Novel codon-optimized mini-intronic plasmid for efficient, inexpensive, and xeno-free induction of pluripotency

The development of human induced pluripotent stem cell (iPSC) technology has revolutionized the regenerative medicine field. This technology provides a powerful tool for disease modeling and drug screening approaches. To circumvent the risk of random integration into the host genome caused by retroviruses, non-integrating reprogramming methods have been developed. However, these techniques are relatively inefficient or expensive. The mini-intronic plasmid (MIP) is an alternative, robust transgene expression vector for reprogramming. Here we developed a single plasmid reprogramming system which carries codon-optimized (Co) sequences of the canonical reprogramming factors (Oct4, Klf4, Sox2, and c-Myc) and short hairpin RNA against p53 ("4-in-1 CoMiP"). We have derived human and mouse iPSC lines from fibroblasts by performing a single transfection. Either independently or together with an additional vector encoding for LIN28, NANOG, and GFP, we were also able to reprogram blood-derived peripheral blood mononuclear cells (PBMCs) into iPSCs. Taken together, the CoMiP system offers a new highly efficient, integration-free, easy to use, and inexpensive methodology for reprogramming. Furthermore, the CoMIP construct is color-labeled, free of any antibiotic selection cassettes, and independent of the requirement for expression of the Epstein-Barr Virus nuclear antigen (EBNA), making it particularly beneficial for future applications in regenerative medicine

A pilot study to investigate the effects of electrical stimulation on recovery of hand function and sensation in subacute stroke patients

Objectives: 1) To compare the effect of cyclic neuromuscular electrical stimulation (NMES) of the forearm and elbow extensor muscles with passive stretching exercises on hand function and sensation following stroke. 2) To inform sample size for a larger randomized controlled trial (RCT).Materials and Methods: Twenty-two subjects with hemiplegia resulting from a stroke during the previous 12 months were randomly allocated into stimulation (treatment) and exercise (control) groups. Stimulation was applied to the elbow, and forearm extensor muscle groups of the hemiplegic arm for 12 weeks. Subjects in the control group were taught passive stretching exercises for the same period. The primary outcome measure was the Action Research Arm test (ARAT). Sensation was tested using two-point discrimination. Statistical analysis applied nonparametric analysis of covariance (ancova).Results: Statistically significant between-group differences in change in ARAT scores were shown between the two groups after 12 weeks of treatment (p = 0.003) and following 12 further weeks without intervention (p = 0.012). There were no significant differences in sensation.Conclusions: 1) A significant treatment effect of electrical stimulation over passive exercise has been demonstrated in a group of 22 subacute stroke patients, randomized into two equal groups and further work identified which may help to improve recovery of hand function and sensation following stroke. 2) A sample size of 24 subjects in each group has been estimated assuming a two-sided test significance level of 5% with 80% power, primary outcome variability SD = 6.75, a minimum difference of ten ARAT score units, and a 10% dropout rate

Modeling cardiovascular diseases with patient-specific human pluripotent stem cell-derived cardiomyocytes

The generation of cardiomyocytes from human induced pluripotent stem cells (hiPSCs) provides a source of cells that accurately recapitulate the human cardiac pathophysiology. The application of these cells allows for modeling of cardiovascular diseases, providing a novel understanding of human disease mechanisms and assessment of therapies. Here, we describe a stepwise protocol developed in our laboratory for the generation of hiPSCs from patients with a specific disease phenotype, long-term hiPSC culture and cryopreservation, differentiation of hiPSCs to cardiomyocytes, and assessment of disease phenotypes. Our protocol combines a number of innovative tools that include a codon-optimized mini intronic plasmid (CoMiP), chemically defined culture conditions to achieve high efficiencies of reprogramming and differentiation, and calcium imaging for assessment of cardiomyocyte phenotypes. Thus, this protocol provides a complete guide to use a patient cohort on a testable cardiomyocyte platform for pharmacological drug assessment

Pravastatin reverses obesity-induced dysfunction of induced pluripotent stem cell-derived endothelial cells via a nitric oxide-dependent mechanism

Aims High-fat diet-induced obesity (DIO) is amajor contributor to type II diabetes and micro-andmacro-vascular complications leading to peripheral vascular disease (PVD). Metabolic abnormalities of induced pluripotent stemcell-derived endothelial cells (iPSC-ECs) fromobese individuals could potentially limit their therapeutic efficacy forPVD. The aimof this studywas to compare the function of iPSC-ECs from normal and DIO mice using comprehensive in vitro and in vivo assays.Methods and results Six-week-old C57Bl/6 micewere fed with a normal or high-fat diet. At 24weeks, iPSCs were generated fromtail tip fibroblasts and differentiated into iPSC-ECs using a directed monolayerapproach. In vitro functional analysis revealed that iPSCECs from DIO mice had significantly decreased capacity to form capillary-like networks, diminished migration, and lower proliferation. Microarray and ELISA confirmed elevated apoptotic, inflammatory, and oxidative stress pathways in DIO iPSC-ECs. Following hindlimb ischaemia, mice receiving intramuscular injections of DIO iPSC-ECs had significantly decreased reperfusion compared with mice injected with control healthy iPSC-ECs. Hindlimb sections revealed increased muscle atrophy and presence of inflammatory cells in mice receiving DIO iPSC-ECs. When pravastatin was co-administered to mice receiving DIO iPSC-ECs, a significant increase in reperfusionwas observed; however, this beneficial effect was blunted by co-administration of the nitric oxide synthase inhibitor, Nv-nitro-L-arginine methyl ester.Conclusion This is the first study to provide evidence that iPSC-ECs from DIO mice exhibit signs of endothelial dysfunction and have suboptimal efficacy following transplantation in a hindlimb ischaemia model. These findings may have important implications for future treatment of PVD using iPSC-ECs in the obese population.Vascular Surger

