Small-molecule-based generation of functional cardiomyocytes from human umbilical cord-derived induced pluripotent stem cells.

The purpose of this study was to investigate the cardiac-differentiation potential of induced pluripotent stem cells (iPSCs) generated from human umbilical cord-derived mesenchymal cells. Spontaneous beating colonies were observed at day 7 after the sequential addition of CHIR99021 and IWP-4. The combined use of CHIR99021 and IWP-4 downregulated the expression of pluripotency markers while upregulating cardiac transcription factors and cardiomyocyte-specific markers. The derived cardiomyocytes demonstrated typical sarcomeric structures and action-potential features; most importantly, the derived cells exhibited responsiveness to ß-adrenergic and muscarinic stimulations. The analyses of molecular, structural, and functional properties revealed that the derived cardiomyocytes were similar to cardiomyocytes derived from BJ foreskin fibroblast cells. In summary, our results demonstrate that functional cardiomyocytes can be generated from human umbilical cord-derived cells. The methodology described here has potential as a means for the production of functional cardiomyocytes from discarded human umbilical cord tissue.

Efficient generation of functional cardiomyocytes from human umbilical cord-derived virus-free induced pluripotent stem cells.

We have previously demonstrated that human umbilical cord-derived mesenchymal stem cells (UC-MSCs) can differentiate into cardiomyocyte-like cells. However, no contracting cells were observed during differentiation. In this study, we generated induced pluripotent stem cells (iPSCs) from UC-MSCs using mRNA reprogramming and focused on the differentiation of reprogrammed iPSCs into functional cardiomyocytes. For cardiac differentiation, the spontaneously contracting cell clusters were present on day 8 of differentiation. Immunostaining studies and cardiac-specific gene expression confirmed the cardiomyocyte phenotype of the differentiated cells. Electrophysiology studies indicated that iPSCs derived from UC-MSCs had a capacity for differentiation into nodal-, atrial-, and ventricular-like phenotypes based on action potential characteristics, and the derived cardiomyocytes exhibited responsiveness to ß-adrenergic and muscarinic stimulations. Moreover, the derived cardiomyocytes displayed spontaneous intracellular Ca2+ transients. These results demonstrate that functional cardiomyocytes can be generated from reprogrammed UC-MSCs, and the methodology described here will serve as a useful protocol to obtain functional cardiomyocytes from human mesenchymal stem cells.

Isolation, characterization and cardiac differentiation of human thymus tissue derived mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are promising candidate donor cells for replacement of cardiomyocyte loss during ischemia and in vitro generation of myocardial tissue. We have successfully isolated MSCs from the discarded neonatal thymus gland during cardiac surgery. The thymus MSCs were characterized by cell-surface antigen expression. These cells have high ability for proliferation and are able to differentiate into osteoblasts and adipocytes in vitro. For cardiac differentiation, the cells were divided into 3 groups: untreated control; 5-azacytidine group and sequential exposure to 5-azacytidine, bone morphogenetic protein 4, and basic fibroblast growth factor. Thymus MSCs showed a fibrolast-like morphology and some differentiated cells increased in size, formed a ball-like appearance over time and spontaneously contracting cells were observed in sequential exposure group. Immunostaining studies, cardiac specific genes/protein expression confirmed the cardiomyocyte phenotype of the differentiated cells. These results demonstrate that thymus MSCs can be a promising cellular source for cardiac cell therapy and tissue engineering.

Preoperative serum cortisone levels are associated with cognition in preschool-aged children with tetralogy of Fallot after corrective surgery: new evidence from human populations and mice.

