Akinori Hirano

A Massive Suspension Culture System With Metabolic Purification for Human Pluripotent Stem Cell-Derived Cardiomyocytes

Cardiac regenerative therapy with human pluripotent stem cells (hPSCs), such as human embryonic stem cells and induced pluripotent stem cells, has been hampered by the lack of efficient strategies for expanding functional cardiomyocytes (CMs) to clinically relevant numbers. The development of the massive suspension culture system (MSCS) has shed light on this critical issue, although it remains unclear how hPSCs could differentiate into functional CMs using a MSCS. The proliferative rate of differentiating hPSCs in the MSCS was equivalent to that in suspension cultures using nonadherent culture dishes, although the MSCS provided more homogeneous embryoid bodies (EBs), eventually reducing apoptosis. However, pluripotent markers such as Oct3/4 and Tra‐1‐60 were still expressed in EBs 2 weeks after differentiation, even in the MSCS. The remaining undifferentiated stem cells in such cultures could retain a strong potential for teratoma formation, which is the worst scenario for clinical applications of hPSC‐derived CMs. The metabolic purification of CMs in glucose‐depleted and lactate‐enriched medium successfully eliminated the residual undifferentiated stem cells, resulting in a refined hPSC‐derived CM population. In colony formation assays, no Tra‐1‐60‐positive colonies appeared after purification. The nonpurified CMs in the MSCS produced teratomas at a rate of 60%. However, purified CMs never induced teratomas, and enriched CMs showed proper electrophysiological properties and calcium transients. Overall, the combination of a MSCS and metabolic selection is a highly effective and practical approach to purify and enrich massive numbers of functional CMs and provides an essential technique for cardiac regenerative therapy with hPSC‐derived CMs.

Glutamine Oxidation Is Indispensable for Survival of Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) are uniquely dependent on aerobic glycolysis to generate ATP. However, the importance of oxidative phosphorylation (OXPHOS) has not been elucidated. Detailed amino acid profiling has revealed that glutamine is indispensable for the survival of hPSCs. Under glucose- and glutamine-depleted conditions, hPSCs quickly died due to the loss of ATP. Metabolome analyses showed that hPSCs oxidized pyruvate poorly and that glutamine was the main energy source for OXPHOS. hPSCs were unable to utilize pyruvate-derived citrate due to negligible expression of aconitase 2 (ACO2) and isocitrate dehydrogenase 2/3 (IDH2/3) and high expression of ATP-citrate lyase. Cardiomyocytes with mature mitochondria were not able to survive without glucose and glutamine, although they were able to use lactate to synthesize pyruvate and glutamate. This distinguishing feature of hPSC metabolism allows preparation of clinical-grade cell sources free of undifferentiated hPSCs, which prevents tumor formation during stem cell therapy.

Heterotopic transplantation of a decellularized and recellularized whole porcine heart

OBJECTIVES One of the final treatments for end-stage heart failure is heart transplantation. However, a shortage of donor hearts has created a long waiting list and limited benefits. Our ultimate goal is to create a whole beating heart fabricated on an organ scaffold for human heart transplantation. Here, we successfully performed the first transplantation using a decellularized whole porcine heart with mesenchymal stem cells. METHODS A porcine heart was harvested following cardiac arrest induced by a high-potassium solution and stored at -80°C for 24 h. The porcine heart was completely decellularized with 1% sodium dodecyl sulphate and 1% Triton X-100 under the control of perfusion pressure (100 mmHg) and maintained at 37°C. A decellularized whole-heart scaffold was sterilized with gamma irradiation. Cultured mesenchymal stem cells were collected and either infused into the ascending aorta or injected directly into the left ventricular wall. Finally, recellularized whole-heart scaffolds were transplanted into pigs under systemic anticoagulation treatment with heparin. Coronary artery angiography of the transplanted heart graft was performed. RESULTS In our decellularization method, all cellular components were removed, preserving the heart extracellular matrix. Heterotopic transplantations were successfully performed using a decellularized heart and a recellularized heart. The scaffolds were well perfused, without bleeding from the surface or anastomosis site. Coronary angiography revealed a patent coronary artery in both scaffolds. The transplanted decellularized heart was harvested on Day 3. Haematoxylin and eosin staining showed thrombosis in the coronary arteries and migrated inflammatory cells. Haematoxylin and eosin staining of the transplanted recellularized heart showed similar findings, with the exception of injected mesenchymal stem cells. CONCLUSIONS To the best of our knowledge, this is the first report of heterotopic transplantation of a decellularized whole porcine heart with mesenchymal stem cells. The scaffolds endured surgical procedures. We detected short-term coronary artery perfusion in the transplanted scaffolds by angiography. Future studies should analyse the histological features of transplanted decellularized scaffolds and optimize the system for recellularization to apply this unique technology clinically.

