Generation of Induced Pluripotent Stem Cells from CD34+ Cells across Blood Drawn from Multiple Donors with Non-Integrating Episomal Vectors

The methodology to create induced pluripotent stem cells (iPSCs) affords the opportunity to generate cells specific to the individual providing the host tissue. However, existing methods of reprogramming as well as the types of source tissue have significant limitations that preclude the ability to generate iPSCs in a scalable manner from a readily available tissue source. We present the first study whereby iPSCs are derived in parallel from multiple donors using episomal, non-integrating, oriP/EBNA1-based plasmids from freshly drawn blood. Specifically, successful reprogramming was demonstrated from a single vial of blood or less using cells expressing the early lineage marker CD34 as well as from unpurified peripheral blood mononuclear cells. From these experiments, we also show that proliferation and cell identity play a role in the number of iPSCs per input cell number. Resulting iPSCs were further characterized and deemed free of transfected DNA, integrated transgene DNA, and lack detectable gene rearrangements such as those within the immunoglobulin heavy chain and T cell receptor loci of more differentiated cell types. Furthermore, additional improvements were made to incorporate completely defined media and matrices in an effort to facilitate a scalable transition for the production of clinic-grade iPSCs

Automated feature extraction from large cardiac electrophysiological data sets

RATIONALE: A new multi-electrode array-based application for the long-term recording of action potentials from electrogenic cells makes possible exciting cardiac electrophysiology studies in health and disease. With hundreds of simultaneous electrode recordings being acquired over a period of days, the main challenge becomes achieving reliable signal identification and quantification. OBJECTIVE: We set out to develop an algorithm capable of automatically extracting regions of high-quality action potentials from terabyte size experimental results and to map the trains of action potentials into a low-dimensional feature space for analysis. METHODS AND RESULTS: Our automatic segmentation algorithm finds regions of acceptable action potentials in large data sets of electrophysiological readings. We use spectral methods and support vector machines to classify our readings and to extract relevant features. We are able to show that action potentials from the same cell site can be recorded over days without detrimental effects to the cell membrane. The variability between measurements 24 h apart is comparable to the natural variability of the features at a single time point. CONCLUSIONS: Our work contributes towards a non-invasive approach for cardiomyocyte functional maturation, as well as developmental, pathological and pharmacological studies. As the human-derived cardiac model tissue has the genetic makeup of its donor, a powerful tool for individual drug toxicity screening emerges

Seroprevalence of SARS-CoV-2 Antibody in Echocardiography and Stress Laboratory

Purpose: Transesophageal echocardiography is an aerosol-generating procedure, and exercise stress testing is a potentially aerosol-generating activity. Concern has been raised about heightened risk of transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among health care personnel participating in these procedures. We aimed to investigate the prevalence of past coronavirus disease 2019 (COVID-19) infection in echocardiography and stress laboratory staff. Methods: All staff who worked in the echocardiography and stress laboratories of one high-traffic urban hospital from March 15, 2020, to June 15, 2020, were asked to voluntarily participate. Those willing to participate were consented, and past COVID-19 infection was confirmed by a SARS-CoV-2 IgG antibody test (ARCHITECT, Abbott Laboratories) from June 15, 2020, to July 3, 2020. Clinical data were collected from the electronic medical record, and self-reported symptoms were documented with a participant survey. Results: A total of 43 staff members (86.0% of 50 total laboratory staff) participated. A majority of participants were less than 40 years old (69.8%), were White (86.0%), and were women (79.1%); mean body mass index was 24.9 ± 4.7 kg/m2. Of the 43 staff members tested for past COVID-19 infection, 3 (7.0%) had a positive SARS-CoV-2 IgG antibody result. There were no unique features in the 3 SARS-CoV-2 antibody-positive subjects; of these, 2 had known prior COVID-19 infection and 1 was asymptomatic. Conclusions: This study provides clinical data on the seroprevalence of SARS-CoV-2 antibody in echocardiography and stress laboratory staff who regularly participate in a variety of procedures that are or may be aerosol-generating

Seroprevalence of SARS-CoV-2 Antibody in Echocardiography and Stress Laboratory

Purpose: Transesophageal echocardiography is an aerosol-generating procedure, and exercise stress testing is a potentially aerosol-generating activity. Concern has been raised about heightened risk of transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among health care personnel participating in these procedures. We aimed to investigate the prevalence of past coronavirus disease 2019 (COVID-19) infection in echocardiography and stress laboratory staff. Methods: All staff who worked in the echocardiography and stress laboratories of one high-traffic urban hospital from March 15, 2020, to June 15, 2020, were asked to voluntarily participate. Those willing to participate were consented, and past COVID-19 infection was confirmed by a SARS-CoV-2 IgG antibody test (ARCHITECT, Abbott Laboratories) from June 15, 2020, to July 3, 2020. Clinical data were collected from the electronic medical record, and self-reported symptoms were documented with a participant survey. Results: A total of 43 staff members (86.0% of 50 total laboratory staff) participated. A majority of participants were less than 40 years old (69.8%), were White (86.0%), and were women (79.1%); mean body mass index was 24.9 ± 4.7 kg/m2. Of the 43 staff members tested for past COVID-19 infection, 3 (7.0%) had a positive SARS-CoV-2 IgG antibody result. There were no unique features in the 3 SARS-CoV-2 antibody-positive subjects; of these, 2 had known prior COVID-19 infection and 1 was asymptomatic. Conclusions: This study provides clinical data on the seroprevalence of SARS-CoV-2 antibody in echocardiography and stress laboratory staff who regularly participate in a variety of procedures that are or may be aerosol-generating

Human iPSC-derived cardiomyocyte networks on multiwell micro-electrode arrays for recurrent action potential recordings

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 µs. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity

Statin therapy alters the transcriptome of ventricular fibroblasts from human failing heart

Introduction: The mechanical and electrical dysfunction in heart failure (HF) is associated with excessive cardiac fibrosis (CF). Activation of human ventricular fibroblasts (hVF) and transdifferentiation to myofibroblasts underlies the increased CF. We recently reported that statin therapy reduced differentiation of hVF in HF patients. However, the underlying mechanism is not known. Therefore, we studied the effect of statin therapy on the transcriptome of hVF from HF patients. Hypothesis: We tested the hypothesis that statin therapy alters the expression of differentiation associated transcription factors (TF) in hVFs from HF patients. Methods:Primary cultures of hVF obtained from HF patients undergoing left ventricular assist device implantation either under statin therapy for at least 1 year (n=3) or not (n=3). The extent of transcriptomic changes induced by statin therapy in hVFs was studied from total RNA using RT2 ProfilerTM PCR array - human transcription factors (Qiagen, Catalog No: PAHS-075Z ) run on Roche LightCycler 96-well block. Fold change was calculated by 2-ΔΔCt method. Data were analyzed by Student’s t test, and P value Results:Out of the 84 related genes profiled, statin therapy upregulated significantly (P Conclusion: Statin therapy mitigates differentiation of hVFs from human failing heart patients by associated changes in the transcriptome. Selective targeting of hVF transcription factor may be a potential therapeutic strategy to de-differentiate myofibroblasts and mitigate the progression of CF and HF