Pre-clinical evaluation of the efficacy and safety of human induced pluripotent stem cell-derived cardiomyocyte patch.

BACKGROUND: Cell- or tissue-based regenerative therapy is an attractive approach to treat heart failure. A tissue patch that can safely and effectively repair damaged heart muscle would greatly improve outcomes for patients with heart failure. In this study, we conducted a preclinical proof-of-concept analysis of the efficacy and safety of clinical-grade human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) patches. METHODS: A clinical-grade hiPSC line was established using peripheral blood mononuclear cells from a healthy volunteer that was homozygous for human leukocyte antigens. The hiPSCs were differentiated into cardiomyocytes. The obtained hiPSC-CMs were cultured on temperature-responsive culture dishes for patch fabrication. The cellular characteristics, safety, and efficacy of hiPSCs, hiPSC-CMs, and hiPSC-CM patches were analyzed. RESULTS: The hiPSC-CMs expressed cardiomyocyte-specific genes and proteins, and electrophysiological analyses revealed that hiPSC-CMs exhibit similar properties to human primary myocardial cells. In vitro and in vivo safety studies indicated that tumorigenic cells were absent. Moreover, whole-genome and exome sequencing revealed no genomic mutations. General toxicity tests also showed no adverse events posttransplantation. A porcine model of myocardial infarction demonstrated significantly improved cardiac function and angiogenesis in response to cytokine secretion from hiPSC-CM patches. No lethal arrhythmias were observed. CONCLUSIONS: hiPSC-CM patches are promising for future translational research and may have clinical application potential for the treatment of heart failure.

Percutaneous drainage and staged valve replacement followed by laparoscopic splenectomy in infective endocarditis with splenic abscess.

Splenic abscess is a severe complication of infective endocarditis. The need for splenectomy to control prosthetic valve infection remains controversial. Here, we present the case of a 49-year-old man who complained of fever and general fatigue. Blood cultures grew Group G Streptococcus, and intravenous antibiotics were started. Abdominal computed tomography showed splenic abscess; thus, percutaneous drainage was performed. Two-dimensional transthoracic echocardiogram revealed a mobile vegetation on the right coronary cusp of the aortic valve with mild aortic regurgitation. The patient underwent aortic valve replacement using a 23-mm SJM Regent mechanic valve, followed by laparoscopic splenectomy 3 days later. The patient was asymptomatic without recurrence of infection 13 months postoperatively. Current guidelines recommend that splenectomy should be performed first, followed by valve replacement. However, we performed valve surgery first because of the risk of embolism. Depending on the patient's condition, performing splenic drainage and valve replacement first may be considered.

Human induced pluripotent stem cell-derived three-dimensional cardiomyocyte tissues ameliorate the rat ischemic myocardium by remodeling the extracellular matrix and cardiac protein phenotype.

The extracellular matrix (ECM) plays a key role in the viability and survival of implanted human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We hypothesized that coating of three-dimensional (3D) cardiac tissue-derived hiPSC-CMs with the ECM protein fibronectin (FN) would improve the survival of transplanted cells in the heart and improve heart function in a rat model of ischemic heart failure. To test this hypothesis, we first explored the tolerance of FN-coated hiPSC-CMs to hypoxia in an in vitro study. For in vivo assessments, we constructed 3D-hiPSC cardiac tissues (3D-hiPSC-CTs) using a layer-by-layer technique, and then the cells were implanted in the hearts of a myocardial infarction rat model (3D-hiPSC-CTs, n = 10; sham surgery control group (without implant), n = 10). Heart function and histology were analyzed 4 weeks after transplantation. In the in vitro assessment, cell viability and lactate dehydrogenase assays showed that FN-coated hiPSC-CMs had improved tolerance to hypoxia compared with the control cells. In vivo, the left ventricular ejection fraction of hearts implanted with 3D-hiPSC-CT was significantly better than that of the sham control hearts. Histological analysis showed clear expression of collagen type IV and plasma membrane markers such as desmin and dystrophin in vivo after implantation of 3D-hiPSC-CT, which were not detected in 3D-hiPSC-CMs in vitro. Overall, these results indicated that FN-coated 3D-hiPSC-CT could improve distressed heart function in a rat myocardial infarction model with a well-expressed cytoskeletal or basement membrane matrix. Therefore, FN-coated 3D-hiPSC-CT may serve as a promising replacement for heart transplantation and left ventricular assist devices and has the potential to improve survivability and therapeutic efficacy in cases of ischemic heart disease.

