Nano-structural analysis of engrafted human induced pluripotent stem cell-derived cardiomyocytes in mouse hearts using a genetic-probe APEX2.

Many studies have shown the feasibility of in vivo cardiac transplantation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in animal experiments. However, nano-structural confirmation of the successful incorporation of the engrafted iPSC-CMs including electron microscopy (EM) has not been accomplished, partly because identification of graft cells in EM has proven to be difficult. Using APEX2, an engineered ascorbate peroxidase imaging tag, we successfully localized and analyzed the fine structure of sarcomeres and the excitation contraction machinery of iPSC-CMs 6 months after their engraftment in infarcted mouse hearts. APEX2 made iPSC-CMs visible in multiple imaging modalities including light microscopy, X-ray microscopic tomography, transmission EM, and scanning EM. EM tomography allowed assessment of the differentiation state of APEX2-positive iPSC-CMs and analysis of the fine structure of the sarcomeres including T-tubules and dyads.

Engineering the AAVS1 locus for consistent and scalable transgene expression in human iPSCs and their differentiated derivatives.

The potential use of induced pluripotent stem cells (iPSCs) in personalized regenerative medicine applications may be augmented by transgenics, including the expression of constitutive cell labels, differentiation reporters, or modulators of disease phenotypes. Thus, there is precedence for reproducible transgene expression amongst iPSC sub-clones with isogenic or diverse genetic backgrounds. Using virus or transposon vectors, transgene integration sites and copy numbers are difficult to control, and nearly impossible to reproduce across multiple cell lines. Moreover, randomly integrated transgenes are often subject to pleiotropic position effects as a consequence of epigenetic changes inherent in differentiation, undermining applications in iPSCs. To address this, we have adapted popular TALEN and CRISPR/Cas9 nuclease technologies in order to introduce transgenes into pre-defined loci and overcome random position effects. AAVS1 is an exemplary locus within the PPP1R12C gene that permits robust expression of CAG promoter-driven transgenes. Gene targeting controls transgene copy number such that reporter expression patterns are reproducible and scalable by ∼2-fold. Furthermore, gene expression is maintained during long-term human iPSC culture and in vitro differentiation along multiple lineages. Here, we outline our AAVS1 targeting protocol using standardized donor vectors and construction methods, as well as provide practical considerations for iPSC culture, drug selection, and genotyping.

Septic pulmonary embolism due to periodontal disease.

BACKGROUND AND OBJECTIVE: Septic pulmonary embolism due to periodontal disease (SPE-PD) is rarely reported and little is known about its clinical features. The purpose of this study was to evaluate the clinical and radiological features, as well as outcome, in SPE-PD. METHODS: Patients' records were retrospectively reviewed and 12 patients with SPE-PD were identified (10 men, mean age 60.5 years). The patients' demographic features, laboratory data, physical and radiological findings, and clinical outcomes were evaluated. RESULTS: All but one patient were smokers. Eight of the 12 patients had comorbidities including hypertension (58%) and/or diabetes mellitus (17%). Prevalent symptoms were fever (67%) and chest pain (58%). Only two patients fulfilled the criteria of systemic inflammatory response syndrome; most of the subjects were not clinically severely ill. Blood cultures were negative in all cases. Contrast-enhanced chest computed tomography (CT) showed multiple peripheral nodules in all 12 patients, wedge-shaped peripheral lesions abutting on the pleura in 10 (83%) and a feeding-vessel sign in 9 (75%). All patients recovered from their illness after antimicrobial therapy concomitant with tooth extraction or periodontal care. The median duration of antibiotic administration was 51 days. CONCLUSIONS: Most patients with SPE-PD were not seriously ill. Contrast-enhanced chest CT appeared to be useful to diagnose SPE-PD.

Am80, a retinoic acid receptor agonist, activates the cardiomyocyte cell cycle and enhances engraftment in the heart.

Human induced pluripotent stem cell-derived (hiPSC) cardiomyocytes are a promising source for regenerative therapy. To realize this therapy, however, their engraftment potential after their injection into the host heart should be improved. Here, we established an efficient method to analyze the cell cycle activity of hiPSC cardiomyocytes using a fluorescence ubiquitination-based cell cycle indicator (FUCCI) system. In vitro high-throughput screening using FUCCI identified a retinoic acid receptor (RAR) agonist, Am80, as an effective cell cycle activator in hiPSC cardiomyocytes. The transplantation of hiPSC cardiomyocytes treated with Am80 before the injection significantly enhanced the engraftment in damaged mouse heart for 6 months. Finally, we revealed that the activation of endogenous Wnt pathways through both RARA and RARB underlies the Am80-mediated cell cycle activation. Collectively, this study highlights an efficient method to activate cell cycle in hiPSC cardiomyocytes by Am80 as a means to increase the graft size after cell transplantation into a damaged heart.

A versatile and robust cell purification system with an RNA-only circuit composed of microRNA-responsive ON and OFF switches.