BACKGROUND: Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease. Children with TOF would be confronted with neurological impairment across their lifetime. Our study aimed to identify the risk factors for cerebral morphology changes and cognition in postoperative preschool-aged children with TOF. METHODS: We used mass spectrometry (MS) technology to assess the levels of serum metabolites, Wechsler preschool and primary scale of intelligence-Fourth edition (WPPSI-IV) index scores to evaluate neurodevelopmental levels and multimodal magnetic resonance imaging (MRI) to detect cortical morphological changes. RESULTS: Multiple linear regression showed that preoperative levels of serum cortisone were positively correlated with the gyrification index of the left inferior parietal gyrus in children with TOF and negatively related to their lower visual spaces index and nonverbal index. Meanwhile, preoperative SpO2 was negatively correlated with levels of serum cortisone after adjusting for all covariates. Furthermore, after intervening levels of cortisone in chronic hypoxic model mice, total brain volumes were reduced at both postnatal (P) 11.5 and P30 days. CONCLUSIONS: Our results suggest that preoperative serum cortisone levels could be used as a biomarker of neurodevelopmental impairment in children with TOF. Our study findings emphasized that preoperative levels of cortisone could influence cerebral development and cognition abilities in children with TOF.

Atoh1 mediated disturbance of neuronal maturation by perinatal hypoxia induces cognitive deficits.

Neurodevelopmental disorders are currently one of the major complications faced by patients with congenital heart disease (CHD). Chronic hypoxia in the prenatal and postnatal preoperative brain may be associated with neurological damage and impaired long-term cognitive function, but the exact mechanisms are unknown. In this study, we find that delayed neuronal migration and impaired synaptic development are attributed to altered Atoh1 under chronic hypoxia. This is due to the fact that excessive Atoh1 facilitates expression of Kif21b, which causes excess in free-state α-tubulin, leading to disrupted microtubule dynamic stability. Furthermore, the delay in neonatal brain maturation induces cognitive disabilities in adult mice. Then, by down-regulating Atoh1 we alleviate the impairment of cell migration and synaptic development, improving the cognitive behavior of mice to some extent. Taken together, our work unveil that Atoh1 may be one of the targets to ameliorate hypoxia-induced neurodevelopmental disabilities and cognitive impairment in CHD.

Perioperative extracorporeal membrane oxygenation in pediatric congenital heart disease: Chinese expert consensus.

BACKGROUND: Congenital heart disease (CHD) is one of the main supportive diseases of extracorporeal membrane oxygenation in children. The management of extracorporeal membrane oxygenation (ECMO) for pediatric CHD faces more severe challenges due to the complex anatomical structure of the heart, special pathophysiology, perioperative complications and various concomitant malformations. The survival rate of ECMO for CHD was significantly lower than other classifications of diseases according to the Extracorporeal Life Support Organization database. This expert consensus aims to improve the survival rate and reduce the morbidity of this patient population by standardizing the clinical strategy. METHODS: The editing group of this consensus gathered 11 well-known experts in pediatric cardiac surgery and ECMO field in China to develop clinical recommendations formulated on the basis of existing evidences and expert opinions. RESULTS: The primary concern of ECMO management in the perioperative period of CHD are patient selection, cannulation strategy, pump flow/ventilator parameters/vasoactive drug dosage setting, anticoagulation management, residual lesion screening, fluid and wound management and weaning or transition strategy. Prevention and treatment of complications of bleeding, thromboembolism and brain injury are emphatically discussed here. Special conditions of ECMO management related to the cardiovascular anatomy, haemodynamics and the surgical procedures of common complex CHD should be considered. CONCLUSIONS: The consensus could provide a reference for patient selection, management and risk identification of perioperative ECMO in children with CHD. Video abstract (MP4 104726 kb).

Cardiac cell therapy: pre-conditioning effects in cell-delivery strategies.

Stem-cell therapy holds great promise for the treatment of ischemic heart disease. However, the benefit of cardiac cell therapy has not yet been proven in long-term clinical trials. Poor engraftment and survival of transplanted cells is one of the major concerns for the successful application of stem cells in cardiac cell therapy. Cell and cardiac pre-conditioning are now being explored as new approaches to support cell survival and enhance the therapeutic efficacy. In this paper, we summarize the state-of-the-art methods of cell delivery and cell survival post-delivery, with a focus on the pre-conditioning approaches that have been attempted to support the survival of transplanted cells.

Image quality and radiation exposure in pediatric cardiovascular CT angiography from different injection sites.

