Hirohiko Suzuki

Enhanced Angiogenesis by Transplantation of Mesenchymal Stem Cell Sheet Created by a Novel Magnetic Tissue Engineering Method

Objective—Therapeutic angiogenesis with cell transplantation represents a novel strategy for severe ischemic diseases. However, some patients have poor response to such conventional injection-based angiogenic cell therapy. Here, we investigated a therapeutic potential of mesenchymal stem cell (MSC) sheet created by a novel magnetite tissue engineering technology for reparative angiogenesis. Methods and Results—Human MSCs incubated with magnetic nanoparticle-containing liposomes were cultured, and a magnet was placed on the reverse side. Magnetized MSCs formed multilayered cell sheets according to magnetic force. Nude mice were subjected to unilateral hind limb ischemia and separated into 3 groups. For the control group, saline was injected into ischemic tissue. In the MSC-injected group, mice received magnetized MSCs by conventional needle injections without sheet formula as a control cell group. In the MSC-sheet group, MSC sheet was layered onto the ischemic tissues before skin closure. Blood flow recovery and the extent of angiogenesis were assessed by a laser Doppler blood flowmetry and histological capillary density, respectively. The MSC-sheet group had a greater angiogenesis in ischemic tissues compared to the control and MSC-injected groups. The angiogenic and tissue-preserving effects of MSC sheets were attributable to an increased expression of vascular endothelial growth factor and reduced apoptosis in ischemic tissues. In cultured MSCs, magnetic labeling itself inhibited apoptosis via a catalase-like antioxidative mechanism. Conclusion—MSC sheet created by the novel magnetic nanoparticle-based tissue engineering technology would represent a new modality for therapeutic angiogenesis and tissue regeneration.

Therapeutic angiogenesis by transplantation of induced pluripotent stem cell-derived Flk-1 positive cells

BackgroundInduced pluripotent stem (iPS) cells are the novel stem cell population induced from somatic cells. It is anticipated that iPS will be used in the expanding field of regenerative medicine. Here, we investigated whether implantation of fetal liver kinase-1 positive (Flk-1+) cells derived from iPS cells could improve angiogenesis in a mouse hind limb model of ischemia.ResultsFlk-1+ cells were induced from iPS cells after four to five days of culture. Hind limb ischemia was surgically induced and sorted Flk-1+ cells were directly injected into ischemic hind limbs of athymic nude mice. Revascularization of the ischemic hind limb was accelerated in mice that were transplanted with Flk-1+ cells compared with control mice, which were transplanted with vehicle, as evaluated by laser Doppler blood flowmetry. Transplantation of Flk-1+ cells also increased expression of VEGF mRNA in ischemic tissue compared to controls.ConclusionsDirect local implantation of iPS cell-derived Flk-1+ cells would salvage tissues from ischemia. These data indicate that iPS cells could be valuable in the therapeutic induction of angiogenesis.

iPS cell sheets created by a novel magnetite tissue engineering method for reparative angiogenesis

Angiogenic cell therapy represents a novel strategy for ischemic diseases, but some patients show poor responses. We investigated the therapeutic potential of an induced pluripotent stem (iPS) cell sheet created by a novel magnetite tissue engineering technology (Mag-TE) for reparative angiogenesis. Mouse iPS cell-derived Flk-1+ cells were incubated with magnetic nanoparticle-containing liposomes (MCLs). MCL-labeled Flk-1+ cells were mixed with diluted extracellular matrix (ECM) precursor and a magnet was placed on the reverse side. Magnetized Flk-1+ cells formed multi-layered cell sheets according to magnetic force. Implantation of the Flk-1+ cell sheet accelerated revascularization of ischemic hindlimbs relative to the contralateral limbs in nude mice as measured by laser Doppler blood flow and capillary density analyses. The Flk-1+ cell sheet also increased the expressions of VEGF and bFGF in ischemic tissue. iPS cell-derived Flk-1+ cell sheets created by this novel Mag-TE method represent a promising new modality for therapeutic angiogenesis.

Maximum Derivative of Left Ventricular Pressure Predicts Cardiac Mortality After Cardiac Resynchronization Therapy

Cardiac resynchronization therapy (CRT) has been reported to improve cardiac performance. However, CRT in patients with advanced heart failure is not always accompanied by an improvement in survival rates. We investigated the association between hemodynamic studies and long‐term prognosis after CRT.

