Optogenetic Reporters Delivered as mRNA Facilitate Repeatable Action Potential and Calcium Handling Assessment in Human iPSC-Derived Cardiomyocytes.

Electrical activity and intracellular Ca2+ transients are key features of cardiomyocytes. They can be measured using organic voltage- and Ca2+-sensitive dyes but their photostability and phototoxicity mean they are unsuitable for long-term measurements. Here, we investigated whether genetically encoded voltage and Ca2+ indicators (GEVIs and GECIs) delivered as modified mRNA (modRNA) into human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) would be accurate alternatives allowing measurements over long periods. These indicators were detected in hiPSC-CMs for up to 7 days after transfection and did not affect responses to proarrhythmic compounds. Furthermore, using the GEVI ASAP2f we observed action potential prolongation in long QT syndrome models, while the GECI jRCaMP1b facilitated the repeated evaluation of Ca2+ handling responses for various tyrosine kinase inhibitors. This study demonstrated that modRNAs encoding optogenetic constructs report cardiac physiology in hiPSC-CMs without toxicity or the need for stable integration, illustrating their value as alternatives to organic dyes or other gene delivery methods for expressing transgenes.

Transforming youth mental health services in a large urban centre: ACCESS Open Minds Edmonton.

AIM: This paper outlines the transformation of youth mental health services in Edmonton, Alberta, a large city in Western Canada. We describe the processes and challenges involved in restructuring how services and care are delivered to youth (11-25 years old) with mental health needs based on the objectives of the pan-Canadian ACCESS Open Minds network. METHODS: We provide a narrative review of how youth mental health services have developed since our engagement with the ACCESS Open Minds initiative, based on its five central objectives of early identification, rapid access, appropriate care, continuity of care, and youth and family engagement. RESULTS: Building on an initial community mapping exercise, a service network has been developed; teams that were previously age-oriented have been integrated together to seamlessly cover the age 11 to 25 range; early identification has thus far focused on high-school populations; and an actual drop-in space facilitates rapid access and linkages to appropriate care within the 30-day benchmark. CONCLUSIONS: Initial aspects of the transformation have relied on restructuring and partnerships that have generated early successes. However, further transformation over the longer term will depend on data demonstrating how this has impacted clinical outcomes and service utilization. Ultimately, sustainability in a large urban centre will likely involve scaling up to a network of similar services to cover the entire population of the city.

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs).

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Assessment of functional competence of endothelial cells from human pluripotent stem cells in zebrafish embryos.

Human pluripotent stem cells (hPSCs) are proving to be a valuable source of endothelial cells (ECs), pericytes, and vascular smooth muscle cells (vSMCs). Although an increasing number of phenotypic markers are becoming available to determine the phenotypes of these cells in vitro, the ability to integrate and form functional vessels in the host organism, typically mouse, remains critical for the assessment of EC functional competence. However, current mouse models require relatively large numbers of cells that might be difficult to derive simultaneously from multiple hPSCs lines. Therefore, there is an urgent need for new functional assays that are robust and can be performed with small numbers of cells. Here we describe a novel zebrafish xenograft model to test functionality of hPSC-derived ECs. The assay can be performed in 10 days and requires only ~100-400 human cells per embryo. Thus, the zebrafish xenograft model can be useful for the accurate and rapid assessment of functionality of hPSC-derived ECs in a lower vertebrate model that is widely viewed by regulatory authorities as a more acceptable alternative to adult mice.

Monitoring of cell therapy and assessment of cardiac function using magnetic resonance imaging in a mouse model of myocardial infarction.

We have developed a mouse severe combined immunodeficient (SCID) model of myocardial infarction based on permanent coronary artery occlusion that allows long-term functional analysis of engrafted human embryonic stem cell-derived cardiomyocytes, genetically marked with green fluorescent protein (GFP), in the mouse heart. We describe methods for delivery of dissociated cardiomyocytes to the left ventricle that minimize scar formation and visualization and validation of the identity of the engrafted cells using the GFP emission spectrum, and histological techniques compatible with GFP epifluorescence, for monitoring phenotypic changes in the grafts in vivo. In addition, we describe how magnetic resonance imaging can be adapted for use in mice to monitor cardiac function non-invasively and repeatedly. The model can be adapted to include multiple control or other cell populations. The procedure for a cohort of six mice can be completed in a maximum of 13 weeks, depending on follow-up, with 30 h of hands-on time.