hPSC gene editing for cardiac disease therapy.

Cardiovascular diseases (CVDs) are the leading cause of mortality worldwide. However, the lack of human cardiomyocytes with proper genetic backgrounds limits the study of disease mechanisms. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have significantly advanced the study of these conditions. Moreover, hPSC-CMs made it easy to study CVDs using genome-editing techniques. This article discusses the applications of these techniques in hPSC for studying CVDs. Recently, several genome-editing systems have been used to modify hPSCs, including zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9). We focused on the recent advancement of genome editing in hPSCs, which dramatically improved the efficiency of the cell-based mechanism study and therapy for cardiac diseases.

Generation of a COL1A2 homozygous knockout stem cell line via CRISPR/Cas9 system.

The loss of function of the COL1A2 gene can result in osteogenesis imperfecta (OI) types I, II, III, and IV and Ehlers-Danlos syndrome (cardiac valvular and arthrochalasia type).To further investigate the significance of COL1A2 in osteogenesis imperfecta and cardiac valve disease, we created a homozygous COL1A2-/- human embryonic stem cell line (WAe009-A-72) using CRISPR/Cas9. In vivo, the WAe009-A-72 cell line retained typical colony form, a normal karyotype, and robustly expressed pluripotency markers while differentiating into all three germ layers. This cell line is a potential tool for investigating the role of the COL1A2 gene in associated disorders.

SiO2/Al2O3/C grafted 3-n propylpyridinium silsesquioxane chloride-based non-enzymatic electrochemical sensor for determination of carcinogenic nitrite in food products.

The excessive uptake of nitrite is perilous and detrimental for human health that prone to cancer disease. Herein, described the synthesis of SiO2/Al2O3/C material through the sol-gel procedure followed by grafting with 3-n propylpyridinium silsesquioxane chloride organic ligand for enhancing electrochemical activity. H-NMR, 13C NMR, and 29Si studies were performed for confirmation of surface functionalization through the grafting technique. The surface morphology was evaluated through SEM and TEM techniques. The material showed an irregular and flakes-like structure that exhibited more compactness and conglomerate structure with no segregation in phase was observed after grafting. The elemental composition was confirmed from EDX analysis. The electrochemical measurements were performed with cyclic voltammetry, electrochemical impedance spectroscopy (EIS), and chronoamperometry. The prepared hybrid inorganic-organic composite Si/C/Al/SiPy+Cl- was applied for the modification of the glassy carbon (GC) electrode and assessed as a sensor for nitrite determination. The sensor showed the low limit of detection (0.01 µM), low limit of quantification (0.08 µM), wide linear response range (0.2-280 µM), and high sensitivity (410 µA·µM-1). It gave a quick response time of <1 s in the presence of 70 µM nitrite. The fabricated sensor showed high sensitivity, chemical stability, and insignificant interference from co-existing species present in sausage meat and food industry discharges. The repeatability of the sensor was evaluated as 2.5 % R.S.D.; for n = 10 at 50 µM nitrite.

hERG-deficient human embryonic stem cell-derived cardiomyocytes for modelling QT prolongation.

BACKGROUND: Long-QT syndrome type 2 (LQT2) is a common malignant hereditary arrhythmia. Due to the lack of suitable animal and human models, the pathogenesis of LQT2 caused by human ether-a-go-go-related gene (hERG) deficiency is still unclear. In this study, we generated an hERG-deficient human cardiomyocyte (CM) model that simulates 'human homozygous hERG mutations' to explore the underlying impact of hERG dysfunction and the genotype-phenotype relationship of hERG deficiency. METHODS: The KCNH2 was knocked out in the human embryonic stem cell (hESC) H9 line using the CRISPR/Cas9 system. Using a chemically defined differentiation protocol, we obtained and verified hERG-deficient CMs. Subsequently, high-throughput microelectrode array (MEA) assays and drug interventions were performed to characterise the electrophysiological signatures of hERG-deficient cell lines. RESULTS: Our results showed that KCNH2 knockout did not affect the pluripotency or differentiation efficiency of H9 cells. Using high-throughput MEA assays, we found that the electric field potential duration and action potential duration of hERG-deficient CMs were significantly longer than those of normal CMs. The hERG-deficient lines also exhibited irregular rhythm and some early afterdepolarisations. Moreover, we used the hERG-deficient human CM model to evaluate the potency of agents (nifedipine and magnesium chloride) that may ameliorate the phenotype. CONCLUSIONS: We established an hERG-deficient human CM model that exhibited QT prolongation, irregular rhythm and sensitivity to other ion channel blockers. This model serves as an important tool that can aid in understanding the fundamental impact of hERG dysfunction, elucidate the genotype-phenotype relationship of hERG deficiency and facilitate drug development.

Microscale grooves regulate maturation development of hPSC-CMs by the transient receptor potential channels (TRP channels).

The use of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is limited in drug discovery and cardiac disease mechanism studies due to cell immaturity. Micro-scaled grooves can promote the maturation of cardiomyocytes by aligning them in order, but the mechanism of cardiomyocytes alignment has not been studied. From the level of calcium activity, gene expression and cell morphology, we verified that the W20H5 grooves can effectively promote the maturation of cardiomyocytes. The transient receptor potential channels (TRP channels) also play an important role in the maturation and development of cardiomyocytes. These findings support the engineered hPSC-CMs as a powerful model to study cardiac disease mechanism and partly mimic the myocardial morphological development. The important role of the TRP channels in the maturation and development of myocardium is first revealed.

