Junsheng Mu

Differentiation of mouse-induced pluripotent stem cells into cardiomyocytes in vitro

Abstract In order to investigate the effective method to induce mice-induced pluripotent stem (miPS) cells into cardiomyocytes in vitro and to investigate the effect of vitamin C on cardiomyocyte differentiation from miPS cells to find a highly efficient and clinically safe method. MiPS cells were isolated and expanded to form embryoid bodies (EBs) using the hanging drop way. EBs were induced using differentiation medium containing vitamin C (10−4 mmol/ml). The control group did not receive any form of inducer. The time and frequency at which beating cardiomyocytes appeared and the percentage of beating colonies were determined to investigate the function of vitamin C on cardiac myocytes differentiation from miPS cells. Beating cell areas were found in (62.5 ± 1.7%) of EBs when using differentiation medium containing vitamin C, which was at a significantly greater frequency than in the control group (7.6 ± 2.6%). Beating cardiomyocytes within the two groups were positive for troponin (cTnT) staining. Vitamin C markedly increased the productivity of miPS cell differentiation into cardiomyocytes, as supported by expression of the unique cardiac protein cTnT. The vitamin C is suitable candidate for the induction of miPS cell differentiation into cardiomyocytes in vitro.

Comparative study of directional differentiation of human and mouse embryonic stem cells into cardiomyocytes

This comparative study investigates the method, efficiency, and anti‐hypoxic ability of cardiomyocytes, directionally induced from human (h) and mouse (m) embryonic stem cells (ESCs). hESCs were induced into cardiomyocytes by suspension culture, without inducers, or adherent culture using the inducers activin A and BMP4. mESCs were induced into cardiomyocytes by hanging‐drop method, without inducers or induced with vitamin C. All four methods successfully induced ESCs to differentiate into cardiomyocytes. There was a significant difference between groups with and without inducers. A significant difference was found between mESC and hESC groups with inducers. The average beating frequency of cardiomyocytes differentiated from hESC was lower than cardiomyocytes differentiated from mESC, while the average beating frequency of cardiomyocytes differentiated from the same cell line, despite different culture methods, did not differ. Beating cardiomyocytes of each group were positive for cTnT staining. Spontaneous action potentials of beating cardiomyocytes were detected by patch‐clamp experiments in each group. Different apoptotic ratios were detected in beating cardiomyocytes in each group and the difference between cardiomyocytes induced from mESCs and hESCs was statistically significant. The differentiation efficiencies in the groups without inducers were significantly higher than those without inducers. The induction of mESCs was more simple and efficient compared with hESCs. Without the presence of other protective factors, the anti‐hypoxic ability of cardiomyocytes induced from hESCs was stronger and the beating times were longer in vitro compared with mESCs.

Examination of bone marrow mesenchymal stem cells seeded onto poly(3-hydroxybutyrate-co-4-hydroxybutyrate) biological materials for myocardial patch

The implantation of bone marrow mesenchymal stem cells (BMSCs) into the heart has been reported to be effective for myocardial infarction; however, it is unknown what methods are most suitable for supporting stem cell growth in a myocardial patch. This study used a new polymer material composed of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] co-cultured with BMSCs to create a myocardial patch. Bone marrow mesenchymal stem cells were obtained from healthy male BSL-C57 mice. The cells were treated with 5-azacytidine to investigate their differentiation into cardiomyocytes. The cells were seeded for 24-hours onto P(3HB-co-4HB) biological material films (n = 8). Cell-biomaterial constructs were fixed and analyzed using different methods. Bone marrow mesenchymal stem cells were CD34, CD45− , CD90+ (low), and CD73+. The cells were stained with anti-cardiac troponin T (anti-cTnT) and anti-connexin 43 (anti-CX43) antibodies after 5-azacytidine treatment. Scanning electron microscopy showed that BMSC morphology was normal, and cell numbers were more abundant on the P(3HB-co-4HB) material surfaces. The growth curve of BMSCs on the biomaterial patches showed that the P(3HB-co-4HB) material permitted good stem cell growth. Owing to its excellent biocompatibility and biodegradability properties, in particular its porosity, P(3HB-co-4HB) is highlighted as an optimal material to support myocardial cell growth and myocardial patch formation in patients with myocardial infarction.

Sudden Thrombosis in Coronary Artery Bypass Grafting Surgery.