OBJECTIVE: The purpose of this article is to evaluate the effect of different injection sites (i.e., head, arm, or leg vein) on image quality and radiation exposure in pediatric cardiovascular CT angiography (CTA) with 64-MDCT. MATERIALS AND METHODS: CTA was performed in 61 children with suspected extracardiac abnormalities. Patients were assigned to three groups according to the different injection sites: head, arm, or leg vein. Enhancement of heart chamber and great vessels and background noise were quantified. Signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), dose-length product (DLP), and effective dose (ED) were calculated. Subjective image quality was assessed by two radiologists in consensus. RESULTS: There was no significant difference among all groups in the mean attenuation of the heart chamber, pulmonary artery (PA), and aorta. There was also no significant difference in their mean attenuation, background noise, SNR, and CNR. However, there were significant differences among the three groups for aorta image quality (p = 0.006), despite the nonsignificant differences in heart chamber and PA image quality. There also were significant differences among the three groups for total DLP and ED (p = 0.01 for both), with prescanning DLPs of 17.6%, 20.2%, and 24.5%, respectively, of the total DLP for each group. CONCLUSION: Although all injection sites can yield diagnostic-quality images with a low radiation dose in pediatric cardiovascular CTA, the injection site has a slight impact on the image quality of different targeted areas with a significantly different radiation dose. The optimization of a prescanning protocol may open an avenue to reduce the radiation dose associated with cardiovascular CTA.

Serum vitamin E concentration is negatively associated with body mass index change in girls not boys during adolescence.

BACKGROUND: Vitamin E is the most abundant lipid-soluble antioxidants present in plasma; however, the relationship between serum vitamin E and change in body mass index (BMI)-for-age Z scores in adolescents has not been well described. METHODS: This study is a cross-sectional study. Data were analyzed from 4014 adolescents who participated in the National Health and Nutrition Examination Survey. The nutritional status was calculated by BMI Z scores and was classified into normal weight, overweight, and obese. Multivariable-adjusted logistic regression was used to examine the association between serum vitamin E levels with overweight/obesity. Besides, the interaction effects between potential confounders and vitamin E on obesity were further evaluated. RESULTS: After adjusting potential confounders, serum vitamin E levels were negatively associated with overweight/obesity in girls but not in boys. Per standard deviation increment in vitamin E concentrations was associated with a 92% decreased risk of obesity in females. Besides, lower quartiles of serum vitamin E were associated with a higher risk of overweight/obesity in girls. Moreover, the inverse association between serum vitamin E levels and obesity was also found in most subgroups through subgroup analysis. CONCLUSIONS: Our study supports the negative association between serum vitamin E levels and overweight/obesity in adolescents. A higher serum vitamin E level may be associated with a reduced probability of obesity in girls, but not in boys.

[Diagnostic value of 64 multislice CT in typing of congenital aortic anomaly in neonates and infants].

OBJECTIVE: To study diagnostic value of 64 multislice CT in typing of congenital aortic anomaly in neonates and infants. METHODS: 120 pediatric patients (under one year of age) with congenital heart disease (CHD) underwent 64 contrast-enhanced MSCT before a corrective operations. The diagnostic sensitivity, specificity and accuracy of 64 MSCT were evaluated and also compared with those of echocardiography with Doppler. The patients were randomly assigned to two groups (72 and 48 persons) respectively according to tube tension of 80 and 100 kV. The differences of the image qualities were compared between them. RESULTS: 36 congenital aortic anomalies were found (36/120, 30%) by 64 MSCT, which were furtherly distinguished into 2 cases in double aortic arch, 2 cases in left-sided aortic arch with aberrant right subclavian artery, 22 cases in right aortic arch (6 cases in right-sided aortic arch with aberrant left subclavian artery, 12 cases in right aortic arch with mirror image branching), 10 cases in coarctation of aorta. Diagnostic sensitivity, specificity and accuracy of 64 MSCT were all 100%. By contrast, those of echocardiography with Doppler were 27.8%, 97.6%, 76.7%, respectively. The quality scores were 4.69 ± 0.52, 4.58 ± 0.58 at 80 kV and 100 kV, respectively. No significant statistical difference was found between them (t = 1.08, P = 0.28). CONCLUSION: MSCT allows a detailed assessment of the anatomy of congenital aortic anomaly, which can be used as an important supplementary method in diagnosing CHD and offer important information for operation. 80 kV should be selected in CHD patients less than one year old for CT examination to reduce radiation exposure.

