Posts Tagged: cardiovascular disease modeling

Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes: Insights Into Molecular, Cellular, and Functional Phenotypes

Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes: Insights Into Molecular, Cellular, and Functional Phenotypes

Ioannis Karakikes, Mohamed Ameen, Vittavat Termglinchan, Joseph C. Wu

Expression of key structural and functional genes in induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs). A, Schematic of the major structural and functional features of iPSC-CMs. In adult CMs, on membrane depolarization a small amount of Ca2+ influx induced by activation of voltage-dependent L-type Ca2+ channels (CACNAC1) triggers the release of Ca2+ from the sarcoplasmic reticulum (SR) through the ryanodine receptors (ryanodine receptor 2 [RYR2]), termed Ca2+-induced Ca2+-release mechanism. The released Ca2+ ions diffuse through the cytosolic space and bind to troponin C (TNNC1), resulting in the release of inhibition induced by troponin I (TNNI3), which activates the sliding of thin and thick filaments, and lead to cardiac contraction. Recovery occurs as Ca2+ is extruded by the Na2+/Ca2+ exchanger (NCX1) and returned to the SR by the sarco(endo)plasmic Ca2+-ATPase pumps on the nonjunctional region of the SR that are regulated by phospholamban (PLN). This process is conserved in iPSC-CMs, but major differences exist, such as a nascent SR, the presence of an inositol 1,4,5-trisphosphate–releasable Ca2+ pool, and the complete absence of T-tubules. The genes encoding the major transmembrane ion channels involved in the generation of action potential are also shown. B, Immunofluorescence staining of cardiac troponin T and α-sarcomeric actinin in iPSC-CMs. C, Line-scan images and spontaneous Ca2+ transients in iPSC-CMs. ACTC1 indicates actin, α, cardiac muscle 1; CACNA1C, calcium channel, voltage-dependent, L type, α1C subunit; IPTR3, inositol 1,4,5-trisphosphate receptor, type 3; KCNH2, potassium voltage-gated channel, subfamily H (eag-related), member 2; KCNIP2, potassium channel–interacting protein 2; KCNJ2, potassium inwardly rectifying channel, subfamily J, member 2; KCNQ1, potassium voltage-gated channel, KQT-like subfamily, member 1; MYBPC3, myosin binding protein C; MYH6, myosin, heavy chain 6, α; MYH7, myosin, heavy chain 7, β; MYL2, myosin, light chain 2; MYL3, myosin, light chain 3; SCN5A, sodium channel, voltage-gated, type V, α-subunit; SERCA2, SR Ca2+-ATPase 2; TNNI3, troponin I type 3; TNNT2, troponin T type 2; TTN, titin; and TPM1, tropomyosin 1 (α). [Powerpoint File]

Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes: Insights Into Molecular, Cellular, and Functional Phenotypes

Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes: Insights Into Molecular, Cellular, and Functional Phenotypes

Ioannis Karakikes, Mohamed Ameen, Vittavat Termglinchan, Joseph C. Wu

Current applications of patient-specific induced pluripotent stem cell–derived cardiomyocyte (iPSC-CM) technology. iPSC-CMs have been used for disease modeling of inherited cardiomyopathies and channelopathies, regenerative therapies, drug discovery, and cardiotoxicity testing, as well as for studying metabolic abnormalities and cardiac development. [Powerpoint File]

New and TALENted Genome Engineering Toolbox

New and TALENted Genome Engineering Toolbox

Jarryd M. Campbell*, Katherine A. Hartjes*, Timothy J. Nelson, Xiaolei Xu, Stephen C. Ekker

Genome engineering in induced pluripotent (iPS) cells: modeling the cell-autonomous nature of gene-linked cardiovascular disease. 1, Fibroblasts are derived from skin biopsy of patient with an inherited cardiovascular disease. Nuclear reprogramming generates induced pluripotent stem cells that are genetically engineered via transcription activator-like effector nucleases. 2, Error-prone or programmed gene repair mechanisms followed by in vitro cardiac differentiation yield patient-specific cardiomyocytes with different experimental phenotypes. 3, Engineered cardiomyocytes provide patient-specific insights into the cell-autonomous mechanism of disease. 4a, Gene-corrected cardiomyocytes offer an autologous cellular therapy and (4b) safety profiling screens enable the identification of potential arrhythmogenic pharmaceutical agents. 5, Cellular therapies are delivered back to the patient to complete the individualized medicine loop. [Powerpoint File]