Mesenchymal Stem Cell-Like Cells Derived from Mouse Induced Pluripotent Stem Cells Ameliorate Diabetic Polyneuropathy in Mice

Background. Although pathological involvements of diabetic polyneuropathy (DPN) have been reported, no dependable treatment of DPN has been achieved. Recent studies have shown that mesenchymal stem cells (MSCs) ameliorate DPN. Here we demonstrate a differentiation of induced pluripotent stem cells (iPSCs) into MSC-like cells and investigate the therapeutic potential of the MSC-like cell transplantation on DPN. Research Design and Methods. For induction into MSC-like cells, GFP-expressing iPSCs were cultured with retinoic acid, followed by adherent culture for 4 months. The MSC-like cells, characterized with flow cytometry and RT-PCR analyses, were transplanted into muscles of streptozotocin-diabetic mice. Three weeks after the transplantation, neurophysiological functions were evaluated. Results. The MSC-like cells expressed MSC markers and angiogenic/neurotrophic factors. The transplanted cells resided in hindlimb muscles and peripheral nerves, and some transplanted cells expressed S100β in the nerves. Impairments of current perception thresholds, nerve conduction velocities, and plantar skin blood flow in the diabetic mice were ameliorated in limbs with the transplanted cells. The capillary number-to-muscle fiber ratios were increased in transplanted hindlimbs of diabetic mice. Conclusions. These results suggest that MSC-like cell transplantation might have therapeutic effects on DPN through secreting angiogenic/neurotrophic factors and differentiation to Schwann cell-like cells.

Transplantation of Neural Crest-Like Cells Derived from Induced Pluripotent Stem Cells Improves Diabetic Polyneuropathy in Mice:

Impaired vascularity and nerve degeneration are the most important pathophysiological abnormalities of diabetic polyneuropathy (DPN). Therefore, regeneration of both the vascular and nervous systems is required for the treatment of DPN. The neural crest (NC) is a transient embryonic structure in vertebrates that differentiates into a vast range of cells, including peripheral neurons, Schwann cells, and vascular smooth muscle cells. In this study, we investigated the ability of transplantation of NC-like (NCL) cells derived from aged mouse induced pluripotent stem (iPS) cells in the treatment of DPN. iPS cells were induced to differentiate into neural cells by stromal cell-derived inducing activity (SDIA) and subsequently supplemented with bone morphogenetic protein 4 to promote differentiation of NC lineage. After the induction, p75 neurotrophin receptor-positive NCL cells were purified using magnetic-activated cell sorting. Sorted NCL cells differentiated to peripheral neurons, glial cells, and smooth muscle cells by additional SDIA. NCL cells were transplanted into hind limb skeletal muscles of 16-week streptozotocin-diabetic mice. Nerve conduction velocity, current perception threshold, intraepidermal nerve fiber density, sensitivity to thermal stimuli, sciatic nerve blood flow, plantar skin blood flow, and capillary number-to-muscle fiber ratio were evaluated. Four weeks after transplantation, the engrafted cells produced growth factors: nerve growth factor, neurotrophin 3, vascular endothelial growth factor, and basic fibroblast growth factor. It was also confirmed that some engrafted cells differentiated into vascular smooth muscle cells or Schwann cell-like cells at each intrinsic site. The transplantation improved the impaired nerve and vascular functions. These results suggest that transplantation of NCL cells derived from iPS cells could have therapeutic effects on DPN through paracrine actions of growth factors and differentiation into Schwann cell-like cells and vascular smooth muscle cells.

Synthesis of iron-dispersed carbons by pressure pyrolysis of divinylbenzene-vinylferrocene copolymer

Versatile carbons with finely dispersed iron were synthesized by pressure pyrolysis of a copolymer prepared from divinylbenzene and vinylferrocene at temperatures below 680‡ C and pressures of 125 MPa. The pyrolysis conditions of the copolymer were found to influence the final morphology of carbons to give fibrils, spheres and polyhedra. The resulting carbons contained uniformly fine particles of cementite (Fe3C) which were less than 30 nm in size, whereas the magnetite was dispersed in the carbon matrix by pressure pyrolysis in the presence of water. Highly dispersed cementite in carbon was found to decompose into metallic iron by further heat treatment above 850‡ C. Porous spherulitic carbons were also synthesized by heat treatment of magnetite containing carbon spherulites.