[The individualized surgical treatment of transposition of the great arteries].

OBJECTIVE: To discuss the effectiveness of individualized strategy of surgical management on the great arteries (TGA). METHODS: From March 1998 to October 2009, 127 cases (97 males and 30 females) with TGA were treated. There were 97 male and 30 female, aged from 4 hours old to 17 years old with a mean of (25 ± 37) months, weighted from 2.7 to 47.5 kg with a mean of (8 ± 8) kg. The palliative operations included Glenn operation in 14 cases (3 cases double Glenn procedure), Balalock-Taussing shunt in 14 cases, Banding operation in 8 cases, and atrial septal defect enlarge/Banding/Balalock-Taussing shunt in 15 cases. The end-stage operation included Senning procedure in 5 cases, Switch procedure in 32 cases, 2(nd)-stage Switch procedure in 11 cases, Switch procedure with VSD repairing in 20 cases, Switch procedure with Hybrid in 1 case, Nikaidoh procedure in 3 cases, Rastelli procedure in 13 cases, Fonton procedure in 18 cases, other procedure in 4 cases. Twenty-one cases underwent 2 operations, and 5 cases underwent 3 or more operations. Sixty-six cases underwent delayed sternal closure. RESULTS: There were 12 cases of death operatively in 127 cases. The total operative mortality was 9.4%. There were 5 cases dying of low cardiac output during the operation, 2 of pulmonary hypertension crisis, 2 of hemorrhage, 1 of grafting problem of coronary artery deformation, 1 of renal failure after Fonton procedure and 1 case of newborn dying of spontaneous rupture of liver post-operatively. The patients were followed up for 1 month to 12 years. There were 10 patients with vary degrees complications such as pulmonary stenosis, residual shunt and narrow channel. Three cases underwent reoperation. The rest of survived cases had normal heart function, good growth and development state. CONCLUSIONS: Individualized strategy of surgical management based on anatomical conditions of TGA can significantly improve the success rate of surgery and long-term survival.

[Effect of diazoxide on oxygen free radicals and cell apoptosis in brain tissue after deep hypothermia cerebral ischemia reperfusion injury in young rats].

OBJECTIVE: To determine the effects of diazoxide on oxygen free radicals and cell apoptosis in brain tissue after deep hypothermia cerebral ischemia reperfusion injury in young rats. METHODS: Fifty-four 3-week-old Sprague-Dawley rats were randomly and equitably divided into sham-operated group, model group and diazoxide group respectively (n = 18). The model of hypothermia cerebral ischemia reperfusion injury was made. After 24 hours of operation, the brains of rats were removed and preserved. The content of superoxide dismutase (SOD) and malonaldehyde (MDA) in brain tissue were detected. Cytosolic C release of cytochrome was confirmed by Western Blot. The protein expression of Caspase-3 was determined by immunohistochemistry. RESULTS: In the model group, the content of SOD was (198 +/- 41) U/mg, lower than the sham-operated group's (321 +/- 36) U/mg (P < 0.01). The content of MDA was (212 +/- 21) nmol/mg, was higher than the sham-operated group's (100 +/- 23) nmol/mg (P < 0.01), and the expressions of cytochrome C (0.72 +/- 0.09) and Caspase-3 (83 +/- 10) were all significantly higher than those in the sham-operated group (0.17 +/- 0.02 and 115 +/- 9) (P < 0.01). Compared with the model group, the content of SOD in the diazoxide group [(264 +/- 34) U/mg] was markedly increased (P < 0.05). In addition, diazoxide provided significant reductions in the content of MDA [(174 +/- 19) nmol/mg] and the expressions of cytochrome C (0.41 +/- 0.05) and Caspase-3 (99 +/- 11) (P < 0.05). CONCLUSIONS: The neuroprotective effects of diazoxide against brain injury induced by deep hypothermia cerebral ischemia reperfusion through inhibiting oxygen free radicals and cell apoptosis. Diazoxide may become a new neuroprotective drug after infant complicated congenital cardiac operation.

