomyocytes were cultured on temperature-responsive culture dishes and showed spontaneous and synchronous beating, even after cell sheets were detached from culture dishes. Furthermore, extracellular action potential propagation was observed between cell sheets when two cardiac cell sheets were partially overlaid. These findings suggest that cardiac cell sheets formed by hiPSC-derived cardiomyocytes might have sufficient properties for the creation of thickened cardiac tissue.

Critical Role of Bone Marrow Apoptosis-Associated Speck-Like Protein, an Inflammasome Adaptor Molecule, in Neointimal Formation After Vascular Injury in Mice

Background— Inflammatory cytokines such as interleukin (IL)-1β and IL-18 play an important role in the development of atherosclerosis and restenosis. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein that regulates caspase-1–dependent IL-1β and IL-18 generation; however, the role of ASC in vascular injury remains undefined. Here, we investigated the contribution of ASC to neointimal formation after vascular injury in ASC-deficient (ASC−/−) mice. Methods and Results— Wire-mediated vascular injury was produced in the femoral artery of ASC−/− and wild-type mice. Immunohistochemical analysis revealed that ASC was markedly expressed at the site of vascular injury. Neointimal formation was significantly attenuated in ASC−/− mice after injury. IL-1β and IL-18 were expressed in the neointimal lesion in wild-type mice but showed decreased expression in the lesion of ASC−/− mice. To investigate the contribution of bone marrow–derived cells, we developed bone marrow–transplanted mice and found that neointimal formation was significantly decreased in wild-type mice in which bone marrow was replaced with ASC−/− bone marrow cells. Furthermore, in vitro experiments showed that the proliferation activity of ASC−/− vascular smooth muscle cells was not impaired. Conclusions— These findings suggest that bone marrow–derived ASC is critical for neointimal formation after vascular injury and identify ASC as a novel therapeutic target for atherosclerosis and restenosis.

M-CSF Accelerates Neointimal Formation in the Early Phase After Vascular Injury in Mice. The Critical Role of the SDF-1-CXCR4 System

Objective—Since the macrophage colony-stimulating factor (M-CSF) has been shown to stimulate differentiation and proliferation of monocyte/macrophage lineage and to be involved in the process of neointimal formation after vascular injury, we tested the effects of M-CSF on the recruitment of bone marrow–derived progenitor cells in neointimal formation after vascular injury in mice. Methods and Results—Wire-mediated vascular injury was produced in the femoral artery of C57BL/6 mice. Recombinant human M-CSF [500 &mgr;g/(kg·day)] or saline (control) was administered for 10 consecutive days, starting 4 days before the injury. Treatment with M-CSF accelerated neointimal formation in the early phase after injury, and this neointimal lesion mainly consisted of bone marrow–derived cells. M-CSF treatment had no effect on the mobilization of endothelial progenitor cells (EPCs: CD34+/Flk-1+) and reendothelialization after injury. The stromal cell-derived factor-1 (SDF-1) was markedly expressed in the neointima and media after injury, whereas CXCR4+ cells were observed in the neointima. Further, a novel CXCR4 antagonist, AMD3100, significantly attenuated the M-CSF–induced neointimal formation. Conclusions—These findings suggest that M-CSF accelerated neointimal formation after vascular injury via the SDF-1–CXCR4 system, and the inhibition of this system has therapeutic potential for the treatment of cardiovascular diseases.

Electrical integration of human embryonic stem cell-derived cardiomyocytes in a guinea pig chronic infarct model

Background: Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) were recently shown to be capable of electromechanical integration following direct injection into intact or recently injured guinea pig hearts, and hESC-CM transplantation in recently injured hearts correlated with improvements in contractile function and a reduction in the incidence of arrhythmias. The present study was aimed at determining the ability of hESC-CMs to integrate and modulate electrical stability following transplantation in a chronic model of cardiac injury. Methods and Results: At 28 days following cardiac cryoinjury, guinea pigs underwent intracardiac injection of hESC-CMs, noncardiac hESC derivatives (non-CMs), or vehicle. Histology confirmed partial remuscularization of the infarct zone in hESC-CM recipients while non-CM recipients showed heterogeneous xenografts. The 3 experimental groups showed no significant difference in the left ventricular dimensions or fractional shortening by echocardiography or in the incidence of spontaneous arrhythmias by telemetric monitoring. Although recipients of hESC-CMs and vehicle showed a similar incidence of arrhythmias induced by programmed electrical stimulation at 4 weeks posttransplantation, non-CM recipients proved to be highly inducible, with a ∼3-fold greater incidence of induced arrhythmias. In parallel studies, we investigated the ability of hESC-CMs to couple with host myocardium in chronically injured hearts by the intravital imaging of hESC-CM grafts that stably expressed a fluorescent reporter of graft activation, the genetically encoded calcium sensor GCaMP3. In this work, we found that only ∼38% (5 of 13) of recipients of GCaMP3+ hESC-CMs showed fluorescent transients that were coupled to the host electrocardiogram. Conclusions: Human embryonic stem cell-derived cardiomyocytes engraft in chronically injured hearts without increasing the incidence of arrhythmias, but their electromechanical integration is more limited than previously reported following their transplantation in a subacute injury model. Moreover, non-CM grafts may promote arrhythmias under certain conditions, a finding that underscores the need for input preparations of high cardiac purity.

Notch signaling regulates the differentiation of bone marrow-derived cells into smooth muscle-like cells during arterial lesion formation.

