Posts Tagged: induced pluripotent stem cells

Induced Pluripotent Stem Cells 10 Years Later For Cardiac Applications

Induced Pluripotent Stem Cells 10 Years Later: For Cardiac Applications

Yoshinori Yoshida, Shinya Yamanaka

Factors which possibly cause clonal differences of induced pluripotent stem cells (iPSCs). [Powerpoint File]

Induced Pluripotent Stem Cells 10 Years Later: For Cardiac Applications

Induced Pluripotent Stem Cells 10 Years Later: For Cardiac Applications

Yoshinori Yoshida, Shinya Yamanaka

Patient stratification based on drug responsiveness using induced pluripotent stem cells–derived cardiac myocytes. [Powerpoint File]

Exosomes Generated From iPSC-Derivatives: New Direction for Stem Cell Therapy in Human Heart Diseases

Exosomes Generated From iPSC-Derivatives: New Direction for Stem Cell Therapy in Human Heart Diseases

Ji-Hye Jung, Xuebin Fu, Phillip C. Yang

Biogenesis of exosome. Exosomes are compartmentalized into the multivesicular bodies (MVBs), which fuse with the cell membrane and released to the extracellular space. This process prevents exosomes from degradation by the lysosomes. Exosomes comprise of various transmembrane and cytosolic proteins, such as integrins, CD9, CD63, and CD81. Furthermore, exosomes retain donor cells’ proteins, DNA fragments, miRNAs, and noncoding RNAs within the bilipid membrane. As such, exosomes preserve the genetic information of donor cells from enzymatic degradation while enabling targeted delivery of specific cargo. [Powerpoint File]

Exosomes Generated From iPSC-Derivatives: New Direction for Stem Cell Therapy in Human Heart Diseases

Exosomes Generated From iPSC-Derivatives: New Direction for Stem Cell Therapy in Human Heart Diseases

Ji-Hye Jung, Xuebin Fu, Phillip C. Yang

Exosomes mediated intercellular communication in heart. Exosomes facilitate communication among cardiomyocytes, endothelial cells, and vascular smooth muscle cells in the infarcted area of the heart. Exosomes transfer signaling molecules, such as miRNAs, mRNAs, and proteins to confer paracrine effects on the neighboring cells. In addition, pathological and physiological influence on the heart stimulates exosome secretion. Therefore, cardiac exosomes under pathological conditions could be used as ideal markers for diagnostic tools in the clinic. [Powerpoint FIle]

Exosomes Generated From iPSC-Derivatives: New Direction for Stem Cell Therapy in Human Heart Diseases

Exosomes Generated From iPSC-Derivatives: New Direction for Stem Cell Therapy in Human Heart Diseases

Ji-Hye Jung, Xuebin Fu, Phillip C. Yang

Personalized therapy with the exosomes generated from induced pluripotent stem cell (iPSC)–derived cardiomyocytes (iCMs). Exosomes from patient-specific iPSC derivatives provide a platform for personalized therapy by simulating endogenous repair. Exosomes can be directed to specific injured site of each individual patient to promote salvage of the existing injured cardiac cells. Exosomes isolated from the supernatant of iCM culture undergo ultracentrifugation methods, as well as advanced characterization and quantification by transmission electronical microscopy, Nanosight, and dynamic light scattering. [Powerpoint File]

Mending a Faltering Heart

Mending a Faltering Heart

Mo Li, Juan Carlos Izpisua Belmonte

Recent strategies for cardiomyocyte regeneration. Cardiomyocytes can be induced to re-enter the cell cycle through modulation of endogenous cardiomyocyte proliferation programs. Reprogramming to induced pluripotent stem cell (iPSCs) followed by directed differentiation could provide a large number of immature cardiomyocytes. Other noncardiomyocyte cells, such as fibroblasts, can be directly converted to induced cardiomyocytes (iCMs). These in vitro derived cardiomyocytes may be further matured using tissue engineering technologies. In situ conversion of cardiac fibroblasts also represents a promising strategy for heart repair. MI indicates myocardial infarction. [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

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]

Finding the Rhythm of Sudden Cardiac Death: New Opportunities Using Induced Pluripotent Stem Cell–Derived Cardiomyocytes

Finding the Rhythm of Sudden Cardiac Death: New Opportunities Using Induced Pluripotent Stem Cell–Derived Cardiomyocytes

Karim Sallam*, Yingxin Li*, Philip T. Sager, Steven R. Houser, Joseph C. Wu

Potential role for iPSC-CMs in evaluation of patients with known or at risk for arrhythmic disorders. Clinical genetic testing attempts to identify a rare variant in genes commonly associated with arrhythmic disorders. When a known variant is identified, this may aid the clinical diagnosis and genetic screening can be offered to family members to identify those at risk for developing the disorder. If a variant of uncertain significance (VUS) is identified, genome-edited lines with VUS can be developed, and cellular EP testing can be done on the resultant iPSC-CMs to detect electrophysiological abnormalities and compare them to proband iPSC-CMs. This may help re-characterize the VUS as possibly disease causing and place it in a similar category as a known variant. In cases where EP testing of the VUS is negative or inconclusive or no variants are identified on genetic testing, one can ignore the genetic component and use iPSC-CMs as the cellular functional testing platform (EP study in a dish). iPSC-CMs from the proband would be created and undergo comprehensive EP testing; abnormalities thus identified may be used to aid in the diagnosis and management of the patient. Furthermore, family members could be offered the opportunity to have iPSC-CMs generated and screened for their arrhythmic predisposition. Much like genetic testing, iPSC-CM testing may not identify all arrhythmic abnormalities, and those patients are left to rely on clinical testing and screening (Illustration credit: Ben Smith). [Powerpoint File]

Direct Cardiac Reprogramming: Progress and Challenges in Basic Biology and Clinical Applications

Direct Cardiac Reprogramming: Progress and Challenges in Basic Biology and Clinical Applications

Taketaro Sadahiro, Shinya Yamanaka, Masaki Ieda

Future cardiac regenerative therapy. The lost myocardial tissue is replaced by fibrous tissue that mainly consists of cardiac fibroblasts and extracellular matrix. The cell transplantation–based approach using induced pluripotent stem cell (iPSC)–derived cardiomyocytes is shown (upper). Direct cardiac reprogramming approach may convert endogenous cardiac fibroblasts directly into cardiomyocytes by defined factors in situ (lower). [Powerpoint File]

Perspectives for Induced Pluripotent Stem Cell Technology: New Insights Into Human Physiology Involved in Somatic Mosaicism

Perspectives for Induced Pluripotent Stem Cell Technology: New Insights Into Human Physiology Involved in Somatic Mosaicism

Naoki Nagata, Shinya Yamanaka

Generation of induced pluripotent stem (iPS) cell lines from patient with mosaic Down syndrome (DS) [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]