Gelatin Hydrogel Enhances the Engraftment of Transplanted Cardiomyocytes and Angiogenesis to Ameliorate Cardiac Function after Myocardial Infarction

Cell transplantation therapy will mean a breakthrough in resolving the donor shortage in cardiac transplantation. Cardiomyocyte (CM) transplantation, however, has been relatively inefficient in restoring cardiac function after myocardial infarction (MI) due to low engraftment of transplanted CM. In order to ameliorate engraftment of CM, the novel transplantation strategy must be invented. Gelatin hydrogel (GH) is a biodegradable water-soluble polymer gel. Gelatin is made of collagen. Although we observed that collagen strongly induced the aggregation of platelets to potentially cause coronary microembolization, GH did not enhance thrombogenicity. Therefore, GH is a suitable biomaterial in the cell therapy after heart failure. To assess the effect of GH on the improvement of cardiac function, fetal rat CM (5×106 or 1x106 cells) were transplanted with GH (10 mg/ml) to infarcted hearts. We compared this group with sham operated rats, CM in phosphate buffered saline (PBS), only PBS, and only GH-transplanted groups. Three weeks after transplantation, cardiac function was evaluated by echocardiography. The echocardiography confirmed that transplantation of 5×106 CM with GH significantly improved cardiac systolic function, compared with the CM+PBS group (fractional area change: 75.1±3.4% vs. 60.7±5.9%, p<0.05), only PBS, and only GH groups (60.1±6.5%, 65.0±2.8%, p<0.05). Pathological analyses demonstrated that in the CM+GH group, CM were efficiently engrafted in infarcted myocardium (p<0.01) and angiogenesis was significantly enhanced (p<0.05) in both central and peripheral areas of the scar. Moreover, quantitative RT-PCR revealed that angiogenic cytokines, such as basic fibroblast growth factor, vascular endothelial growth factor, and hepatocyte growth factor, were significantly enriched in the CM+GH group (p<0.05). Here, we report that GH confined the CM effectively in infarcted myocardium after transplantation, and that CM transplanted with GH improved cardiac function with a direct contraction effect and enhanced angiogenesis.

Derivation of transgene-free human induced pluripotent stem cells from human peripheral T cells in defined culture conditions.

Recently, induced pluripotent stem cells (iPSCs) were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer, both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore, the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front, although donor cell types for generating iPSCs are wide-ranging, T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs) have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells, which can be applied to novel immunotherapies. In the present study, we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining, had a normal karyotype, and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined, animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application.

Efficient Large-Scale 2D Culture System for Human Induced Pluripotent Stem Cells and Differentiated Cardiomyocytes

Summary Cardiac regenerative therapies utilizing human induced pluripotent stem cells (hiPSCs) are hampered by ineffective large-scale culture. hiPSCs were cultured in multilayer culture plates (CPs) with active gas ventilation (AGV), resulting in stable proliferation and pluripotency. Seeding of 1 × 106 hiPSCs per layer yielded 7.2 × 108 hiPSCs in 4-layer CPs and 1.7 × 109 hiPSCs in 10-layer CPs with pluripotency. hiPSCs were sequentially differentiated into cardiomyocytes (CMs) in a two-dimensional (2D) differentiation protocol. The efficiency of cardiac differentiation using 10-layer CPs with AGV was 66%–87%. Approximately 6.2–7.0 × 108 cells (4-layer) and 1.5–2.8 × 109 cells (10-layer) were obtained with AGV. After metabolic purification with glucose- and glutamine-depleted and lactate-supplemented media, a massive amount of purified CMs was prepared. Here, we present a scalable 2D culture system using multilayer CPs with AGV for hiPSC-derived CMs, which will facilitate clinical applications for severe heart failure in the near future.