Human induced pluripotent stem cells (iPSCs) are promising cell resources for cell therapy and drug discovery. However, iPSC-derived differentiated cells are often heterogenous and need purification using a flow cytometer, which has high cost and time consumption for large-scale purification. MicroRNAs (miRNAs) can be used as cell selection markers, because their activity differs between cell types. Here, we show miRNA-responsive ON and OFF switch mRNAs for robust cell purification. The ON switch contains a miRNA-target sequence after the polyadenylate tail, triggering translational activation by sensing the target miRNA. By designing RNA-only circuits with miRNA-ON and -OFF switch mRNAs that encode a lethal ribonuclease, Barnase, and its inhibitor, Barstar, we efficiently purified specific cell types, including human iPSCs and differentiated cardiomyocytes, without flow cytometry. Synthetic mRNA circuits composed of ON and OFF switches provide a safe, versatile, and time-saving method to purify various cell types for biological and clinical applications.

Purification of human iPSC-derived cells at large scale using microRNA switch and magnetic-activated cell sorting.

For regenerative cell therapies using pluripotent stem cell (PSC)-derived cells, large quantities of purified cells are required. Magnetic-activated cell sorting (MACS) is a powerful approach to collect target antigen-positive cells; however, it remains a challenge to purify various cell types efficiently at large scale without using antibodies specific to the desired cell type. Here we develop a technology that combines microRNA (miRNA)-responsive mRNA switch (miR-switch) with MACS (miR-switch-MACS) to purify large amounts of PSC-derived cells rapidly and effectively. We designed miR-switches that detect specific miRNAs expressed in target cells and controlled the translation of a CD4-coding transgene as a selection marker for MACS. For the large-scale purification of induced PSC-derived cardiomyocytes (iPSC-CMs), we transferred miR-208a-CD4 switch-MACS and obtained purified iPSC-CMs efficiently. Moreover, miR-375-CD4 switch-MACS highly purified pancreatic insulin-producing cells and their progenitors expressing Chromogranin A. Overall, the miR-switch-MACS method can efficiently purify target PSC-derived cells for cell replacement therapy.

Transplantation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in a Mouse Myocardial Infarction Model.

Myocardial infarction is caused by a lack of oxygen due to blockage of a coronary artery and is a common cause of heart failure. Despite therapeutic advances, the prognosis of patients with heart failure is poor. One of the reasons is that present therapeutic approaches do not restore the loss of cardiac tissue. Stem cell-based therapies have the potential to regenerate the myocardium, and numerous studies using stem cells have shown improved cardiac function and reduced infarct size. In this chapter, we describe our methodology for transplanting human induced pluripotent stem cell-derived cardiomyocytes into immunodeficient mouse hearts with myocardial infarction.

ERRγ enhances cardiac maturation with T-tubule formation in human iPSC-derived cardiomyocytes.

One of the earliest maturation steps in cardiomyocytes (CMs) is the sarcomere protein isoform switch between TNNI1 and TNNI3 (fetal and neonatal/adult troponin I). Here, we generate human induced pluripotent stem cells (hiPSCs) carrying a TNNI1EmGFP and TNNI3mCherry double reporter to monitor and isolate mature sub-populations during cardiac differentiation. Extensive drug screening identifies two compounds, an estrogen-related receptor gamma (ERRγ) agonist and an S-phase kinase-associated protein 2 inhibitor, that enhances cardiac maturation and a significant change to TNNI3 expression. Expression, morphological, functional, and molecular analyses indicate that hiPSC-CMs treated with the ERRγ agonist show a larger cell size, longer sarcomere length, the presence of transverse tubules, and enhanced metabolic function and contractile and electrical properties. Here, we show that ERRγ-treated hiPSC-CMs have a mature cellular property consistent with neonatal CMs and are useful for disease modeling and regenerative medicine.

Lionheart LincRNA alleviates cardiac systolic dysfunction under pressure overload.

Recent high-throughput approaches have revealed a vast number of transcripts with unknown functions. Many of these transcripts are long noncoding RNAs (lncRNAs), and intergenic region-derived lncRNAs are classified as long intergenic noncoding RNAs (lincRNAs). Although Myosin heavy chain 6 (Myh6) encoding primary contractile protein is down-regulated in stressed hearts, the underlying mechanisms are not fully clarified especially in terms of lincRNAs. Here, we screen upregulated lincRNAs in pressure overloaded hearts and identify a muscle-abundant lincRNA termed Lionheart. Compared with controls, deletion of the Lionheart in mice leads to decreased systolic function and a reduction in MYH6 protein levels following pressure overload. We reveal decreased MYH6 results from an interaction between Lionheart and Purine-rich element-binding protein A after pressure overload. Furthermore, human LIONHEART levels in left ventricular biopsy specimens positively correlate with cardiac systolic function. Our results demonstrate Lionheart plays a pivotal role in cardiac remodeling via regulation of MYH6.