To the Editor: A 47-year-old male patient was admitted because of chest discomfort for 2 weeks. He had no obvious incentive precordial discomfort 2 weeks ago, accompanied by palpitation. He immediately went to the town hospital. Electrocardiogram (ECG) showed myocardial infarction. Coronary angiography showed coronary artery disease accompanied by a ventricular aneurysm. The patient was transferred to Beijing Anzhen Hospital. ECG showed sinus rhythm, heart axis deviation + 111°, anterior septal, anterior lateral, anterior myocardial infarction, and complete right bundle branch block. Chest X-ray showed no obvious abnormalities in heart and lung. Echocardiography showed abnormal motion of segmental ventricular wall; formation of a ventricular aneurysm in apex area of the heart, the diameter of the ventricular aneurysm was 2 cm, the diastolic function of left ventricular was reduced. Coronary angiography showed left anterior descending artery filling slowly and its intima was not smooth; the stenosis rate was 90%. The stenosis rate of the circumflex artery was 90% [Figure ​[Figure1a1a and ​andb].b]. He was diagnosed with acute anterior myocardial infarction, left ventricular aneurysm, and hypertension. Figure 1 Coronary angiography right anterior oblique 30° display. (a) The circumflex artery was filled and stenosis. Left anterior descending artery was not completely filled; (b) After the circumflex artery was filled, left anterior descending artery ... The patient received treatment of dilation of a coronary artery, anticoagulation, nourishing myocardium, and other symptomatic and supportive treatment. He underwent off-pump coronary artery bypass surgery with median incision of the sternum. Take the left internal mammary artery and the right saphenous vein in reserve. Activating clotting time (ACT) value was 391 s after intravenous injection of 75 mg heparin. Left internal mammary artery - left anterior descending artery; aorta - saphenous vein - obtuse marginal branch vascular anastomosis were performed using external fixator. Anastomotic stoma was unobstructed and nonhemorrhage. Flow meter displayed satisfactory flow in grafts. Heparin was neutralized followed by routine chest shut surgery. The patient broke out in the sudden reduction of blood pressure and ventricular fibrillation. Defibrillation (200 Ws) and emergency chest compressions were performed followed by exploratory thoracotomy surgery. Intra-aortic balloon counterpulsation (IABP) was used as an assistive device. Extracorporeal circulation was established. Heparin (250 mg) was intravenously injected. ACT value after heparin injection was 486 s, intraoperative ACT value was 610 s. Flow meter displayed no flow in grafts. A redo coronary artery bypass grafting surgery was performed. Thrombosis was found in the left internal mammary artery distal anastomosis and the left anterior descending artery, saphenous vein proximal and distal anastomosis, and the circumflex artery. The thrombus in anastomosis and grafts was carefully removed and the proximal and the distal grafts was spied. The distal circumflex artery was not satisfying. The distal left anterior descending artery was satisfying. Aorta - saphenous vein - anterior descending artery, aorta - saphenous vein - obtuse marginal branch vascular anastomosis were performed again. Anastomotic stoma was unobstructed. Extracorporeal circulation device could not stop so as to install extracorporeal membrane oxygenation (ECMO) assist device. Stop extracorporeal circulation device followed by routine chest shut surgery. The patients vital signs were closely surveilled in Intensive Care Unit (ICU). The patient accepted cardiotonic agents, anti-infection, nourishing myocardium, and other symptomatic treatment. The patient condition gradually improved, ECMO device was removed on the fourth postoperative day. Trachea cannula was removed on the fifth postoperative day. IABP device was removed on the seventh postoperative day. The patient went back to the ward on the eighth postoperative day. Heart rhythm was regular. Cardiac murmur did not exist. Chest X-ray, echocardiography, and ECG showed no abnormalities. The patient was discharged 15 days after the operation. Thrombosis is a common complication of coronary artery bypass graft surgery. The thrombosis is classified as arterial thrombosis and venous thrombosis. Venous thrombosis is common, arterial thrombosis is rare. The main causes of venous thrombosis are vascular intimal injury and blood clotting disorders. The intimal injury may activate platelet function and make platelets release thromboxane A and other clotting factors to promote thrombosis. Coagulation abnormalities may lead to increased activity of clotting factor which could cause thrombosis. Arterial thrombosis is rare and its mechanism is unknown. Currently, it is known that heparin resistance is one of the reasons for arterial thrombosis. Heparin is an acidic mucopolysaccharide composed of glucosamine L-iduronic glucoside, N-acetyl glucosamine, and D-glucuronic acid. It is mainly produced by mast cells and basophils. Despite its little content in plasma under normal physiological conditions, it has rich content in the lung, liver, and other tissues. The anticoagulant effect of heparin is strong. It enhances the affinity of antithrombin III and thrombin, accelerates thrombin inactivation, inhibits clotting factor activation, inhibits platelet adhesion and aggregation, increases protein C activation, and stimulates vascular endothelial cells to release anticoagulant and fibrinolytic substances. Heparin is widely used in cardiovascular surgery. Studies have shown that patients undergoing coronary artery bypass surgery shows heparin resistance. The incidence of this phenomenon is rising.[1] Heparin resistance refers to the phenomena that ACT value is <400 s under the use of a standard dose of heparin.[2] If ACT value is <400 s, it is not sufficient for the patient to have plasma anticoagulant capacity in vivo. Meanwhile, the use of extracorporeal circulation system will lead to excessive activation of the coagulation system.[3] Lower activity of antithrombin III may be the cause of heparin resistance.[4] However, some reports showed that there is no direct relationship between antithrombin III and heparin.[5] Patients show symptoms of heparin resistance with the high levels of platelet in the blood. The mechanism is that platelets can release platelet factor 4 which could inhibit the function of heparin. The patients with the high levels of platelet in the blood will be able to inhibit the effect of heparin to a certain extent. The reason for heparin resistance is very complex. ACT value is not a specific indicator for the anticoagulant capacity of heparin. ACT value is affected by many variables in cardiac surgery. However, clinicians still use ACT value as the detection indicator of heparin anticoagulant capabilities. Heparin resistance can lead to a variety of serious complications such as bleeding, cardiac arrhythmia, ventricular fibrillation, prolonged intubation time, and prolonged residence time in ICU. This patient who had coronary artery bypass grafting surgery may have heparin resistance which lead to thrombosis in a coronary artery, artery, and vein grafts. The mechanism of heparin resistance is unclear. It is hoped that medical workers gain experience in future clinical work. Hence, heparin resistance phenomenon can be reduced. Financial support and sponsorship This work was supported by grants from 2014 Chaoyang District, Beijing Science and Technology Program (No. SF1417); 2015 Capital Medical University Student Technological Innovation Program (No. XSKY2015192). Conflicts of interest There are no conflicts of interest.