Bone marrow- (BM-) derived cells can differentiate into smooth muscle-like cells (SMLC), resulting in vascular pathogenesis. However, the molecular mechanism of the differentiation remains unknown. We have recently reported that Notch signaling promotes while a Notch target HERP1 inhibit the differentiation of mesenchymal cells to SMC. During the differentiation of BM-derived mononuclear cells into smooth muscle alpha-actin (SMA)-positive cells, expression of Jagged1 and SMC-specific Notch3 was increased. Blocking Notch with gamma-secretase inhibitor prevented the induction of SMA. Wire-mediated vascular injury was produced in femoral arteries in mice transplanted with green fluorescent protein (GFP)-positive cells. Many double-positive cells for GFP/Jagged1 or GFP/Notch3 were detected in the thickened neointima. In contrast, only a few SMA-positive cells were positive for GFP in neointima where HERP1, a suppressor for Notch, were abundantly expressed. In conclusion, Notch-HERP1 pathway plays an important role in differentiation of BM-derived mononuclear cells into SMLC.

Crucial Role of Hyaluronan in Neointimal Formation after Vascular Injury

Background Hyaluronan (HA) is a primary component of the extracellular matrix of cells, and it is involved in the pathogenesis of atherosclerosis. The purpose of this study was to investigate the role of HA in neointimal formation after vascular injury and determine its tissue-specific role in vascular smooth muscle cells (VSMCs) by using a cre-lox conditional transgenic (cTg) strategy. Methods and Results HA was found to be expressed in neointimal lesions in humans with atherosclerosis and after wire-mediated vascular injury in mice. Inhibition of HA synthesis using 4-methylumbelliferone markedly inhibited neointimal formation after injury. In vitro experiments revealed that low-molecular-weight HA (LMW-HA) induced VSMC activation, including migration, proliferation, and production of inflammatory cytokines, and reactive oxygen species (ROS). The migration and proliferation of VSMCs were mediated by the CD44/RhoA and CD44/ERK1/2 pathways, respectively. Because HA synthase 2 (HAS2) is predominantly expressed in injured arteries, we generated cTg mice that overexpress the murine HAS2 gene specifically in VSMCs (cHAS2/CreSM22α mice) and showed that HA overexpression markedly enhanced neointimal formation after cuff-mediated vascular injury. Further, HA-overexpressing VSMCs isolated from cHAS2/CreSM22α mice showed augmented migration, proliferation, and production of inflammatory cytokines and ROS. Conclusion VSMC-derived HA promotes neointimal formation after vascular injury, and HA may be a potential therapeutic target for cardiovascular disease.

Bone marrow CXCR4 induction by cultivation enhances therapeutic angiogenesis

AIMS The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor (CXCR4, CXC chemokine receptor 4) play a critical role in the process of post-natal neovascularization. Here, we investigated the role of CXCR4(+) bone marrow cells (BMCs) in neovascularization in a murine hindlimb ischaemia model. METHODS AND RESULTS We found that the expression of CXCR4 in BMCs was specifically upregulated by cultivation; therefore, we used freshly isolated BMCs and cultivated BMCs, designated as BMC(Fr) and BMC(Cul), respectively. The increased CXCR4 expression corresponded to the migratory capacity in response to SDF-1 alpha. Real-time reverse transcription-polymerase chain reaction and immunohistochemical analyses revealed that SDF-1 alpha expression was significantly increased in the ischaemic limbs of mice. Blood flow perfusion and capillary density were significantly accelerated in mice implanted with BMC(Cul) as compared with those in mice implanted with BMC(Fr). The stimulatory effect of BMC(Cul) on neovascularization was significantly impaired when BMC(Cul) derived from CXCR4(+/-) mice were implanted. The implanted BMC(Cul) showed high retention in the ischaemic limbs. Further, the implantation of BMC(Cul) significantly increased the expression of interleukin (IL)-1 beta and vascular endothelial growth factor-A in the ischaemic limbs. CONCLUSION The upregulation of CXCR4 expression by cultivation may serve as a useful source of BMCs for accelerating therapeutic angiogenesis in ischaemic cardiovascular diseases.

Transplantation of adipose tissue-derived stem cells improves cardiac contractile function and electrical stability in a rat myocardial infarction model

The transplantation of adipose tissue-derived stem cells (ADSCs) improves cardiac contractility after myocardial infarction (MI); however, little is known about the electrophysiological consequences of transplantation. The purpose of this study was to clarify whether the transplantation of ADSCs increases or decreases the incidence of ventricular tachyarrhythmias (VT) in a rat model of MI. MI was induced experimentally by permanent occlusion of the left anterior descending artery of Lewis rats. ADSCs were harvested from GFP-transgenic rats, and were cultured until passage four. ADSCs (10×10(6)) resuspended in 100μL saline or pro-survival cocktail (PSC), which enhances cardiac graft survival, were injected directly into syngeneic rat hearts 1week after MI. The recipients of ADSCs suspended in PSC had a larger graft area compared with those receiving ASDCs suspended in saline at 1week post-transplantation (number of graft cells/section: 148.7±10.6 vs. 22.4±3.4, p<0.05, n=5/group). Thereafter, all ADSC recipients were transplanted with ASDCs in PSC. ADSCs were transplanted into infarcted hearts, and the mechanical and electrophysiological functions were assessed. Echocardiography revealed that ADSC recipients had improved contractile function compared with those receiving PSC vehicle (fractional shortening: 21.1±0.9 vs. 14.1±1.2, p<0.05, n≥12/group). Four weeks post-transplantation, VT was induced via in vivo programmed electrical stimulation. The recipients of ADSCs showed a significantly lower incidence of induced VT compared with the control (31.3% vs. 83.3%, p<0.05, n≥12/group). To understand the electrical activity following transplantation, we performed ex vivo optical mapping using a voltage sensitive dye, and found that ADSC transplantation decreased conduction velocity and its dispersion in the peri-infarct area. These results suggest that ADSC transplantation improved cardiac mechanical and electrophysiological functions in subacute MI.