Induction of Human Induced Pluripotent Stem Cells to Cardiomyocytes Using Embryoid Bodies.

Specific cell lineages differentiated from human induced pluripotent stem cells (iPSCs) are promising sources for cell replacement therapy and are useful biomedical research tools for research on disease mechanisms and drug discovery. Among the different lineages, cardiac lineage has been one of the most efficiently differentiated through several established protocols. In this chapter, we describe our reproducible and highly efficient methodology for differentiating iPSCs into cardiomyocytes using embryoid bodies. We also describe methods to dissociate iPSC-derived cardiomyocytes and to evaluate iPSC-derived cardiomyocytes.

Identification of Cardiomyocyte-Fated Progenitors from Human-Induced Pluripotent Stem Cells Marked with CD82.

Here, we find that human-induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM)-fated progenitors (CFPs) that express a tetraspanin family glycoprotein, CD82, almost exclusively differentiate into CMs both in vitro and in vivo. CD82 is transiently expressed in late-stage mesoderm cells during hiPSC differentiation. Purified CD82+ cells gave rise to CMs under nonspecific in vitro culture conditions with serum, as well as in vivo after transplantation to the subrenal space or injured hearts in mice, indicating that CD82 successfully marks CFPs. CD82 overexpression in mesoderm cells as well as in undifferentiated hiPSCs increased the secretion of exosomes containing ß-catenin and reduced nuclear ß-catenin protein, suggesting that CD82 is involved in fated restriction to CMs through Wnt signaling inhibition. This study may contribute to the understanding of CM differentiation mechanisms and to cardiac regeneration strategies.

Associations of residual left ventricular and left atrial remodeling with clinical outcomes in patients after aortic valve replacement for severe aortic stenosis.

BACKGROUND: Aortic valve replacement (AVR) is currently the standard therapy for severe aortic stenosis (AS), and regression of left ventricular (LV) hypertrophy after AVR has been reported. However, data regarding a temporal relation between LV mass and left atrial (LA) volume are limited, and their prognostic impacts have not been fully elucidated. We aimed to clarify the temporal patterns of LA and LV reverse remodeling and their associations with clinical outcomes. METHODS: We retrospectively reviewed 198 consecutive patients who underwent AVR for severe AS. After excluding patients with prior cardiac surgery, atrial fibrillation, concomitant moderate to severe aortic regurgitation, or concurrent mitral valve surgery, 83 patients with echocardiographic LV mass index (LVMI) and LA volume index (LAVI) data before and 1 year after AVR were eligible for the outcome analysis and 29 patients with these 2 measures before surgery, 1 month, 1 year, and 3 years after surgery were eligible for the analysis of time-dependent change of LVMI and LAVI. RESULTS: Significant reductions in LVMI and LAVI (both p<0.001) after surgery were observed over time. LA dilatation improved and reached a plateau 1 month after surgery, whereas LV hypertrophy improved more gradually and reached a plateau at 1 year. The presence of both LV hypertrophy and LA dilatation 1 year after surgery was associated with significantly higher mortality (patients with both conditions vs. patients with neither or one condition=22.6% vs. 7.3% at 3 years; p=0.031) and major adverse cardiac and cerebrovascular events (38.9% vs. 12.6% at 3 years; p=0.021). CONCLUSIONS: LA reverse remodeling occurred rapidly after AVR for severe AS, and regression of LV hypertrophy was more gradual. The presence of both residual LV hypertrophy and LA dilatation 1 year after AVR was associated with poor long-term outcomes.

Enhanced engraftment, proliferation, and therapeutic potential in heart using optimized human iPSC-derived cardiomyocytes.

Human pluripotent stem cell-derived cardiomyocytes (CMs) are a promising tool for cardiac cell therapy. Although transplantation of induced pluripotent stem cell (iPSC)-derived CMs have been reported in several animal models, the treatment effect was limited, probably due to poor optimization of the injected cells. To optimize graft cells for cardiac reconstruction, we compared the engraftment efficiency of intramyocardially-injected undifferentiated-iPSCs, day 4 mesodermal cells, and day 8, day 20, and day 30 purified iPSC-CMs after initial differentiation by tracing the engraftment ratio (ER) using in vivo bioluminescence imaging. This analysis revealed the ER of day 20 CMs was significantly higher compared to other cells. Transplantation of day 20 CMs into the infarcted hearts of immunodeficient mice showed good engraftment, and echocardiography showed significant functional improvement by cell therapy. Moreover, the imaging signal and ratio of Ki67-positive CMs at 3 months post injection indicated engrafted CMs proliferated in the host heart. Although this graft growth reached a plateau at 3 months, histological analysis confirmed progressive maturation from 3 to 6 months. These results suggested that day 20 CMs had very high engraftment, proliferation, and therapeutic potential in host mouse hearts. They also demonstrate this model can be used to track the fate of transplanted cells over a long time.