Laminin-221 Enhances Therapeutic Effects of Human-Induced Pluripotent Stem Cell-Derived 3-Dimensional Engineered Cardiac Tissue Transplantation in a Rat Ischemic Cardiomyopathy Model.

Background Extracellular matrix, especially laminin-221, may play crucial roles in viability and survival of human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) after in vivo transplant. Then, we hypothesized laminin-221 may have an adjuvant effect on therapeutic efficacy by enhancing cell viability and survival after transplantation of 3-dimensional engineered cardiac tissue (ECT) to a rat model of myocardial infarction. Methods and Results In vitro study indicates the impacts of laminin-221 on hiPS-CMs were analyzed on the basis of mechanical function, mitochondrial function, and tolerance to hypoxia. We constructed 3-dimensional ECT containing hiPS-CMs and fibrin gel conjugated with laminin-221. Heart function and in vivo behavior were assessed after engraftment of 3-dimensional ECT (laminin-conjugated ECT, n=10; ECT, n=10; control, n=10) in a rat model of myocardial infarction. In vitro assessment indicated that laminin-221 improves systolic velocity, diastolic velocity, and maximum capacity of oxidative metabolism of hiPS-CMs. Cell viability and lactate dehydrogenase production revealed that laminin-221 improved tolerance to hypoxia. Furthermore, analysis of mRNA expression revealed that antiapoptotic genes were upregulated in the laminin group under hypoxic conditions. Left ventricular ejection fraction of the laminin-conjugated ECT group was significantly better than that of other groups 4 weeks after transplantation. Laminin-conjugated ECT transplantation was associated with significant improvements in expression levels of rat vascular endothelial growth factor. In early assessments, cell survival was also improved in laminin-conjugated ECTs compared with ECT transplantation without laminin-221. Conclusions In vitro laminin-221 enhanced mechanical and metabolic function of hiPS-CMs and improved the therapeutic impact of 3-dimensional ECT in a rat ischemic cardiomyopathy model. These findings suggest that adjuvant laminin-221 may provide a clinical benefit to hiPS-CM constructs.

Surgical Results for Infective Endocarditis Complicated With Cardiogenic Shock.

BACKGROUND: Infective endocarditis remains associated with substantial mortality and morbidity rates, and the presence of acute heart failure (AHF) compromises clinical results after valve surgery; however, little is known in cardiogenic shock (CGS) patients. This study evaluated the clinical results and risk of mortality in CGS patients after valve surgery.Methods and Results:This study enrolled 585 patients who underwent valve surgery for active endocarditis at 14 institutions between 2009 and 2017. Of these patients, 69 (12%) were in CGS, which was defined as systolic blood pressure <80 mmHg and severe pulmonary congestion, requiring mechanical ventilation and/or mechanical circulatory support, preoperatively. The predictors of CGS were analyzed, and clinical results of patients with non-CGS AHF (n=215) were evaluated and compared.Staphylococcus aureusinfection (odds ratio [OR] 2.19; P=0.044), double valve involvement (OR 3.37; P=0.003), and larger vegetation (OR 1.05; P=0.036) were risk factors for CGS. Hospital mortality occurred in 27 (13%) non-CGS AHF patients and in 15 (22%) CGS patients (P=0.079). Overall survival at 1 and 5 years in CGS patients was 76% and 69%, respectively, and there were no significant differences in overall survival compared with non-CGS AHF patients (P=1.000). CONCLUSIONS: Clinical results after valve surgery in CGS patients remain challenging; however, mid-term results were equivalent to those of non-CGS AHF patients.