Directional differentiation of human embryonic stem cells into cardiomyocytes by direct adherent culture

Abstract Myocardial infarction is a serious and common disease in clinics, with a poor prognosis and a high mortality in the long term. The latest research progress in stem cell research, which has been at the frontier of medical research in recent years, gives hope to the patients with heart disease. Researchers have been trying to find an efficient and practical differentiation procedure to induce embryonic stem cells (ESCs) to differentiate into cardiomyocytes. This study used a direct adherent-culture method to induce the differentiation of human ESCs (hESCs) into cardiomyocytes in vitro and its differentiation efficiency was detected. The hESCs were induced into cardiomyocytes by adherence culture using the inducers activin A and BMP4. The time of appearance of beating cardiomyocytes, the percentage of beating colonies, and the beating frequency of cardiomyocytes under the microscope were observed and counted; the specific cardiomyocyte marker cTnT was started by immunofluorescence, and the electrophysiological function of cardiomyocytes was detected by patch clamp experiment. An apoptosis-Hoechst staining kit was used to detect the apoptosis ratio of beating of cardiomyocytes which had been treated by hypoxia for 24 hours. Widespread spontaneous beating cardiomyocytes was typically observed by day 13 after differentiation. The statistical result was that the average time of appearance of beating cardiomyocytes was 13·0±1·1 days, the percentage of beating colonies was 66·7%, and the beating frequency of cardiomyocytes 63·0±7·0 times/min; beating cardiomyocytes were positive to cTnT staining. Spontaneous action potentials of beating cardiomyocytes were detected, and the apoptosis ratio of beating cardiomyocytes which had been treated by hypoxia for 24 hours was 8·0±0·5%. The direct adherent-culture method was successfully used to induce the differentiation of hESCs into cardiomyocytes. The adherent method of hESC induced with activin A+BMP4 was determined to be more simple and effective than other methods. The differentiation efficiency reached 66·7%, and the differentiation time was about 13 days.