ABSTRACT
AIMS: Human embryonic stem cells (hESCs) can be used to generate scalable numbers of cardiomyocytes (CMs) for studying cardiac biology, disease modelling, drug screens, and potentially for regenerative therapies. A fluorescence-based reporter line will significantly enhance our capacities to visualize the derivation, survival, and function of hESC-derived CMs. Our goal was to develop a reporter cell line for real-time monitoring of live hESC-derived CMs. METHODS AND RESULTS: We used CRISPR/Cas9 to knock a mCherry reporter gene into the MYH6 locus of hESC lines, H1 and H9, enabling real-time monitoring of the generation of CMs. MYH6:mCherry+ cells express atrial or ventricular markers and display a range of cardiomyocyte action potential morphologies. At 20 days of differentiation, MYH6:mCherry+ cells show features characteristic of human CMs and can be used successfully to monitor drug-induced cardiotoxicity and oleic acid-induced cardiac arrhythmia. CONCLUSION: We created two MYH6:mCherry hESC reporter lines and documented the application of these lines for disease modelling relevant to cardiomyocyte biology.
Subject(s)
Arrhythmias, Cardiac/chemically induced , Cell Differentiation , Doxorubicin/toxicity , Heart Diseases/chemically induced , Human Embryonic Stem Cells/drug effects , Myocytes, Cardiac/drug effects , Oleic Acid/toxicity , Action Potentials/drug effects , Arrhythmias, Cardiac/metabolism , Arrhythmias, Cardiac/physiopathology , Biomarkers/metabolism , CRISPR-Cas Systems , Cardiac Myosins/genetics , Cardiotoxicity , Cell Line , Gene Knock-In Techniques , Genes, Reporter , Heart Diseases/genetics , Heart Diseases/metabolism , Heart Diseases/pathology , Human Embryonic Stem Cells/metabolism , Human Embryonic Stem Cells/pathology , Humans , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Myosin Heavy Chains/genetics , Time Factors , Red Fluorescent ProteinABSTRACT
Chemically modified RNA (cmRNA) has potential as a safe and efficient tool for nucleic acid-based therapies and regenerative medicine. Modifications in the chemistry of mRNA can enhance stability, reduce immunogenicity, and thus facilitate mRNA-based nucleic acid therapy, which eliminates risk of insertional mutagenesis. In addition to these valuable advantages, the mRNA-based method showed significantly higher efficacy for reprogramming somatic cells to pluripotency compared with DNA- or protein-based methods. These findings suggest cmRNA can provide a powerful and safe tool for cell programming and reprogramming. Delivery methods, particularly using lipid nanoparticles, provide strategies for cell and organ-specific targeting. The present study comprehensively compares studies that have used cmRNAs for cell fate conversion and tissue engineering. The information should be useful for investigators looking to choose the most efficient and straightforward cmRNA-based strategy and protocol for tissue engineering and regenerative medicine research. Stem Cells Translational Medicine 2019;8:833&843.
Subject(s)
Cellular Reprogramming Techniques/methods , RNA Processing, Post-Transcriptional , RNA, Messenger/chemistry , Tissue Engineering/methods , Animals , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/transplantation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Regenerative Medicine/methodsABSTRACT
Cancer has been ranked as the second leading cause of death in the United States. To reduce cancer mortality, immunotherapy is gaining momentum among other therapeutic modalities, due to its impressive results in clinical trials. The genetically engineered T cells expressing chimeric antigen receptors (CARs) are emerging as a new approach in cancer immunotherapy, with the most successful outcomes in the refractory/relapse hematologic malignancies. However, the widespread clinical applications are limited by adverse effects some of which are life-threatening. Strategies to reduce the chance of side effects as well as close monitoring, rapid diagnosis and proper treatment of side effects are necessary to take the most advantages of this valuable therapy. Here we review the reported toxicities associated with CAR engineered T cells, the strategies to ameliorate the toxicity, and further techniques and designs leading to a safer CAR T-cell therapy.