Chemically defined generation of human cardiomyocytes

Existing methods for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed an optimized cardiac differentiation strategy, using a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate and rice-derived recombinant human albumin. Along with small molecule-based induction of differentiation, this protocol produced contractile sheets of up to 95% TNNT2(+) cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell and was effective in 11 hiPSC lines tested. This chemically defined platform for cardiac specification of hiPSCs will allow the elucidation of cardiomyocyte macromolecular and metabolic requirements and will provide a minimal system for the study of maturation and subtype specification

Characterization of the molecular mechanisms underlying increased ischemic damage in the aldehyde dehydrogenase 2 genetic polymorphism using a human induced pluripotent stem cell model system

Nearly 8% of the human population carries an inactivating point mutation in the gene that encodes the cardioprotective enzyme aldehyde dehydrogenase 2 (ALDH2). This genetic polymorphism (ALDH2*2) is linked to more severe outcomes from ischemic heart damage and an increased risk of coronary artery disease (CAD), but the underlying molecular bases are unknown. We investigated the ALDH2*2 mechanisms in a human model system of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from individuals carrying the most common heterozygous form of the ALDH2*2 genotype. We showed that the ALDH2*2 mutation gave rise to elevated amounts of reactive oxygen species and toxic aldehydes, thereby inducing cell cycle arrest and activation of apoptotic signaling pathways, especially during ischemic injury. We established that ALDH2 controls cell survival decisions by modulating oxidative stress levels and that this regulatory circuitry was dysfunctional in the loss-of-function ALDH2*2 genotype, causing up-regulation of apoptosis in cardiomyocytes after ischemic insult. These results reveal a new function for the metabolic enzyme ALDH2 in modulation of cell survival decisions. Insight into the molecular mechanisms that mediate ALDH2*2-related increased ischemic damage is important for the development of specific diagnostic methods and improved risk management of CAD and may lead to patient-specific cardiac therapies

Human induced pluripotent stem cell-derived cardiomyocytes as an in vitro model for coxsackievirus B3-induced myocarditis and antiviral drug screening platform

RATIONALE: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection. OBJECTIVE: This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. METHODS AND RESULTS: hiPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were used to characterize virally infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferonß1, ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after interferonß1 treatment. CONCLUSIONS: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion

Genetic Mosaicism in Calmodulinopathy.

CaM (calmodulin) mutations are associated with congenital arrhythmia susceptibility (calmodulinopathy) and are most often de novo. In this report, we sought to broaden the genotype-phenotype spectrum of calmodulinopathies with 2 novel calmodulin mutations and to investigate mosaicism in 2 affected families. CaM mutations were identified in 4 independent cases by DNA sequencing. Biochemical and electrophysiological studies were performed to determine functional consequences of each mutation. Genetic studies identified 2 novel CaM variants (CALM3-E141K in 2 cases; CALM1-E141V) and one previously reported CaM pathogenic variant (CALM3-D130G) among 4 probands with shared clinical features of prolonged QTc interval (range 505-725 ms) and documented ventricular arrhythmia. A fatal outcome occurred for 2 of the cases. The parents of all probands were asymptomatic with normal QTc duration. However, 2 of the families had multiple affected offspring or multiple occurrences of intrauterine fetal demise. The mother from the family with recurrent intrauterine fetal demise exhibited the CALM3-E141K mutant allele in 25% of next-generation sequencing reads indicating somatic mosaicism, whereas CALM3-D130G was present in 6% of captured molecules of the paternal DNA sample, also indicating mosaicism. Two novel mutations (E141K and E141V) impaired Ca <sup>2+</sup> binding affinity to the C-domain of CaM. Human-induced pluripotent stem cell-derived cardiomyocytes overexpressing mutant or wild-type CaM showed that both mutants impaired Ca <sup>2+</sup> -dependent inactivation of L-type Ca <sup>2+</sup> channels and prolonged action potential duration. We report 2 families with somatic mosaicism associated with arrhythmogenic calmodulinopathy, and demonstrate dysregulation of L-type Ca <sup>2+</sup> channels by 2 novel CaM mutations affecting the same residue. Parental mosaicism should be suspected in families with unexplained fetal arrhythmia or fetal demise combined with a documented CaM mutation