Comparison of aortic arch repair using the endovascular technique, total arch replacement and staged surgery

OBJECTIVES We evaluated the operative and long-term outcomes of various approaches for aortic arch repair. METHODS A total of 436 consecutive patients who underwent aortic arch repair from January 2001 to March 2016 in our centre were evaluated. Of these, 276 underwent conventional total arch replacement (TAR), and 118 underwent thoracic endovascular repair (TEVAR). The remaining 42 patients underwent staged thoracic endovascular repair (STEVAR). A total of 72 patients in the TEVAR group were matched to 72 patients who underwent open surgery including TAR or STEVAR by using propensity score analysis. RESULTS Surgical outcomes showed shorter ICU and hospital stay in the TEVAR group ( P  < 0.001 and P  < 0.001, respectively). The 30-day mortality and neurologic dysfunction showed no significant difference among the three groups (2.8 and 5.4% in TAR group, 1.7 and 8.5% in TEVAR group and 0 and 2.4% in STEVAR group; P  = 0.500 and P  = 0.297, respectively). Long-term survival was not significantly different among the three groups (78% in TAR group, 67% in TEVAR group and 81% in STEVAR group at 5 years; P  = 0.123). Freedom from aortic reintervention was lower in the TEVAR group than in other groups (98% in TAR, 92% in TEVAR and 97% in STEVAR at 5 years, P  = 0.040). CONCLUSIONS Operative outcomes showed no significant differences between the groups except for early recovery after TEVAR. Long-term survival was similar between groups; however, TEVAR had inferior reintervention free rate.

Hybrid Thoracic Endovascular Aortic Repair for Intercostal Patch Aneurysm after Thoracoabdominal Aortic Replacement

We report a case of hybrid thoracic endovascular aortic repair for intercostal patch aneurysm after thoracoabdominal aortic replacement. Eighteen years ago, a 63-year-old woman with Marfan syndrome had undergone thoracoabdominal aortic replacement with reimplantation of the intercostal artery in an island fashion. Follow-up computed tomography (CT) revealed a remaining intercostal patch aneurysm of diameter 60 mm 17 years after the last operation. Hybrid thoracic endovascular aortic repair for exclusion of this intercostal patch aneurysm was successfully performed, with visceral artery bypasses. Postoperative CT showed no anastomotic stenosis or endoleak.

Stonehenge technique is associated with faster aortic clamp time in group of minimally invasive aortic valve replacement via right infra-axillary thoracotomy

BackgroundTrans-right axillary aortic valve replacement (TAX-AVR) remains uncommon. We developed a special method to pull the heart closer to the right chest wall to make the surgery as easy and safe as aortic valve replacement via median sternotomy. Because the retraction sutures lifting the ascending aorta and aortic root are arranged circularly around the wound, we named this technique “Stonehenge technique”.MethodsWe examined 47 patients who underwent aortic valve replacement through a small right infra-axillary thoracotomy as the initial surgical therapy. These patients were divided into two groups: the conventional TAX-AVR group that underwent AVR via the conventional small right axillary incision approach (n = 20) and the TAX-AVR with SH group that underwent AVR with the Stonehenge technique (n = 27).ResultsThe aortic cross-clamp and the extracorporeal circulation time were significantly shorter in the TAX-AVR with SH group than in the conventional TAX-AVR group (conventional TAX-AVR group: 125.5 ± 47.9; TAX-AVR with SH group: 96.0 ± 14.0, p = 0.004, and conventional TAX-AVR group: 163.8 ± 55.9; TAX-AVR with SH group: 140.0 ± 16.8, p = 0.04).ConclusionThe outcomes of this technique depend on the site of the retraction sutures in the opened pericardium, direction of pull, amount of force applied, and precautions taken. If performed correctly, the ascending aorta and the root can be pulled from the wound to within the surgeon’s fingers’ reach, thereby reducing aortic cross-clamp and extracorporeal circulation times in group of minimally invasive aortic valve replacement via right infra-axillary thoracotomy.

Left atrioventricular valve replacement in a patient with hereditary haemorrhagic telangiectasia

Hereditary haemorrhagic telangiectasia, or Osler-Rendu-Weber disease, is characterized by multiple arteriovenous malformations and telangiectasia which develop in the brain, lungs or liver. We report the case of a patient who had severe left atrioventricular valve regurgitation with atrial fibrillation after atrioventricular septal defect repair in her childhood and was diagnosed as having hereditary haemorrhagic telangiectasia by chance. The patient underwent successful left atrioventricular valve replacement and maze operation. The postoperative course was uneventful without nose bleeding worsening.