Knockout of MYOM1 in human cardiomyocytes leads to myocardial atrophy via impairing calcium homeostasis.

Myomesin-1 (encoded by MYOM1 gene) is expressed in almost all cross-striated muscles, whose family (together with myomesin-2 and myomesin-3) helps to cross-link adjacent myosin to form the M-line in myofibrils. However, little is known about its biological function, causal relationship and mechanisms underlying the MYOM1-related myopathies (especially in the heart). Regrettably, there is no MYMO1 knockout model for its study so far. A better and further understanding of MYOM1 biology is urgently needed. Here, we used CRISPR/Cas9 gene-editing technology to establish an MYOM1 knockout human embryonic stem cell line (MYOM1-/- hESC), which was then differentiated into myomesin-1 deficient cardiomyocytes (MYOM1-/- hESC-CMs) in vitro. We found that myomesin-1 plays an important role in sarcomere assembly, contractility regulation and cardiomyocytes development. Moreover, myomesin-1-deficient hESC-CMs can recapitulate myocardial atrophy phenotype in vitro. Based on this model, not only the biological function of MYOM1, but also the aetiology, pathogenesis, and potential treatments of myocardial atrophy caused by myomesin-1 deficiency can be studied.

Generation of a homozygous COX6A2 knockout human embryonic stem cell line (WAe009-A-47) via an epiCRISPR/Cas9 system.

COX6A2 protein is a structural subunit of Complex IV (CIV/Cytochrome c oxidase/COX) in the mitochondrial respiratory chain. It is mainly expressed in the heart and skeletal muscle, also in some interneurons, regulating the assembly and catalytic activity of CIV. Its mutations can lead to COX deficiency, causing human myopathies, and maybe a potential cause of neurological abnormalities. Here, we used the CRISPR/Cas9 editing system to establish a homozygous COX6A2 knockout (COX6A2-KO) human embryonic stem cell (hESC) line. This COX6A2-KO hESC has normal morphology, pluripotency, and karyotype, which can differentiate into three germ layers in vivo.

Ascorbic acid can promote the generation and expansion of neuroepithelial-like stem cells derived from hiPS/ES cells under chemically defined conditions through promoting collagen synthesis.

INTRODUCTION: Spinal cord injury (SCI) is a neurological, medically incurable disorder. Human pluripotent stem cells (hPSCs) have the potential to generate neural stem/progenitor cells (NS/PCs), which hold promise in the treatment of SCI by transplantation. In our study, we aimed to establish a chemically defined culture system using serum-free medium and ascorbic acid (AA) to generate and expand long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) differentiated from hPSCs effectively and stably. METHODS: We induced human embryonic stem cells (hESCs)/induced PSCs (iPSCs) to neurospheres using a newly established in vitro induction system. Moreover, lt-NES cells were derived from hESC/iPSC-neurospheres using two induction systems, i.e., conventional N2 medium with gelatin-coated plates (coated) and N2+AA medium without pre-coated plates (AA), and were characterized by reverse transcription polymerase chain reaction (RT-PCR) analysis and immunocytochemistry staining. Subsequently, lt-NES cells were induced to neurons. A microelectrode array (MEA) recording system was used to evaluate the functionality of the neurons differentiated from lt-NES cells. Finally, the mechanism underlying the induction of lt-NES cells by AA was explored through RNA-seq and the use of inhibitors. RESULTS: HESCs/iPSCs were efficiently induced to neurospheres using a newly established induction system in vitro. lt-NES cells derived from hESC/iPSC-neurospheres using the two induction systems (coated vs. AA) both expressed the neural pluripotency-associated genes PAX6, NESTIN, SOX1, and SOX2. After long-term cultivation, we found that they both exhibited long-term expansion for more than a dozen generations while maintaining neuropluripotency. Moreover, the lt-NES cells retained the ability to differentiate into general functional neurons that express ß-tubulin at high levels. We also demonstrated that AA promotes the generation and long-term expansion of lt-NES cells by promoting collagen synthesis via the MEK-ERK1/2 pathway. CONCLUSIONS: This new chemically defined culture system was stable and effective regarding the generation and culture of lt-NES cells induced from hESCs/iPSCs using serum-free medium combined with AA. The lt-NES cells induced under this culture system maintained their long-term expansion and neural pluripotency, with the potential to differentiate into functional neurons.

Design of synthetic microenvironments to promote the maturation of human pluripotent stem cell derived cardiomyocytes.

Cardiomyocyte like cells derived from human pluripotent stem cells (hPSC-CMs) have a good application perspective in many fields such as disease modeling, drug screening and clinical treatment. However, these are severely hampered by the fact that hPSC-CMs are immature compared to adult human cardiomyocytes. Therefore, many approaches such as genetic manipulation, biochemical factors supplement, mechanical stress, electrical stimulation and three-dimensional culture have been developed to promote the maturation of hPSC-CMs. Recently, establishing in vitro synthetic artificial microenvironments based on the in vivo development program of cardiomyocytes has achieved much attention due to their inherent properties such as stiffness, plasticity, nanotopography and chemical functionality. In this review, the achievements and deficiency of reported synthetic microenvironments that mainly discussed comprehensive biological, chemical, and physical factors, as well as three-dimensional culture were mainly discussed, which have significance to improve the microenvironment design and accelerate the maturation of hPSC-CMs.