Cardiac potential of stem cells from whole human umbilical cord tissue.

We investigated the role of stem cells from human umbilical cord tissue in cardiomyocyte regeneration. The umbilical cord stem cells were initially characterized and differentiated in a myocardial differentiation medium containing 5-azacytidine for 24 h. Differentiation into cardiomyocytes was determined by expression of cardiac specific markers, like cardiac alpha-actin, connexin43, myosin, Troponin T, and ultrastructural analysis. In vivo, the transplanted umbilical cord stem cells were sprouting from local injection and differentiated into cardiomyocyte-like cells in a rat myocardial infarction model. Echocardiography revealed increasing left ventricular function after umbilical cord stem cell transplantation. These results demonstrate that umbilical cord stem cells can differentiate into cardiomyocyte-like cells both in vitro and in vivo. Therefore, human umbilical cord might represent a source of stem cells useful for cellular therapy and myocardial tissue engineering. Future studies are required to determine the molecular signaling mechanisms responsible for this phenomenon.

Clinical analysis and surgical results of cardiac myxoma in pediatric patients.

BACKGROUND: Cardiac myxoma, the most common primary tumor in the adult, is less often encountered in infants and children. We reviewed our series of children with cardiac myxoma over a 22-year period and present the long-term results of surgical resection of the tumor. PATIENTS AND METHODS: From January 1985 to December 2006, 15 children between the ages of 5 months and 14 years (mean age, 6.5 years) with cardiac myxoma were reviewed. Follow-up information from every patient was obtained from outpatient records and direct patient contact. RESULTS: Cardiac signs appeared in 86.7% of the patients. Most myxomas originated from the left atrium. There was no operative death. No patients were lost to follow-up, and the mean follow-up was 9.6 years. There were no late deaths during the follow-up. To date, 13 of 15 operative survivors were in New York Heart Association (NYHA) functional class I without medication and the other 2 were in class II. All patients demonstrated sinus rhythm and no child required reexcision of tumor. CONCLUSIONS: Our clinical experience showed surgical resection was a safe and effective treatment for cardiac myxoma in pediatric patients and the risk of recurrence was low during the long term follow-up.

[Diagnosis of 22q11 deletion and duplication in congenital heart disease by multiplex ligation dependent probe amplification].

OBJECTIVE: To investigate the clinical utility of multiplex ligation-dependent probe amplification (MLPA) for detecting 22q11 deletion and duplication in congenital heart disease (CHD) cases and to study the incidence of 22q11 deletion and duplicaton in different kinds of CHD. METHODS: Forty eight probes of which 25 located in 22q11 low copy number region (LCR 22s A-H), 7 in 22q11 surrounding region (CES, 22q13) and 16 in chromosomes 4, 8, 10 and 17 were selected to detect 22q11 deletion and duplication in 181 preoperative children with CHD and 14 fetuses with serious CHD or CHD with multiple malformations. In these cases, karyotype analysis was also performed. RESULTS: MLPA demonstrated that 7 cases had 22q11 deletion [6 cases from CLTCL1 to LZTR1(LCR A-D) and 1 case from CLTCL1 to PCQAP (LCR A-C)] and that 1 case had 22q11 duplication,spanning from ZNF74 to LZTR1(LCR B-D). The phenotypes of heart defect included ventricular septal defect, atrioventricular septal defect, pulmonary stenosis and tetralogy of Fallot. Karyotype analysis showed that 1 case had 21q deletion [46, XY, 21q], 1 case had mosaic trisomy 8 [47,XY, +8/46, XY(1:2)] and 4 cases had trisomy 21. One of the 4 cases with trisomy 21 had concurrent 22q11 duplication. CONCLUSIONS: MLPA is a rapid, sensitive, site specific and relatively inexpensive method for diagnosis of 22q11 deletion and duplication in CHD. 22q11 deletion and duplication may cause various kinds of CHD, suggesting that genetic detection should be performed routinely in CHD patients.

Cell sheet engineering for the injured heart.

