Effect of Xenogeneic Substances on the Glycan Profiles and Electrophysiological Properties of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Background and Objectives@#Human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM) hold greatpromise as a cellular source of CM for cardiac function restoration in ischemic heart disease. However, the use of animal-derived xenogeneic substances during the biomanufacturing of hiPSC-CM can induce inadvertent immune responses or chronic inflammation, followed by tumorigenicity. In this study, we aimed to reveal the effects of xenogeneic substances on the functional properties and potential immunogenicity of hiPSC-CM during differentiation, demonstrating the quality and safety of hiPSC-based cell therapy. @*Methods@#and Results: We successfully generated hiPSC-CM in the presence and absence of xenogeneic substances(xeno-containing (XC) and xeno-free (XF) conditions, respectively), and compared their characteristics, including the contractile functions and glycan profiles. Compared to XC-hiPSC-CM, XF-hiPSC-CM showed early onset of myocyte contractile beating and maturation, with a high expression of cardiac lineage-specific genes (ACTC1, TNNT2, and RYR2) by using MEA and RT-qPCR. We quantified N-glycolylneuraminic acid (Neu5Gc), a xenogeneic sialic acid, in hiPSC-CM using an indirect enzyme-linked immunosorbent assay and liquid chromatography-multiple reaction monitoring-mass spectrometry. Neu5Gc was incorporated into the glycans of hiPSC-CM during xeno-containing differentiation, whereas it was barely detected in XF-hiPSC-CM. @*Conclusions@#To the best of our knowledge, this is the first study to show that the electrophysiological function andglycan profiles of hiPSC-CM can be affected by the presence of xenogeneic substances during their differentiation and maturation. To ensure quality control and safety in hiPSC-based cell therapy, xenogeneic substances should be excluded from the biomanufacturing process.

Trends in the development of human stem cell-based non-animal drug testing models

In vivo animal models are limited in their ability to mimic the extremely complex systems of the human body, and there is increasing disquiet about the ethics of animal research. Many authorities in different geographical areas are considering implementing a ban on animal testing, including testing for cosmetics and pharmaceuticals. Therefore, there is a need for research into systems that can replicate the responses of laboratory animals and simulate environments similar to the human body in a laboratory. An in vitro two-dimensional cell culture model is widely used, because such a system is relatively inexpensive, easy to implement, and can gather considerable amounts of reference data. However, these models lack a real physiological extracellular environment. Recent advances in stem cell biology, tissue engineering, and microfabrication techniques have facilitated the development of various 3D cell culture models. These include multicellular spheroids, organoids, and organs-on-chips, each of which has its own advantages and limitations. Organoids are organ-specific cell clusters created by aggregating cells derived from pluripotent, adult, and cancer stem cells. Patient-derived organoids can be used as models of human disease in a culture dish. Biomimetic organ chips are models that replicate the physiological and mechanical functions of human organs. Many organoids and organ-on-a-chips have been developed for drug screening and testing, so competition for patents between countries is also intensifying. We analyzed the scientific and technological trends underlying these cutting-edge models, which are developed for use as non-animal models for testing safety and efficacy at the nonclinical stages of drug development.

Trends in the development of human stem cell-based non-animal drug testing models

In vivo animal models are limited in their ability to mimic the extremely complex systems of the human body, and there is increasing disquiet about the ethics of animal research. Many authorities in different geographical areas are considering implementing a ban on animal testing, including testing for cosmetics and pharmaceuticals. Therefore, there is a need for research into systems that can replicate the responses of laboratory animals and simulate environments similar to the human body in a laboratory. An in vitro two-dimensional cell culture model is widely used, because such a system is relatively inexpensive, easy to implement, and can gather considerable amounts of reference data. However, these models lack a real physiological extracellular environment. Recent advances in stem cell biology, tissue engineering, and microfabrication techniques have facilitated the development of various 3D cell culture models. These include multicellular spheroids, organoids, and organs-on-chips, each of which has its own advantages and limitations. Organoids are organ-specific cell clusters created by aggregating cells derived from pluripotent, adult, and cancer stem cells. Patient-derived organoids can be used as models of human disease in a culture dish. Biomimetic organ chips are models that replicate the physiological and mechanical functions of human organs. Many organoids and organ-on-a-chips have been developed for drug screening and testing, so competition for patents between countries is also intensifying. We analyzed the scientific and technological trends underlying these cutting-edge models, which are developed for use as non-animal models for testing safety and efficacy at the nonclinical stages of drug development.

