Platform for the interdisciplinary study of cardiovascular, metabolic and neurovascular diseases (PICMAN) protocol.

Through extensive multisystem phenotyping, the central aim of Project PICMAN is to correlate metabolic flexibility to measures of cardiometabolic health, including myocardial diastolic dysfunction, coronary and cerebral atherosclerosis, body fat distribution and severity of non-alcoholic fatty liver disease. This cohort will form the basis of larger interventional trials targeting metabolic inflexibility in the prevention of cardiovascular disease. Participants aged 21-72 years with no prior manifest atherosclerotic cardiovascular disease (ASCVD) are being recruited from a preventive cardiology clinic and an existing cohort of non-alcoholic fatty liver disease (NAFLD) in an academic medical centre. A total of 120 patients will be recruited in the pilot phase of this study and followed up for 5 years. Those with 10-year ASCVD risk ≥ 5% as per the QRISK3 calculator are eligible. Those with established diabetes mellitus are excluded. Participants recruited undergo a detailed assessment of health behaviours and physical measurements. Participants also undergo a series of multimodality clinical phenotyping comprising cardiac tests, vascular assessments, metabolic tests, liver and neurovascular testing. Blood samples are also being collected and banked for plasma biomarkers, 'multi-omics analyses' and for generation of induced pluripotent stem cells (iPSC). Extensive evidence points to metabolic dysregulation as an early precursor of cardiovascular disease, particularly in Asia. We hypothesise that quantifiable metabolic inflexibility may be representative of an individual in his/her silent, but high-risk progression towards insulin resistance, diabetes and cardiovascular disease. The platform for interdisciplinary cardiovascular-metabolic-neurovascular diseases (PICMAN) is a pilot, prospective, multi-ethnic cohort study.

Pluripotent stem cell-derived committed cardiac progenitors remuscularize damaged ischemic hearts and improve their function in pigs.

Ischemic heart disease, which is often associated with irreversibly damaged heart muscle, is a major global health burden. Here, we report the potential of stem cell-derived committed cardiac progenitors (CCPs) have in regenerative cardiology. Human pluripotent embryonic stem cells were differentiated to CCPs on a laminin 521 + 221 matrix, characterized with bulk and single-cell RNA sequencing, and transplanted into infarcted pig hearts. CCPs differentiated for eleven days expressed a set of genes showing higher expression than cells differentiated for seven days. Functional heart studies revealed significant improvement in left ventricular ejection fraction at four and twelve weeks following transplantation. We also observed significant improvements in ventricular wall thickness and a reduction in infarction size after CCP transplantation (p-value < 0.05). Immunohistology analyses revealed in vivo maturation of the CCPs into cardiomyocytes (CM). We observed temporary episodes of ventricular tachyarrhythmia (VT) in four pigs and persistent VT in one pig, but the remaining five pigs exhibited normal sinus rhythm. Importantly, all pigs survived without the formation of any tumors or VT-related abnormalities. We conclude that pluripotent stem cell-derived CCPs constitute a promising possibility for myocardial infarction treatment and that they may positively impact regenerative cardiology.

Role of cardiac mitofusins in cardiac conduction following simulated ischemia-reperfusion.

