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.

Blood vessels are essential components for many tissues and organs. Thus, efficient induction of endothelial cells (ECs) from human pluripotent stem cells is a key method for generating higher tissue structures entirely from stem cells. We previously established an EC differentiation system with mouse pluripotent stem cells to show that vascular endothelial growth factor (VEGF) is essential to induce ECs and that cyclic adenosine monophosphate (cAMP) synergistically enhances VEGF effects. Here we report an efficient and robust EC differentiation method from human pluripotent stem cell lines based on a 2D monolayer, serum-free culture. We controlled the direction of differentiation from mesoderm to ECs using stage-specific stimulation with VEGF and cAMP combined with the elimination of non-responder cells at early EC stage. This “stimulation-elimination” method robustly achieved very high efficiency (>99%) and yield (>10 ECs from 1 hiPSC input) of EC differentiation, with no purification of ECs after differentiation. We believe this method will be a valuable technological basis broadly for regenerative medicine and 3D tissue engineering.

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.

To realize human induced pluripotent stem cell (hiPSC)-based cardiac regenerative therapy, evidence of therapeutic advantages in human-sized diseased hearts are indispensable. In combination with an efficient and simultaneous differentiation of various cardiac lineages from hiPSCs and cell sheet technology, we aimed to generate clinical-sized large cardiac tissue sheets (L-CTSs) and to evaluate the therapeutic potential in porcine infarct heart. We simultaneously induced cardiomyocytes (CMs) and vascular cells [vascular endothelial cells (ECs) and vascular mural cells (MCs)] from hiPSCs. We generated L-CTSs using 10cm-sized temperature-responsive culture dishes. We induced myocardial infarction (MI) in micromini-pigs (15–25 kg) and transplanted the L-CTSs (Tx) 2 weeks after MI induction (4 sheets/recipient) under immunosuppression (Tx: n = 5, Sham: n = 5). Self-pulsating L-CTSs were approximately 3.5cm in diameter with 6.8×106±0.8 of cells containing cTnT+-CMs (45.6±13.2%), VE-cadherin+-ECs (5.3±4.4%) and PDGFRβ+-MCs (14.4±20.7%), respectively (n = 5). In Tx group, echocardiogram indicated a significantly higher systolic function of the left ventricle (LV) compared to that in sham control (Sham vs Tx: fractional shortening: 24.2±8.6 vs 40.5±9.7%; p<0.05). Ejection fraction evaluated by left ventriculogram was significantly higher in Tx group (25.3±6.2% vs 39.8±4.2%; p<0.01). Speckle tracking echocardiogram showed a significant increase of circumference strain in infarct and border regions after transplantation. Fibrotic area was significantly lower in Tx group (23.8±4.5 vs 15.9±3.8%; P<0.001). Capillary density in the border region was significantly higher in Tx group (75.9±42.6/mm2 vs 137.4±44.8/mm2, p<0.001). These data indicate that the L-CTS transplantation attenuated LV remodeling. L-CTSs potentially restore cardiac dysfunction of human-sized infarct heart.

SummaryThe blood-brain barrier (BBB) is composed of four cell populations, brain endothelial cells (BECs), pericytes, neurons, and astrocytes. Its role is to precisely regulate the microenvironment of the brain through selective substance crossing. Here we generated an in vitro model of the BBB by differentiating human induced pluripotent stem cells (hiPSCs) into all four populations. When the four hiPSC-derived populations were co-cultured, endothelial cells (ECs) were endowed with features consistent with BECs, including a high expression of nutrient transporters (CAT3, MFSD2A) and efflux transporters (ABCA1, BCRP, PGP, MRP5), and strong barrier function based on tight junctions. Neuron-derived Dll1, which activates Notch signaling in ECs, was essential for the BEC specification. We performed in vitro BBB permeability tests and assessed ten clinical drugs by nanoLC-MS/MS, finding a good correlation with the BBB permeability reported in previous cases. This technology should be useful for research on human BBB physiology, pathology, and drug development.