Emergency valve surgery improves clinical results in patients with infective endocarditis complicated with acute cerebral infarction: analysis using propensity score matching†.

OBJECTIVES: To date, the optimal timing for patients with infective endocarditis (IE) with acute cerebral infarction (CI) to undergo valve surgery is unknown. Although some previous studies have reported that early valve surgery for IE patients within 1 or 2 weeks after CI could be performed safely, an initial strategy has not been identified because of the unmatched cohorts in previous studies. This study aimed to assess the feasibility and safety of early surgery within a few days after cerebral infarction by using propensity score matching. METHODS: Between 2009 and 2017, 585 patients underwent valve surgery for patients with active IE at 14 institutions. Among these, 152 had preoperative acute CI. Early surgery was defined as surgery within 3 days after the diagnosis of CI. Of these 152 patients, 67 underwent early valve surgery (early group), whereas 85 underwent delayed valve surgery (delayed group). Of the patients, 45 in each group were analysed using propensity score matching. The primary outcome was in-hospital death after valve surgery, and secondary outcomes included neurological complications. We compared the clinical results of these matched patients. RESULTS: Hospital mortality was lower in the early group (2% vs 16%, P = 0.058). The rate of postoperative intracranial haemorrhage in the early and delayed groups was 4% in both groups. The postoperative modified Rankin scale was not significantly different [early group: 0 (0-2); delayed group: 0 (0-2)]. Incidence of neurological deterioration did not differ significantly between the groups. The survival rates after the first discharge at 1, 3 and 5 years after valve operation were 100%, 97% and 97% in the early group and 91%, 83% and 80% in the delayed group, respectively (P = 0.029). CONCLUSIONS: Early valve surgery for patients with IE within 3 days after a CI measuring <2 cm in size improved clinical results without increasing the incidence of postoperative neurological complications.

Surgery-first treatment improves clinical results in infective endocarditis complicated with disseminated intravascular coagulation†.

OBJECTIVES: Infective endocarditis (IE) is a critical infection with a high mortality rate, and it usually causes sepsis. Though disseminated intravascular coagulation (DIC) sometimes occurs in IE patients, no definitive treatment strategy for IE patients with DIC as a complication exists. Therefore, we evaluated the prevalence, surgical results and treatment strategy for IE complicated with DIC. METHODS: Between 2009 and 2017, a total of 585 patients undergoing valve surgery for active IE were enrolled at 14 institutions, of whom 116 (20%) had DIC as a complication. For further evaluation, we divided DIC patients into medical treatment-first (n = 45, group M) and valve surgery-first (n = 51, group S) groups after excluding 20 patients with intracranial haemorrhage. RESULTS: The overall survival rates at 1 and 5 years were 91% and 85% in the non-DIC group and 65% and 55% in the DIC group, respectively (P < 0.001). Recurrence-free survival rates at 1 and 5 years were 99% and 95% in the non-DIC group and 94% and 74% in the DIC group, respectively (P < 0.001). The overall survival rates at 1 and 5 years were 77% and 64% in group S and 51% and 46% in group M, respectively (P = 0.032). Multivariable analysis revealed that 'medical treatment first' was an exclusive independent risk factor [hazards ratio 2.26 (1.13-4.75), P = 0.024] for overall mortality. CONCLUSIONS: Mortality and IE recurrence were statistically significantly higher in DIC patients. Valve surgery should not be delayed because most patients proceeding with medical treatment eventually require emergency surgery and their clinical outcomes are worse than those of patients undergoing early surgery.

Verification of pharmacogenomics-based algorithms to predict warfarin maintenance dose using registered data of Japanese patients.

