Identification and Purification of Human Induced Pluripotent Stem Cell-Derived Atrial-Like Cardiomyocytes Based on Sarcolipin Expression

<div><p>The use of human stem cell-derived cardiomyocytes to study atrial biology and disease has been restricted by the lack of a reliable method for stem cell-derived atrial cell labeling and purification. The goal of this study was to generate an atrial-specific reporter construct to identify and purify human stem cell-derived atrial-like cardiomyocytes. We have created a bacterial artificial chromosome (BAC) reporter construct in which fluorescence is driven by expression of the atrial-specific gene sarcolipin (<i>SLN</i>). When purified using flow cytometry, cells with high fluorescence specifically express atrial genes and display functional calcium handling and electrophysiological properties consistent with atrial cardiomyocytes. Our data indicate that <i>SLN</i> can be used as a marker to successfully monitor and isolate hiPSC-derived atrial-like cardiomyocytes. These purified cells may find many applications, including in the study of atrial-specific pathologies and chamber-specific lineage development.</p></div

BAC transgene and expression in hiPSC-derived cardiomyocytes.

<p>(a) Schematic of recombineered BAC containing a tdTomato-<i>Rex1</i>-Neo^R cassette. (b) tdTomato fluorescence in differentiating EBs appeared at Day 10 of differentiation and increased over time, lasting up to 60 days. Scale bars, 1000 µm. (c) qPCR of differentiating EBs showing timeline of expression of <i>cTNT, SLN,</i> and <i>ANP</i> consistent with the appearance of red fluorescence. (d) Dissociated red⁺ cells were positive for cTNT by immunofluorescence. Scale bars, 100 µm.</p

Purification and characterization of red^high and red^low cells.

<p>(a) Cell sorting strategy to purify red^high and red^low cardiomyocytes. Cells were first gated on the total cardiomyocyte population, marked as SIRPα⁺/CD90⁻, and then purified based on high or low levels of red fluorescence. (b) qPCR for relevant atrial (<i>SLN, ANP)</i> and ventricular (<i>MYL2, HRT2)</i> genes in purified red^high (n = 3) and red^low (n = 3) populations. (**p<0.01, ***p<0.001, ****p<0.0001). (c) Immunofluorescence for MLC2v showing exclusive expression in red^low population. Scale bars, 200 µm. (d) Quantification of fraction red^low (n = 143) and red^high (n = 514) cells expressing MLC2v protein from (c).</p

Red^high cells display electrophysiological properties similar to atrial cardiomyocytes.

<p>(a) Representative triggered action potential traces recorded from red^high and red^low cells. (b) Quantification of APD₅₀ and APD₉₀ for red^high (n = 15) and red^low (n = 10) cells (***p<0.001, *p<0.05). (c–e) Expression of depolarization-activated potassium currents. (c) Upper panel: representative instantaneous outward currents. Lower panel: representative sustained potassium currents. (d) Comparison of peak instantaneous outward currents between red^high (n = 7) and red^low (n = 8) cells. (e) Comparison of I–V curves of sustained potassium currents between red^high and red^low cells (***p<0.001). (f) qPCR for gene expression of the I_Kur subunit <i>KCNA5</i> and I_KAch subunit <i>KCNJ3</i>, revealing increased expression in red^high cells (*p<0.05, ****p<0.0001). All genes normalized to expression of <i>GAPDH</i> and relative to gene expression in EBs.</p

Red^high cells possess functional Ca²⁺ handling properties similar to atrial cardiomyocytes.

<p>(a) Representative spontaneous calcium transients recorded from red^high and red^low cells. (b) Quantification of calcium transient decay and interval properties in red^high (n = 13) and red^low (n = 13) cells (**p<0.01). (c) Quantification of beating rate in spontaneously beating red^high (n = 22) and red^low (n = 23) cells (****p<0.0001).</p

Analysis of Transcriptional Variability in a Large Human iPSC Library Reveals Genetic and Non-genetic Determinants of Heterogeneity

Variability in induced pluripotent stem cell (iPSC) lines remains a concern for disease modeling and regenerative medicine. We have used RNA sequencing analysis and linear mixed models to examine the sources of gene expression variability in 317 human iPSC lines from 101 individuals. We found that ~50% of genome-wide expression variability is explained by variation across individuals and identified a set of expression quantitative trait loci that contribute to this variation. These analyses coupled with allele specific expression show that iPSCs retain a donor specific gene expression pattern. Network, pathway and key driver analyses showed that Polycomb targets contribute significantly to the non-genetic variability seen within and across individuals, highlighting this chromatin regulator as a likely source of reprogramming-based variability. Our findings therefore shed light on variation between iPSC lines and illustrate the potential for our dataset and other similar large-scale analyses to identify underlying drivers relevant to iPSC applications

Myeloid Dysregulation in a Human Induced Pluripotent Stem Cell Model of PTPN11 -Associated Juvenile Myelomonocytic Leukemia

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A Library of Induced Pluripotent Stem Cells from Clinically Well-Characterized, Diverse Healthy Human Individuals

Abstract A library of well-characterized human induced pluripotent stem cell (hiPSC) lines from clinically healthy human subjects could serve as a powerful resource of normal controls for in vitro human development, disease modeling, genotype-phenotype association studies, and drug response evaluation. We report generation and extensive characterization of a gender-balanced, racially/ethnically diverse library of hiPSC lines from forty clinically healthy human individuals who range in age from 22-61. The hiPSCs match the karyotype and short tandem repeat identity of their parental fibroblasts, and have a transcription profile characteristic of pluripotent stem cells. We provide whole genome sequencing data for one hiPSC clone from each individual, ancestry determination, and analysis of Mendelian disease genes and risks. We document similar physiology of cardiomyocytes differentiated from multiple independent hiPSC clones derived from two individuals. This extensive characterization makes this hiPSC library a unique and valuable resource for many studies on human biology. Competing Interest Statement The authors have declared no competing interest