Comparative Angiogenic Activities of Induced Pluripotent Stem Cells Derived from Young and Old Mice

Advanced age is associated with decreased stem cell activity. However, the effect of aging on the differentiation capacity of induced pluripotent stem (iPS) cells into cardiovascular cells has not been fully clarified. We investigated whether iPS cells derived from young and old mice are equally capable of differentiating into vascular progenitor cells, and whether these cells regulate vascular responses in vivo. iPS cells from mouse embryonic fibroblasts (young) or 21 month-old mouse bone marrow (old) were used. Fetal liver kinase-1 positive (Flk-1+) cells, as a vascular progenitor marker, were induced after 3 to 4 days of culture from iPS cells derived from young and old mice. These Flk-1+ cells were sorted and shown to differentiate into VE-cadherin+ endothelial cells and α-SMA+ smooth muscle cells. Tube-like formation was also successfully induced in both young and old murine Flk-1+ cells. Next, hindlimb ischemia was surgically induced, and purified Flk-1+ cells were directly injected into ischemic hindlimbs of nude mice. Revascularization of the ischemic hindlimb was significantly accelerated in mice transplanted with Flk-1+ cells derived from iPS cells from either young or old mice, as compared to control mice as evaluated by laser Doppler blood flowmetry. The degree of revascularization was similar in the two groups of ischemic mice injected with iPS cell-derived Flk-1+ cells from young or old mice. Transplantation of Flk-1+ cells from both young and old murine iPS cells also increased the expression of VEGF, HGF and IGF mRNA in ischemic tissue as compared to controls. iPS cell-derived Flk-1+ cells differentiated into vascular progenitor cells, and regulated angiogenic vascular responses both in vitro and in vivo. These properties of iPS cells derived from old mice are essentially the same as those of iPS cells from young mice, suggesting the functionality of generated iPS cells themselves to be unaffected by aging.

Therapeutic Reendothelialization by Induced Pluripotent Stem Cells After Vascular Injury—Brief Report

Objective—Endothelial damage is an early requisite step for atherosclerosis after vascular injury. It has been reported that vascular wall cells can develop from induced pluripotent stem (iPS) cell–derived fetal liver kinase-1–positive (Flk-1+) cells. Here, we investigated the efficacies of intravenously administered iPS cell–derived Flk-1+ cells on reendothelialization and neointimal hyperplasia in a mouse model of vascular injury. Approach and Results—Femoral arteries of KSN nude mice were injured using a steel wire. Mouse iPS cell–derived Flk-1+ or Flk-1− cells were intravenously injected into those mice at 24 hours after vascular injury. Delivery of iPS cell–derived Flk-1+ cells significantly attenuated neointimal hyperplasia compared with controls. Evans blue staining of the injured vessel revealed that administration of iPS cell–derived Flk-1+ significantly enhanced reendothelialization compared with the Flk-1− cell control group. Recruitment of PKH26-labeled iPS cell–derived Flk-1+ cells to the site of injury was also detectable. Expression level of CXCR4 in iPS cell–derived Flk-1+ cells was 7.5-fold higher than that of iPS cell–derived Flk-1− cells. Stromal cell-derived factor-1&agr; treatment significantly enhanced adhesion and migration of iPS cell–derived Flk-1+ cells to the endothelia, but these were not observed in Flk-1− cells. Conclusions—Intravenously administered iPS cell–derived Flk-1+ cells are recruited to the site of vascular injury, thereby enhancing reendothelialization followed by suppression of neointimal hyperplasia. Administration of iPS cell–derived Flk-1+ cells is a potentially useful therapeutic means for vascular dysfunction and prevention of restenosis after angioplasty.

Nifedipine ameliorates ischemia-induced revascularization in diet-induced obese mice

BACKGROUND Obesity is a risk factor for the development of cardiovascular diseases that are associated with impaired angiogenesis. Nifedipine, a calcium-channel blocker, has a number of blood pressure (BP)-independent effects as well, such as improving endothelial function and decreasing oxidative stress. Here, we investigated whether nifedipine could improve the angiogenic responses in a diet-induced obese (DIO) model. METHODS DIO was induced by allowing 8-week-old C57BL/6J mice ad libitum access to a high-fat/high-sucrose (HF/HS) diet. Mice were randomly divided into two groups that were fed either the HF/HS or normal chow. At the age of 12 weeks, the animals were treated/not treated with nifedipine admixed with food at a concentration of 0.001%. Then, 1 week later, the mice were subjected to unilateral hind limb surgery. RESULTS Angiogenic repair of the ischemic hind limb was impaired in the DIO mice as compared with that in the control mice as evaluated by laser Doppler blood flowmetry (LDBF) and capillary density analysis. Treatment with nifedipine accelerated angiogenic repair in the DIO mice to a level equal to that seen in the control mice. DIO mice showed increased reactive oxygen species (ROS) production after hind limb ischemia. The number of endothelial progenitor cells (EPCs), which contribute to blood vessel formation, was also significantly lower in these mice. Nifedipine treatment ameliorated the oxidative status and increased the number of EPCs in the DIO mice. CONCLUSIONS Our observations demonstrated that DIO impaired revascularization in response to tissue ischemia. Nifedipine ameliorated obesity-impaired revascularization through suppressing oxidative stress and enhancing the number of EPCs.