PURPOSE: Large inter-individual differences in warfarin maintenance dose are mostly due to the effect of genetic polymorphisms in multiple genes, including vitamin K epoxide reductase complex 1 (VKORC1), cytochromes P450 2C9 (CYP2C9), and cytochrome P450 4F2 (CYP4F2). Thus, several algorithms for predicting the warfarin dose based on pharmacogenomics data with clinical characteristics have been proposed. Although these algorithms consider these genetic polymorphisms, the formulas have different coefficient values that are critical in this context. In this study, we assessed the mutual validity among these algorithms by specifically considering racial differences. METHODS: Clinical data including actual warfarin dose (AWD) of 125 Japanese patients from our previous study (Eur J Clin Pharmacol 65(11):1097-1103, 2009) were used as registered data that provided patient characteristics, including age, sex, height, weight, and concomitant medications, as well as the genotypes of CYP2C9 and VKORC1. Genotyping for CYP4F2*3 was performed by the PCR method. Five algorithms that included these factors were selected from peer-reviewed articles. The selection covered four populations, Japanese, Chinese, Caucasian, and African-American, and the International Warfarin Pharmacogenetics Consortium (IWPC). RESULTS: For each algorithm, we calculated individual warfarin doses for 125 subjects and statistically evaluated its performance. The algorithm from the IWPC had the statistically highest correlation with the AWD. Importantly, the calculated warfarin dose (CWD) using the algorithm from African-Americans was less correlated with the AWD as compared to those using the other algorithms. The integration of CYP4F2 data into the algorithm did not improve the prediction accuracy. CONCLUSION: The racial difference is a critical factor for warfarin dose predictions based on pharmacogenomics.

Laminin-511 Supplementation Enhances Stem Cell Localization With Suppression in the Decline of Cardiac Function in Acute Infarct Rats.

BACKGROUND: The extracellular matrix, in particular basement membrane components such as laminins (LMs), is essential for stem cell differentiation and self-renewal. LM511 and LM221 are the main extracellular matrix components of the epicardium, where stem cells were abundant. Here, we examined whether LMs affected the regeneration process by modulating stem cell activities. METHODS: In vitro, adhesive, and proliferative activities of mesenchymal stem cells (MSCs) were evaluated on LM511 and LM221. To examine the effects of LMs in vivo, we established an acute myocardial infarction model by ligation of the proximal part of the left anterior descending artery at the height of the left atrial appendage and then placed atelocollagen sheets with or without LM511 and LM221 over the anterolateral surface of the left ventricular wall. Four or 8 weeks later, cardiac function, histology, and cytokine expressions were analyzed. RESULTS: MSCs showed greater proliferation and adhesive properties on LM511 than on LM221. In vivo, at 4 weeks, isolectin B4-positive cells were significantly higher in the LM511-transplanted group than in the control group. Moreover, some isolectin B4-positive cells expressed both platelet-derived growth factor receptor α and CD90, suggesting that LM511 enhanced MSC recruitment and attachment at the implanted site. After 8 weeks, these cells were more abundant than at 4 weeks. Transplantation with LM511-conjugated sheets increased the expression of cardioprotective and angiogenic factors. CONCLUSIONS: Transplantation with LM511-conjugated sheets enhanced MSC localization to the implantation site and modulated stem cells activities, leading to angiogenesis in acute myocardial infarction rat models.

Results of surgical management of infective endocarditis associated with Staphylococcus aureus.

OBJECTIVES: Staphylococcus aureus (SA) is a leading cause of infective endocarditis (IE), and such cases are on the rise. Our objective was to evaluate the clinical outcomes of surgical intervention in patients with SA-associated IE and to identify the factors associated with outcomes. METHODS: Between 2009 and 2017, 585 patients underwent valve surgery for definitive left-sided IE at 14 affiliated hospitals. Their medical records were retrospectively reviewed, and the preoperative variables and clinical results of patients with (n = 117) or without SA infection (n = 468) were compared. RESULTS: The SA group had a more critical preoperative condition with higher rates of chronic haemodialysis, preoperative embolic events and preoperative inflammation levels, as well as worse renal function. In-hospital mortality was 20% and 7% in the patients with or without SA infection, respectively. The overall survival rate at 1 year and 5 years was 72% and 62% in the SA group, and 88% and 81% in the non-SA group, respectively (P < 0.001). The Cox hazard analysis revealed that methicillin-resistant SA infection was an independent risk factor for overall mortality in the SA group. The rate of freedom from recurrence of endocarditis at 1 year and 5 years was 95% and 90% in the SA group and 96% and 92% in the non-SA group, respectively (P = 0.43). CONCLUSIONS: The short- and mid-term outcomes after valve surgery for active IE in patients with SA are still challenging. Methicillin-resistant SA infection is an independent predictor of mid-term mortality.

