Here I discovered the Department of Pharmacology and the enriching environment within it, which provided me with a foundation in receptor theory (Dr. Seva Gurevich and Dr. Terry Kanakin), pharmacokinetics and drug metabolism (Dr. Scott Daniels). They made the rest of my studies more enriching than I could ever have imagined. Dan Roden, Eric Delpire, Charles Hong, and David Weaver for providing guidance and support during my transition to a new laboratory and facilitating the completion of my graduate studies.
25 Table 2: Contractile kinetics of single LMNA DCM hiPSC-CM ..60 Table 3: Calcium kinetics of single LMNA DCM hiPSC-CM. 62 Table 4: Impedance measurements of LMNA DCM compared to population control hiPSC-CM in monolayer ..67 Table 5: Impedance measurements of LMNA DCM compared to isogenic control hiPSC-CM.
Overview
In this introductory chapter, I first review the hiPSC technology and its current applications relevant to cardiomyocytes (CM) and heart disease. The current limitations of existing hiPSC-CM and subsequent implications that reduce their implementation are also discussed. Next, the cardiac physiology relevant to the use of hiPSC-CM is reviewed, followed by a brief overview of dilated cardiomyopathy (DCM) and the use of hiPSC-CM to study it.
Human Induced Pluripotent Stem Cells
The reprogramming of somatic cells into a pluripotent state enables unlimited self-renewal and the possibility of differentiation of three lineages (endoderm, ectoderm and mesoderm). Concerns about the safety of genomic integration and potential tumorigenicity have led to the development of non-integrative methods ranging from the use of recombinant proteins27 to small molecule approaches28 (Figure 1). The generation of hiPSCs from various cell types is becoming a routine laboratory protocol; however, there are concerns about the quality of hiPSCs and.
The current consensus is that proper characterization of hiPSC should include validation of pluripotency as evidenced by self-renewal and expression of associated markers, tri-lineage differentiation capacity, genomic stability via karyotyping and recently proposed assessment of epigenetic stability30. With the rapid adoption of hiPSC technology and the initiation of large-scale banking of disease and gene-edited hiPSC lines, its application in biomedical research is on an upward trajectory.
Gene Editing in hiPSC
Methodologies for gene editing include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9)31, 32. The workflow for using CRISPR/Cas9 technology for gene editing of hiPSC as performed in this thesis is described in Figure 3. A plasmid-based approach is used to deliver gene editing machinery for expression of the Cas9 and the sgRNA in the hiPSC.
Depending on the goal of gene modification (knockout or knockin), either the plasmid alone or the plasmid with a repair template in the form of an ssODN is electroporated into hiPSCs. Since the low efficiency of HDR is a major limitation of the use of CRISPR/Cas9-based gene editing in hiPSCs, methods to increase the efficiency of HDR are needed to advance the use of this technology.
Primary applications of hiPSC-CM and limitations
Although it is a multi-component pipeline combining in vitro and in silico evaluation, it depends on the successful implementation of hiPSC-CM to evaluate the proarrhythmic risk of new compounds. Standardization of hiPSC-CM production and methodology for high-throughput evaluation is critical to the successful implementation of CIPA. For example, contractile measurements were limited by the lack of cellular shortening of hiPSC-CMs.
Improvements in devices that allow for high-throughput evaluation of electrophysiological and contractile properties of hiPSC-CM have been critical to this progress66, 67. Overall, hiPSC-CM technology is growing rapidly and is increasingly being used by academia and the industry adopted.
Cardiac Excitation-Contraction Coupling
Rapid repolarization of the cell occurs by closing the LTCC and opening outward potassium channels (mainly those responsible for IKr and IKs, phase 3). Membrane depolarization activates the L-type calcium channel (LTCC), which allows calcium to enter the immediate vicinity of the ryanodine receptor (RyR2) on the sarcoplasmic reticulum (SR). Note the presence of junctophilin 2 (JPH2), bridging integrator-1 (Bin1), and caveolin 3 (Cav3) at the interface of the LTCC and SR.
These factors are critical for invagination of the plasma membrane such that the LTCC and SR are in close proximity to each other, enabling efficient calcium-induced calcium release (CICR). The location of the LTCC in the t-tubules to RyR2 on the SR is essential for efficient CICR, thus demonstrating the necessity of this structural arrangement.
Structural and Functional Characteristics of HiPSC-CM
Inefficient CICR, slow calcium handling, and electrical immaturity of hiPSC-CM are major limitations of hiPSC-CM that may be contributing factors. Although hiPSC-CM exhibits calcium transients, the rise and fall of calcium is significantly slower than that of the adult human-like CM82. HiPSC-CM demonstrate comparable expression of LTCC to that of the adult human ventricular CM85, and patch clamp techniques show robust LTCC current similar to that of the adult mouse CM82.
Efficient CICR is dependent on adequate expression and organization of RyR2 (clustering and appropriate localization to the z-line) as well as juxtaposition of the LTCC to the RyR2, all of which are poorly developed in the hiPSC-CM. Specifically, insufficient localization of RyR2 at the z-line and/or lack of t-tubules are important structural determinants of the functional deficiencies observed in the hiPSC-CM.