Mitochondrial dysfunction induced by acute cardiac ischemia-reperfusion (IR), may increase susceptibility to arrhythmias by perturbing energetics, oxidative stress production and calcium homeostasis. Although changes in mitochondrial morphology are known to impact on mitochondrial function, their role in cardiac arrhythmogenesis is not known. To assess action potential duration (APD) in cardiomyocytes from the Mitofusins-1/2 (Mfn1/Mfn2)-double-knockout (Mfn-DKO) compared to wild-type (WT) mice, optical-electrophysiology was conducted. To measure conduction velocity (CV) in atrial and ventricular tissue from the Mfn-DKO and WT mice, at both baseline and following simulated acute IR, multi-electrode array (MEA) was employed. Intracellular localization of connexin-43 (Cx43) at baseline was evaluated by immunohistochemistry, while Cx-43 phosphorylation was assessed by Western-blotting. Mfn-DKO cardiomyocytes demonstrated an increased APD. At baseline, CV was significantly lower in the left ventricle of the Mfn-DKO mice. CV decreased with simulated-ischemia and returned to baseline levels during simulated-reperfusion in WT but not in atria of Mfn-DKO mice. Mfn-DKO hearts displayed increased Cx43 lateralization, although phosphorylation of Cx43 at Ser-368 did not differ. In summary, Mfn-DKO mice have increased APD and reduced CV at baseline and impaired alterations in CV following cardiac IR. These findings were associated with increased Cx43 lateralization, suggesting that the mitofusins may impact on post-MI cardiac-arrhythmogenesis.

Magnetic fields modulate metabolism and gut microbiome in correlation with Pgc-1α expression: Follow-up to an in vitro magnetic mitohormetic study.

Exercise modulates metabolism and the gut microbiome. Brief exposure to low mT-range pulsing electromagnetic fields (PEMFs) was previously shown to accentuate in vitro myogenesis and mitochondriogenesis by activating a calcium-mitochondrial axis upstream of PGC-1α transcriptional upregulation, recapitulating a genetic response implicated in exercise-induced metabolic adaptations. We compared the effects of analogous PEMF exposure (1.5 mT, 10 min/week), with and without exercise, on systemic metabolism and gut microbiome in four groups of mice: (a) no intervention; (b) PEMF treatment; (c) exercise; (d) exercise and PEMF treatment. The combination of PEMFs and exercise for 6 weeks enhanced running performance and upregulated muscular and adipose Pgc-1α transcript levels, whereas exercise alone was incapable of elevating Pgc-1α levels. The gut microbiome Firmicutes/Bacteroidetes ratio decreased with exercise and PEMF exposure, alone or in combination, which has been associated in published studies with an increase in lean body mass. After 2 months, brief PEMF treatment alone increased Pgc-1α and mitohormetic gene expression and after >4 months PEMF treatment alone enhanced oxidative muscle expression, fatty acid oxidation, and reduced insulin levels. Hence, short-term PEMF treatment was sufficient to instigate PGC-1α-associated transcriptional cascades governing systemic mitohormetic adaptations, whereas longer-term PEMF treatment was capable of inducing related metabolic adaptations independently of exercise.

Selection of human induced pluripotent stem cells lines optimization of cardiomyocytes differentiation in an integrated suspension microcarrier bioreactor.

BACKGROUND: The production of large quantities of cardiomyocyte is essential for the needs of cellular therapies. This study describes the selection of a human-induced pluripotent cell (hiPSC) line suitable for production of cardiomyocytes in a fully integrated bioprocess of stem cell expansion and differentiation in microcarrier stirred tank reactor. METHODS: Five hiPSC lines were evaluated first for their cardiac differentiation efficiency in monolayer cultures followed by their expansion and differentiation compatibility in microcarrier (MC) cultures under continuous stirring conditions. RESULTS: Three cell lines were highly cardiogenic but only one (FR202) of them was successfully expanded on continuous stirring MC cultures. FR202 was thus selected for cardiac differentiation in a 22-day integrated bioprocess under continuous stirring in a stirred tank bioreactor. In summary, we integrated a MC-based hiPSC expansion (phase 1), CHIR99021-induced cardiomyocyte differentiation step (phase 2), purification using the lactate-based treatment (phase 3) and cell recovery step (phase 4) into one process in one bioreactor, under restricted oxygen control (< 30% DO) and continuous stirring with periodic batch-type media exchanges. High density of undifferentiated hiPSC (2 ± 0.4 × 106 cells/mL) was achieved in the expansion phase. By controlling the stirring speed and DO levels in the bioreactor cultures, 7.36 ± 1.2 × 106 cells/mL cardiomyocytes with > 80% Troponin T were generated in the CHIR99021-induced differentiation phase. By adding lactate in glucose-free purification media, the purity of cardiomyocytes was enhanced (> 90% Troponin T), with minor cell loss as indicated by the increase in sub-G1 phase and the decrease of aggregate sizes. Lastly, we found that the recovery period is important for generating purer and functional cardiomyocytes (> 96% Troponin T). Three independent runs in a 300-ml working volume confirmed the robustness of this process. CONCLUSION: A streamlined and controllable platform for large quantity manufacturing of pure functional atrial, ventricular and nodal cardiomyocytes on MCs in conventional-type stirred tank bioreactors was established, which can be further scaled up and translated to a good manufacturing practice-compliant production process, to fulfill the quantity requirements of the cellular therapeutic industry.