Subject(s)
Cancer Vaccines/immunology , Hematologic Neoplasms/therapy , Immunotherapy, Adoptive/methods , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes/physiology , Animals , Antigens, Neoplasm/immunology , Drug-Related Side Effects and Adverse Reactions , Genetic Engineering , Hematologic Neoplasms/immunology , Humans , Neoplasm Recurrence, Local , T-Cell Antigen Receptor Specificity , T-Lymphocytes/transplantationSubject(s)
Acyclovir/therapeutic use , Antiviral Agents/therapeutic use , Herpes Simplex/drug therapy , Rosacea/drug therapy , Acyclovir/pharmacology , Adult , Anti-Bacterial Agents/therapeutic use , Antiviral Agents/pharmacology , Face , Female , Herpes Simplex/virology , Herpesvirus 1, Human/isolation & purification , Humans , Indoleamine-Pyrrole 2,3,-Dioxygenase/antagonists & inhibitors , Nitric Oxide Synthase Type II/antagonists & inhibitors , Rosacea/diagnosis , Rosacea/etiology , Symptom Flare Up , Toll-Like Receptor 2/antagonists & inhibitors , Treatment Outcome , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitorsABSTRACT
Recently, chemically modified mRNA (cmRNA) therapeutics have been the subject of extensive application-oriented research in both academia and industry as a safer alternative for gene and recombinant protein therapies. However, the lack of an efficient delivery system hinders widespread application. Here we used â¼100-nm lipoplexes and magnetic lipoplexes that can protect cmRNA from RNases and efficiently deliver it into muscle and fat tissues as well as to the endothelium of the carotid artery. Establishing magnetofection for ex vivo cmRNA delivery for the first time, we suggest this method for potential enhanced and targeted delivery of cmRNA. This study introduces optimal cmRNA complexes with high ex vivo efficiency as good candidates for further in vivo cmRNA delivery.
Subject(s)
Lipids/chemistry , Magnetics/methods , Magnetite Nanoparticles/chemistry , RNA, Messenger/administration & dosage , RNA, Messenger/chemistry , Transfection/methods , Adipose Tissue/metabolism , Animals , Endothelial Cells/metabolism , Liposomes/chemistry , Mice , Muscles/metabolism , NIH 3T3 Cells , RNA, Messenger/genetics , Sheep , SwineABSTRACT
Modified nucleotide chemistries that increase the half-life (T1/2) of transfected recombinant mRNA and the use of non-native 5'- and 3'-untranslated region (UTR) sequences that enhance protein translation are advancing the prospects of transcript therapy. To this end, a set of UTR sequences that are present in mRNAs with long cellular T1/2 were synthesized and cloned as five different recombinant sequence set combinations as upstream 5'-UTR and/or downstream 3'-UTR regions flanking a reporter gene. Initial screening in two different cell systems in vitro revealed that cytochrome b-245 alpha chain (CYBA) combinations performed the best among all other UTR combinations and were characterized in detail. The presence or absence of CYBA UTRs had no impact on the mRNA stability of transfected mRNAs, but appeared to enhance the productivity of transfected transcripts based on the measurement of mRNA and protein levels in cells. When CYBA UTRs were fused to human bone morphogenetic protein 2 (hBMP2) coding sequence, the recombinant mRNA transcripts upon transfection produced higher levels of protein as compared to control transcripts. Moreover, transfection of human adipose mesenchymal stem cells with recombinant hBMP2-CYBA UTR transcripts induced bone differentiation demonstrating the osteogenic and therapeutic potential for transcript therapy based on hybrid UTR designs.
Subject(s)
NADPH Oxidases/genetics , RNA, Messenger/metabolism , 3' Untranslated Regions , 5' Untranslated Regions , A549 Cells , Adipose Tissue/cytology , Animals , Area Under Curve , Bone Morphogenetic Protein 2/genetics , Bone Morphogenetic Protein 2/metabolism , Genes, Reporter , Half-Life , Humans , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Mice , NADPH Oxidases/metabolism , NIH 3T3 Cells , Osteogenesis , Protein Biosynthesis , RNA Stability , ROC Curve , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , TransfectionABSTRACT
Transcript therapies using chemically modified messenger RNAs (cmRNAs) are emerging as safe and promising alternatives for gene and recombinant protein therapies. However, their applications have been limited due to transient translation and relatively low stability of cmRNAs compared to DNA. Here we show that vacuum-dried cmRNA-loaded collagen sponges, termed transcript activated matrices (TAMs), can serve as depots for sustained delivery of cmRNA. TAMs provide steady state protein production for up to six days, and substantial residual expression until 11days post transfection. Another advantage of this technology was nearly 100% transfection efficiency as well as low toxicity in vitro. TAMs were stable for at least 6months at room temperature. Human BMP-2-encoding TAMs induced osteogenic differentiation of MC3T3-E1 cells in vitro and bone regeneration in a non-critical rat femoral bone defect model in vivo. In summary, TAMs are a promising tool for bone regeneration and potentially also for other applications in regenerative medicine and tissue engineering.