Effect of the Initial Strategy for Active Endocarditis Complicated With Acute Heart Failure.

BACKGROUND: Early surgery for infective endocarditis (IE) with acute heart failure (AHF) is recommended, despite clinical results being unclear. We investigated the effect of initial treatment in such patients. Methods and Results: Outcomes for 470 patients with active IE who underwent valvular surgery during 2009-2016 were reviewed. Of them, 177 had symptomatic AHF when diagnosed with IE (excluding those with cardiogenic shock or intubated for AHF). They were divided into 2 groups based on initial treatment: Group S (underwent valvular surgery immediately; n=74) and Group M (received initial medical treatment for infection and HF; n=103). The median (interquartile range) waiting period from diagnosis to surgery in Groups S and M was 1 (1-3) and 15 (8-33) days, respectively (P<0.001). The 5-year survival rate was higher in Group S than Group M (80% vs. 64%; P=0.108). Group M was divided into Group P (initial medical treatment was effective and elective surgery was performed; n=62) and Group E (emergency surgery was necessary during medical treatment; n=41); overall 5-year survival was significantly worse in Group E than Group P (42% vs. 79%; P<0.012). In Group M, multivariate analysis indicated that Staphylococcus aureus infection (odds ratio 3.82; 95% confidence interval 1.19-13.3; P=0.024) was a significant risk factor for conversion to emergency surgery. CONCLUSIONS: Considering poor outcomes of emergency surgery for medically refractory HF, early surgery may be a reasonable option for IE patients, especially those with S. aureus infection.

Immunologic targeting of CD30 eliminates tumourigenic human pluripotent stem cells, allowing safer clinical application of hiPSC-based cell therapy.

Induced pluripotent stem cells (iPSCs) are promising candidate cells for cardiomyogenesis in the failing heart. However, teratoma/tumour formation originating from undifferentiated iPSCs contaminating the graft is a critical concern for clinical application. Here, we hypothesized that brentuximab vedotin, which targets CD30, induces apoptosis in tumourigenic cells, thus increasing the safety of iPSC therapy for heart failure. Flow cytometry analysis identified consistent expression of CD30 in undifferentiated human iPSCs. Addition of brentuximab vedotin in vitro for 72 h efficiently induced cell death in human iPSCs, associated with a significant increase in G2/M phase cells. Brentuximab vedotin significantly reduced Lin28 expression in cardiomyogenically differentiated human iPSCs. Transplantation of human iPSC-derived cardiomyocytes (CMs) without treatment into NOG mice consistently induced teratoma/tumour formation, with a substantial number of Ki-67-positive cells in the graft at 4 months post-transplant, whereas iPSC-derived CMs treated with brentuximab vedotin prior to the transplantation did not show teratoma/tumour formation, which was associated with absence of Ki-67-positive cells in the graft over the same period. These findings suggest that in vitro treatment with brentuximab vedotin, targeting the CD30-positive iPSC fraction, reduced tumourigenicity in human iPSC-derived CMs, potentially providing enhanced safety for iPSC-based cardiomyogenesis therapy in clinical scenarios.

Diabetes mellitus adversely affects mortality and recurrence after valve surgery for infective endocarditis.