Myocardial infarction and subsequent heart failure are a leading cause of morbidity and mortality. Tissue engineering is emerging as promising alternative approach to treat these kinds of diseases. However, conventional applications using biodegradable scaffolds have disadvantages, such as biocompatibility, biodegradability, and cytotoxicity, and this limits its efficacy. Cell sheet engineering without artificial scaffolds to form new myocardial constructs avoids the shortcomings of traditional tissue engineering approaches using scaffolds. We hypothesize that cell sheet engineering may be a promising strategy for cardiac tissue reconstruction.

miR­760 mediates hypoxia-induced proliferation and apoptosis of human pulmonary artery smooth muscle cells via targeting TLR4.

MicroRNAs (miRNAs) have a key role in the pathogenesis of pulmonary arterial hypertension (PAH), a disease characterized by enhanced proliferation and reduced apoptosis of pulmonary artery smooth muscle cells. In the present study, miR­760 was demonstrated to be downregulated in PAH lung tissues compared with normal lung tissues, an effect that may be associated with the development of PAH. Hypoxia is an important stimulus for human pulmonary artery smooth muscle cell (hPASMC) proliferation and the occurrence of PAH. Therefore, the effect of miR­760 in hypoxia­treated and normal hPASMCs was investigated. Expression of exogenous miR­760 decreased cell proliferation in hypoxia­induced hPASMCs, and promoted cell apoptosis with an increase in the BCL2 associated X/BCL2 ratio and the expression levels of Caspase­3 and Caspase­9. In addition, overexpression of miR­760 suppressed the migration of hPASMCs under hypoxic conditions. Furthermore, miR­760 was demonstrated to mediate its anti­proliferation effect via the regulation of toll­like receptor 4 (TLR4), a direct target of miR­760. The results revealed that knockdown of TLR4 restrained the hypoxia­induced hPASMC proliferation and induced cell apoptosis. The present study uncovered a novel regulatory pathway involving miR­760 and suggested that miR­760 may be explored as a potential therapy for PAH in the future.

Stem cells for tissue engineering of myocardial constructs.

Cardiovascular diseases are the leading cause of morbidity and mortality. Tissue engineering offers new option in the myocardial repair techniques. The cellular component of this regenerative approach will play a key role in bringing these tissue engineered constructs from the laboratory bench to the clinical bedside. However, the ideal source of cells still remains unclear and may differ depending upon the application. Current research for many applications is focused on the use of stem cells. The combination of stem cell technology and tissue engineering has been investigated and offers promising avenues for myocardial tissue regeneration, and this shows great promise in future reconstructive surgery. We explore the basic concepts and methods for myocardial tissue reconstruction and emphasize the progress made and remaining challenges of stem cells in myocardial tissue engineering.

VEGF C-634G polymorphism is associated with protection from isolated ventricular septal defect: case-control and TDT studies.

The ventricular septal defect (VSD) is the most common congenital heart defect and no candidate susceptibility gene has been identified. Endocardial cushion and outflow septal morphogenesis, malalignment of which induces VSD, have been suggested to be mediated by the vascular endothelial growth factor (VEGF). Three single-nucleotide polymorphism (SNP) variants in promoter and 5'-UTR region of the VEGF gene, C-2578A (rs699947), G-1154A (rs1570360) and G-634C (rs2010963), were reported to alter its expression. We assessed the association in a Chinese population between these SNPs and VSD using a double approach: case-control and TDT designs. Among the three SNPs, only -634C allele was less frequently present in 222 patients compared to 352 controls (odds ratio: 0.76, 95% CI: 0.59-0.97, X(2)=5.06, P=0.024, not significant after a Bonferroni correction). This was significantly less transmitted to VSD patients (trios: 142) (odds ratio: 0.39, 95% CI: 0.25-0.62, X(2)=8.11, df=1, P=0.004, corrected P=0.024). A similar result was observed for haplotype -2578C/-1154G/-634C allele in both studies (in TDT: X(2)=7.51, df=1, P=0.006, corrected P=0.048). All these associations for the first time demonstrated that -634C allele was in a significant protective association against VSD, suggesting that VEGF dysregulation was involved in the pathological processes of VSD.