New and TALENted Genome Engineering Toolbox

New and TALENted Genome Engineering Toolbox

Jarryd M. Campbell*, Katherine A. Hartjes*, Timothy J. Nelson, Xiaolei Xu, Stephen C. Ekker

Genome engineering in zebrafish: modeling the physiology of gene-linked cardiovascular disease. 1, Whole-genome sequencing approaches reveal genetic mutations underlying cardiac disease in an individual patient. Identification of zebrafish orthologs enables transcription activator-like effector nuclease-based gene modification in zebrafish to study the patient-specific defect. 2, Error-prone or programmed gene repair mechanisms facilitate the generation of adult zebrafish with different experimental phenotypes. 3, Engineered zebrafish models provide patient-specific insights into the physiological mechanism of disease. 4, Drug screens offer the potential to identify novel therapeutic agents. 5, Pharmaceuticals are then tested and eventually delivered back to the patient to complete the individualized medicine loop. [Powerpoint File]

New and TALENted Genome Engineering Toolbox

New and TALENted Genome Engineering Toolbox

Jarryd M. Campbell*, Katherine A. Hartjes*, Timothy J. Nelson, Xiaolei Xu, Stephen C. Ekker

Classes of transcription activator-like (TAL) repeat assembly methodologies. Top, Shows a plasmid-based restriction digest and ligation method that moves TAL repeat monomers (in blue, red, yellow, and green) in a predetermined order from one plasmid to a receiver plasmid by one round of restriction digest and ligation, and then into the destination TAL backbone by another round. The second panel from the top shows a PCR-based restriction digest and ligation method that is conceptually similar to a plasmid-based assembly, however a PCR amplicon replaces whole plasmids as the starting material, and PCR amplification can replace bacterial plasmid propagation. The restriction sites necessary for the digest and ligation reactions are built into the primer design. The next panel outlines a ligation-free assembly method, in which T4 DNA polymerase activity in the presence of dNTPs chews back free double-stranded DNA ends to make long single-stranded overhangs. These overhangs anneal efficiently, and bacteria ligate together the DNA backbone if a bacteria origin of replication (grey) and antibiotic resistance gene (black) are included in the reaction. This method eliminates the need for in vitro ligation. Bottom, Demonstrates how solid-state technology can facilitate high-throughput TAL repeat assembly. A biotinylated repeat monomer is first tethered to a plate coated with streptavidin beads, and then monomers are sequentially added until the desired repeat length is achieved. The final assembly is cloned into the final TAL scaffold. TALEN indicates transcription activator-like effector nuclease. [Powerpoint File]

New and TALENted Genome Engineering Toolbox

New and TALENted Genome Engineering Toolbox

Jarryd M. Campbell*, Katherine A. Hartjes*, Timothy J. Nelson, Xiaolei Xu, Stephen C. Ekker

Overview of transcription activator-like effector nuclease (TALEN) genome engineering. Left and right TALEN arms are directed to a specific sequence of genomic DNA by the repeat variable di-residue (RVD) repeats comprising the DNA binding domain. Specificity is achieved by the one-to-one interactions between the RVDs and nucleotides, as well as a 5′T that is recognized by the N-terminus of the TAL protein. The length of the TALEN arms and spacer region contribute to TALEN activity; here is shown a 15-15-15 (left arm-spacer-right arm) design that has been shown to be effective.25 The C-terminus domain brings the FokI obligate heterodimer (or homodimer) endonuclease in proximity to dimerize. The resulting double-strand break (DSB) is repaired by either error-prone nonhomologous end joining (NHEJ) or programmed gene repair by homologous recombination (HR) or homology-directed repair (HDR) facilitated by an exogenous DNA donor. Repair by NHEJ causes unpredictable insertions/deletions (indels) at the cut site, which can lead to protein loss-of-function. Repair by HR requires long, double-stranded DNA donors that can be used to direct large changes in the genome, such as the fluorescent labeling of an endogenous protein by GFP. Conversely, HDR using short, single-stranded oligonucleotides can be used to make small, subtle changes like a single nucleotide polymorphism (SNP). NLS indicates nuclear localization sequence. [Powerpoint File]