T- tubules and hiPSC-CM
HiPSC-CM also have minimal IK, which when combined with deficient IK1 leaves only one major source of repolarization, IKr. Inefficient CICR, slow calcium kinetics, and electrical immaturity are all properties that can be explained by the lack of adult-like myocyte structure in hiPSC-CM. Finally, hiPSC-CM showed a lack of co-localization of LTCCs and RyR2, where LTCCs were diffusely distributed throughout the cell and RyR2 was predominantly perinuclear, consistent with a lack of dyad formation.
Also consistent with the absence of t-tubules is the minimal IK1, which likely contributes to the higher resting membrane potential observed in hiPSC-CM and potentially. Current techniques for hiPSC-CM differentiation have failed to promote t-tubule development and result in inefficient CICR.
Dilated Cardiomyopathy
Because of the anticipated value of this tool, research and development of hiPSC-CMs has grown by leaps and bounds. Calcium decay rates were significantly prolonged in LMNA DCM hiPSC-CMs compared to control (Figure 16D,E). Cardioexcyte96 (Nanion), a high-throughput platform that allows impedance measurements under physiological conditions (37 °C and 60 beats/min), was used to investigate the contractile properties of hiPSC-CM monolayers.
HiPSC-CM from three independent population control lines (Ctrl 1, Ctrl 2 and Ctrl 3) and two LMNA DCM proband lines (LMNA DCM 1, LMNA DCM 2) were generated. Here, LMNA DCM hiPSC-CM were aged for 5 days on Matrigel mattress, allowing cellular shortening (probably increasing activation of mechanotransduction pathways). Because of the severe contractile defects identified clinically, we assessed the contractility of patient-derived LMNA DCM hiPSC-CM.
Combinatorial approaches for hiPSC-CM maturation have not been extensively tested in the literature. Immunostaining: HiPSC-CM was grown on Matrigel mattress-lined cell culture chambers (Falcon 354104) and fixed with 4% paraformaldehyde (Thermo #28906) for 10 min at room temperature.
Summary and Future directions
The advent of gene editing tools and progress in hiPSC-CM technology offers the scientific community an opportunity to interrogate specific genetic changes in a manner specific to human cardiomyocytes. 123, most studies summarize only a few aspects of DCM disease pathogenesis with little or no mechanistic insight. For some mutation-causing DCMs, the most clinically relevant aspects (contractile deficits and pro-arrhythmic features) cannot be identified using hiPSC-CM models.
Using CRISPR/Cas9 gene editing, I corrected the mutation and demonstrated restoration of lamin A/C expression and normalization of the contractile deficits. Furthermore, correction of the predicted pathogenic mutation with gene editing may not provide complete rescue if multiple mutations contribute to the pathogenicity. My studies were largely complete, comparing patient-derived CM with that derived from a population control.
Although the CardioExyte96 allows evaluation of the extracellular field potential to produce an action potential reading (field potential duration [FPD]), early versions of the model allowed only spontaneous measurements. Because LMNA hiPSC-CM appeared to have a bradycardial phenotype, these cells cannot be accurately assessed for such properties. Using hiPSC-CM expressing a light-sensitive cationic channel (Channelrhodopsin-2), cells can be stimulated in... Optical stimulation offers three major advantages: 1) increases the number of measurable wells (Figure 37A), 2) allows synchronous activation of the monolayer (Figure 37B), and 3) allows control of heart rate via stimulation.
Interestingly, LMNA DCM hiPSC-CM plated on plastic or glass culture dishes did not show contractile deficits or nuclear abnormalities, whereas the use of a substrate of physiological stiffness began to unmask the phenotype. However, the single cell contractile phenotype observed is not as dramatic as in other hiPSC-CM models of sarcomeric DCM115. Maturation on a substrate that enables cellular shortening and thus activates mechanosensitive pathways appears to promote the unfolding of the phenotype, as does prolonged culture in monolayer.
Summary and Future directions
Spontaneously beating hiPSC-CM do not all demonstrate measurable signal, however implementation of optical pacing greatly facilitates waveform detection and thus data collection
Optically paced hiPSC-CM in monolayer demonstrate uniform depolarization evident by delayed depolarization on the left and rapid depolarization on the right. Data collected and
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IK1-enhanced human-induced pluripotent stem cell-derived cardiomyocytes: an improved cardiomyocyte model to investigate inherited arrhythmia syndromes. A myosin activator enhances actin assembly and sarcomere function of human induced pluripotent stem cell-derived cardiomyocytes with a troponin T point mutation. Modeling structural and functional defects in RBM20 familial dilated cardiomyopathy using human induced pluripotent stem cells.
Thyroid and glucocorticoid hormones promote functional T-tubule development in human induced pluripotent cardiomyocytes. Functional maturation of human pluripotent stem cell-derived cardiomyocytes in vitro - correlation between contraction force and electrophysiology. Identification and characterization of calcium sparks in cardiomyocytes derived from human induced pluripotent stem cells.