BACKGROUND: Although diabetes mellitus (DM) increases the incidence of infective endocarditis (IE), little is known about the outcome of valve surgery for active IE in patients with DM. We evaluated the clinical outcomes of valve surgery for IE in patients with DM. METHODS: Between 2009 and 2016, 470 patients underwent valve surgery for definitive left-sided active IE at 12 affiliated hospitals. We compared the preoperative variables and clinical outcomes between patients without (n = 374) and with DM (n = 96). RESULTS: Staphylococcus and chronic hemodialysis were more prevalent in patients with DM, and these patients had greater preoperative inflammation levels and worse renal function than patients without DM. In-hospital mortality was 8% in patients without DM and 13% in patients with DM (P = .187). The overall survival rate at 1 and 5 years was 87% and 81% in patients without DM and 72% and 59% in patients with DM (P < .001). The incidence of infection-related death was greater in patients with DM than in patients without DM (P < .001; hazard ratio 3.74 [1.78-7.71]). Freedom from the recurrence of endocarditis at 1 and 5 years postoperatively was 98% and 95% in patients without DM, and 89% and 78% in patients with DM (P < .001), respectively. The Cox hazard analysis revealed that the presence of DM was the only independent risk for recurrence (hazard ratio 3.74 [1.45-9.54], P = .007). CONCLUSIONS: The short- and mid-term outcome after valve surgery for active IE in patients with DM is worse because of the greater prevalence of infection-related death and IE recurrence.

Engraftment and morphological development of vascularized human iPS cell-derived 3D-cardiomyocyte tissue after xenotransplantation.

One of the major challenges in cell-based cardiac regenerative medicine is the in vitro construction of three-dimensional (3D) tissues consisting of induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) and a blood vascular network supplying nutrients and oxygen throughout the tissue after implantation. We have successfully built a vascularized iPSC-CM 3D-tissue using our validated cell manipulation technique. In order to evaluate an availability of the 3D-tissue as a biomaterial, functional morphology of the tissues was examined by light and transmission electron microscopy through their implantation into the rat infarcted heart. Before implantation, the tissues showed distinctive myofibrils within iPSC-CMs and capillary-like endothelial tubes, but their profiles were still like immature. In contrast, engraftment of the tissues to the rat heart led the iPSC-CMs and endothelial tubes into organization of cell organelles and junctional apparatuses and prompt development of capillary network harboring host blood supply, respectively. A number of capillaries in the implanted tissues were derived from host vascular bed, whereas the others were likely to be composed by fusion of host and implanted endothelial cells. Thus, our vascularized iPSC-CM 3D-tissues may be a useful regenerative paradigm which will require additional expanded and long-term studies.

Recent Surgical Results for Active Endocarditis Complicated With Perivalvular Abscess.

BACKGROUND: Surgical treatment for endocarditis patients with a perivalvular abscess is still challenging.Methods and Results:From 2009 to 2016, 470 patients underwent surgery for active endocarditis at 11 hospitals. Of these, 226 patients underwent aortic valve surgery. We compared the clinical results of 162 patients without a perivalvular abscess, 37 patients who required patch reconstruction of the aortic annulus (PR group) and 27 who underwent aortic root replacement (ARR group). Patients with a perivalvular abscess had a greater number ofStaphylococcusspecies and prosthetic valve endocarditis, a greater level of inflammation at diagnosis and symptomatic heart failure before surgery, especially in the ARR group. Nevertheless, the duration between diagnosis and surgery was similar, because of a high prevalence of intracranial hemorrhage in the ARR group. Hospital death occurred in 13 (9%) patients without a perivalvular abscess, in 4 (12%) in the PR and in 7 (32%) in the ARR group. Postoperative inflammation and end-organ function were similar between the groups. Overall survival of patients without a perivalvular abscess and that of the PR group was similar, but was significantly worse in the ARR group (P=0.050, 0.026). Freedom from endocarditis recurrence was similar among all patients. CONCLUSIONS: Patients treated with patch reconstruction showed favorable clinical results. Early surgical intervention is necessary when a refractory invasive infection is suspected.