Troponin destabilization impairs sarcomere-cytoskeleton interactions in iPSC-derived cardiomyocytes from dilated cardiomyopathy patients.

The sarcomeric troponin-tropomyosin complex is a critical mediator of excitation-contraction coupling, sarcomeric stability and force generation. We previously reported that induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from patients with a dilated cardiomyopathy (DCM) mutation, troponin T (TnT)-R173W, display sarcomere protein misalignment and impaired contractility. Yet it is not known how TnT mutation causes dysfunction of sarcomere microdomains and how these events contribute to misalignment of sarcomeric proteins in presence of DCM TnT-R173W. Using a human iPSC-CM model combined with CRISPR/Cas9-engineered isogenic controls, we uncovered that TnT-R173W destabilizes molecular interactions of troponin with tropomyosin, and limits binding of PKA to local sarcomere microdomains. This attenuates troponin phosphorylation and dysregulates local sarcomeric microdomains in DCM iPSC-CMs. Disrupted microdomain signaling impairs MYH7-mediated, AMPK-dependent sarcomere-cytoskeleton filament interactions and plasma membrane attachment. Small molecule-based activation of AMPK can restore TnT microdomain interactions, and partially recovers sarcomere protein misalignment as well as impaired contractility in DCM TnT-R173W iPSC-CMs. Our findings suggest a novel therapeutic direction targeting sarcomere- cytoskeleton interactions to induce sarcomere re-organization and contractile recovery in DCM.

Optogenetic Monitoring of the Glutathione Redox State in Engineered Human Myocardium.

Redox signaling affects all aspects of cardiac function and homeostasis. With the development of genetically encoded fluorescent redox sensors, novel tools for the optogenetic investigation of redox signaling have emerged. Here, we sought to develop a human heart muscle model for in-tissue imaging of redox alterations. For this, we made use of (1) the genetically-encoded Grx1-roGFP2 sensor, which reports changes in cellular glutathione redox status (GSH/GSSG), (2) human embryonic stem cells (HES2), and (3) the engineered heart muscle (EHM) technology. We first generated HES2 lines expressing Grx1-roGFP2 in cytosol or mitochondria compartments by TALEN-guided genomic integration. Grx1-roGFP2 sensor localization and function was verified by fluorescence imaging. Grx1-roGFP2 HES2 were then subjected to directed differentiation to obtain high purity cardiomyocyte populations. Despite being able to report glutathione redox potential from cytosol and mitochondria, we observed dysfunctional sarcomerogenesis in Grx1-roGFP2 expressing cardiomyocytes. Conversely, lentiviral transduction of Grx1-roGFP2 in already differentiated HES2-cardiomyocytes and human foreskin fibroblast was possible, without compromising cell function as determined in EHM from defined Grx1-roGFP2-expressing cardiomyocyte and fibroblast populations. Finally, cell-type specific GSH/GSSG imaging was demonstrated in EHM. Collectively, our observations suggests a crucial role for redox signaling in cardiomyocyte differentiation and provide a solution as to how this apparent limitation can be overcome to enable cell-type specific GSH/GSSG imaging in a human heart muscle context.