Antitumor profiles and cardiac electrophysiological effects of aurora kinase inhibitor ZM447439

Aurora kinases inhibitors, including ZM447439 (ZM), which suppress cell division, have attracted a great deal of attention as potential novel anti-cancer drugs. Several recent studies have confirmed the anti-cancer effects of ZM in various cancer cell lines. However, there have been no studies regarding the cardiac safety of this agent. We performed several cytotoxicity, invasion and migration assays to examine the anti-cancer effects of ZM. To evaluate the potential effects of ZM on cardiac repolarisation, whole-cell patch-clamp experiments were performed with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and cells with heterogeneous cardiac ion channel expression. We also conducted a contractility assay with rat ventricular myocytes to determine the effects of ZM on myocardial contraction and/or relaxation. In tests to determine in vitro efficacy, ZM inhibited the proliferation of A549, H1299 (lung cancer), MCF-7 (breast cancer) and HepG2 (hepatoma) cell lines with IC₅₀ in the submicromolar range, and attenuated the invasive and metastatic capacity of A549 cells. In cardiac toxicity testing, ZM did not significantly affect I(Na), I(Ks) or I(K1), but decreased I(hERG) in a dose-dependent manner (IC₅₀: 6.53 µM). In action potential (AP) assay using hiPSC-CMs, ZM did not induce any changes in AP parameters up to 3 µM, but it at 10 µM induced prolongation of AP duration. In summary, ZM showed potent broad-spectrum anti-tumor activity, but relatively low levels of cardiac side effects compared to the effective doses to tumor. Therefore, ZM has a potential to be a candidate as an anti-cancer with low cardiac toxicity.

Comparison of electrophysiological effects of calcium channel blockers on cardiac repolarization

Dihydropyridine (DHP) calcium channel blockers (CCBs) have been widely used to treat of several cardiovascular diseases. An excessive shortening of action potential duration (APD) due to the reduction of Ca2+ channel current (I(Ca)) might increase the risk of arrhythmia. In this study we investigated the electrophysiological effects of nicardipine (NIC), isradipine (ISR), and amlodipine (AML) on the cardiac APD in rabbit Purkinje fibers, voltage-gated K+ channel currents (I(Kr), I(Ks)) and voltage-gated Na+ channel current (I(Na)). The concentration-dependent inhibition of Ca2+ channel currents (I(Ca)) was examined in rat cardiomyocytes; these CCBs have similar potency on I(Ca) channel blocking with IC50 (the half-maximum inhibiting concentration) values of 0.142, 0.229, and 0.227 nM on NIC, ISR, and AML, respectively. However, ISR shortened both APD50 and APD90 already at 1 microM whereas NIC and AML shortened APD50 but not APD90 up to 30 microM. According to ion channel studies, NIC and AML concentration-dependently inhibited I(Kr) and I(Ks) while ISR had only partial inhibitory effects (<50% at 30 microM). Inhibition of I(Na) was similarly observed in the three CCBs. Since the I(Kr) and I(Ks) mainly contribute to cardiac repolarization, their inhibition by NIC and AML could compensate for the AP shortening effects due to the block of I(Ca).

Wide Spectrum of Inhibitory Effects of Sertraline on Cardiac Ion Channels

Sertraline is a commonly used antidepressant of the selective serotonin reuptake inhibitors (SSRIs) class. In these experiments, we have used the whole cell patch clamp technique to examine the effects of sertraline on the major cardiac ion channels expressed in HEK293 cells and the native voltage-gated Ca2+ channels in rat ventricular myocytes. According to the results, sertraline is a potent blocker of cardiac K+ channels, such as hERG, IKs and IK1. The rank order of inhibitory potency was hERG >IK1> IKs with IC50 values of 0.7, 10.5, and 15.2 microM, respectively. In addition to K+ channels, sertraline also inhibited INa and ICa, and the IC50 values are 6.1 and 2.6 microM, respectively. Modification of these ion channels by sertraline could induce changes of the cardiac action potential duration and QT interval, and might result in cardiac arrhythmia.

A Clinical Study of Frostbite / 대한정형외과학회잡지

This report is a clinical review of 58 patients suffering from frostbite, who were treated at the department orthopedic surgery, Seoul City Dong Bu Hospital from November 1982 to March 1985. The results are as follows: 1. The male to female sex ratio was 13.5:l. 2. The majority of the patients (72%) were decades between 30 and 50. 3. Frostbite began on November and registered the highest incidence in January, the lowest temperature of the year. 4. The most commonly affected region was the lower extremities, bilaterally. 5. The average duration of exposure in cold weather was 23 days, 6. The average hospitalization period was 84 days. 7. Among the 58 patients, 14 patients were treated by conservative treatment, 14 patients by closed amputation and 25 patients by open amputation.

A Clinical Study of The Traumatic Amputation By Railroad Accident / 대한정형외과학회잡지

A clinical study of traumatic amputation by railroad accidents was made an 124 patients with 154 amputee, who had been treated at Department of Orthopaedic Surgery, National Seoul Hospital, during the 4 years period from January 1976 to December 1979. The results of this study are as follows: 1. The ratio of male to female was 9 to 1. The 3rd decade of age group showed the hightest number of accident causalties, as much as 37.1%. 2. Causalties showes a maximum during the sprlng (37.1%) and a minimum during winter. 3. The most common injured sites of the amputee were B–K in 44 cases among 154 cases of the amputee. 4. Considering the number of the multiple amputee in 26 casee among 124 cases, frequently they developed in 2 sites with 22 cases, 3 sites with 4 cases. 5. The frequent fracture sites with combined amputation were 18 cases of the pelvic bone, 12 cases of femur in amputation of the lower extremity. 6. Our methods of treatment were debridement, debridement and primary skin closure, debridement with open amputation, circular amputation, closed amputation with skin graft, and closed amputation. 7. The major complications were phantom limb in 108 cases (70.1%), infection in 44 cases (28.6%). 8. The most frequent infectious agent was pseudomonas (16 cases).