Collagen-based engineered heart muscle.

Cardiac muscle engineering has evolved over nearly 20 years from a scientific oddity to a mainstream technology with a wide range of applications. Of the many published methods it appears that hydrogels constitute the preferred scaffolds for myocardial tissue engineering and support of organotypic development. Here we describe a simple and highly robust protocol for the generation of engineered heart muscle using a collagen-based hydrogel method.

Cardiac differentiation of human embryonic stem cells and their assembly into engineered heart muscle.

The advent of pluripotent human embryonic stem cells has created the unique opportunity for the development of a wide variety of humanized cellular tools for basic research, as well as industrial and clinical applications. It has, however, become apparent that embryonic stem cell derivatives in classical monolayer or embryoid body culture do not resemble bona fide tissues, mainly because of their limited organotypic organization and maturation in these culture formats. This shortcoming may be addressed by tissue engineering technologies aiming at the provision of a "natural" growth environment to facilitate organotypic tissue assembly. In this unit, we provide two harmonized basic protocols for (1) cardiac differentiation of human embryonic stem cells under serum-free conditions and (2) the assembly of the stem cell-derived cardiomyocytes into engineered heart muscle. This protocol can be easily adapted to bioengineer heart muscle also from other stem cell-derived cardiomyocytes, including cardiomyocytes from human-induced pluripotent stem cells.

Teratoma formation by human embryonic stem cells: evaluation of essential parameters for future safety studies.

Transplantation of human embryonic stem cells (hESC) into immune-deficient mice leads to the formation of differentiated tumors comprising all three germ layers, resembling spontaneous human teratomas. Teratoma assays are considered the gold standard for demonstrating differentiation potential of pluripotent hESC and hold promise as a standard for assessing safety among hESC-derived cell populations intended for therapeutic applications. We tested the potency of teratoma formation in seven anatomical transplantation locations (kidney capsule, muscle, subcutaneous space, peritoneal cavity, testis, liver, epididymal fat pad) in SCID mice with and without addition of Matrigel, and found that intramuscular teratoma formation was the most experimentally convenient, reproducible, and quantifiable. In the same experimental setting, we compared undifferentiated hESC and differentiated populations enriched for either beating cardiomyocytes or definitive endoderm derivatives (insulin-secreting beta cells), and showed that all cell preparations rapidly formed teratomas with varying percentages of mesoderm, ectoderm, and endoderm. In limiting dilution experiments, we found that as little as two hESC colonies spiked into feeder fibroblasts produced a teratoma, while a more rigorous single-cell titration achieved a detection limit of 1/4000. In summary, we established core parameters essential for facilitating safety profiling of hESC-derived products for future therapeutic applications.

Directed differentiation of human embryonic stem cells into the pancreatic endocrine lineage.

Human embryonic stem (hES) cells represent a potentially unlimited source of transplantable beta-cells for the treatment of diabetes. Here we describe a differentiation strategy that reproducibly directs HES3, an National Institutes of Health (NIH)-registered hES cell line, into cells of the pancreatic endocrine lineage. HES3 cells are removed from their feeder layer and cultured as embryoid bodies in a three-dimensional matrix in the presence of Activin A and Bmp4 to induce definitive endoderm. Next, growth factors known to promote the proliferation and differentiation of pancreatic ductal epithelial cells to glucose-sensing, insulin-secreting beta-cells are added. Pdx1 expression, which identifies pancreatic progenitors, is detected as early as day 12 of differentiation. By day 34, Pdx1+ cells comprise between 5% and 20% of the total cell population and Insulin gene expression is up-regulated, with release of C-peptide into the culture medium. Unlike another recent report of the induction of insulin+ cells in differentiated hES cell populations, we are unable to detect the expression of other pancreatic hormones in insulin+ cells. When transplanted into severe combined immunodeficiency (SCID) mice, differentiated cell populations retain their endocrine identity and synthesize insulin.