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BACKGROUND Human (h) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) serve as a potential unlimited ex vivo source of cardiomyocytes (CMs). However,a well-accepted roadblock has been their immature phenotype. hESC/iPSC-derived ventricular (v) CMs and their engineered cardiac microtissues (hvCMTs) similarly displayed positive chronotropic but null inotropic responses to $\$-adrenergic stimulation. Given that phospholamban (PLB) is robustly present in adult but poorly expressed in hESC/iPSC-vCMs and its defined biological role in $\$-adrenergic signaling,we investigated the functional consequences of PLB expression in hESC/iPSC-vCMs and hvCMTs. METHODS AND RESULTS First,we confirmed that PLB protein was differentially expressed in hESC (HES2,H9)- and iPSC-derived and adult vCMs. We then transduced hES2-vCMs with the recombinant adenoviruses (Ad) Ad-PLB or Ad-S16E-PLB to overexpress wild-type PLB or the pseudophosphorylated point-mutated variant,respectively. As anticipated from the inhibitory effect of unphosphorylated PLB on sarco/endoplasmic reticulum Ca2+-ATPase,Ad-PLB transduction significantly attenuated electrically evoked Ca2+ transient amplitude and prolonged the 50% decay time. Importantly,Ad-PLB-transduced hES2-vCMs uniquely responded to isoproterenol. Ad-S16E-PLB-transduced hES2-vCMs displayed an intermediate phenotype. The same trends were observed with H9- and iPSC-vCMs. Directionally,similar results were also seen with Ad-PLB-transduced and Ad-S16E-transduced hvCMTs. However,Ad-PLB altered neither the global transcriptome nor ICa,L,implicating a PLB-specific effect. CONCLUSIONS Engineered upregulation of PLB expression in hESC/iPSC-vCMs restores a positive inotropic response to $\$-adrenergic stimulation. These results not only provide a better mechanistic understanding of the immaturity of hESC/iPSC-vCMs but will also lead to improved disease models and transplantable prototypes with adult-like physiological responses. View Publication

Gene- and cell-based therapies are promising strategies for the treatment of degenerative retinal diseases such as age-related macular degeneration,Stargardt disease,and retinitis pigmentosa. Cellular engineering before transplantation may allow the delivery of cellular factors that can promote functional improvements,such as increased engraftment or survival of transplanted cells. A current challenge in traditional DNA-based vector transfection is to find a delivery system that is both safe and efficient,but using mRNA as an alternative to DNA can circumvent these major roadblocks. In this study,we show that both unmodified and modified mRNA can be delivered to retinal pigmented epithelial (RPE) cells with a high efficiency compared with conventional plasmid delivery systems. On the other hand,administration of unmodified mRNA induced a strong innate immune response that was almost absent when using modified mRNA. Importantly,transfection of mRNA encoding a key regulator of RPE gene expression,microphthalmia-associated transcription factor (MITF),confirmed the functionality of the delivered mRNA. Immunostaining showed that transfection with either type of mRNA led to the expression of roughly equal levels of MITF,primarily localized in the nucleus. Despite these findings,quantitative RT-PCR analyses showed that the activation of the expression of MITF target genes was higher following transfection with modified mRNA compared with unmodified mRNA. Our findings,therefore,show that modified mRNA transfection can be applied to human embryonic stem cell-derived RPE cells and that the method is safe,efficient,and functional. View Publication

It is likely that arrhythmias should be avoided for therapies based on human pluripotent stem cell (hPSC)-derived cardiomyocytes (CM) to be effective. Towards achieving this goal,we introduced light-activated channelrhodopsin-2 (ChR2),a cation channel activated with 480 nm light,into human embryonic stem cells (hESC). By using in vitro approaches,hESC-CM are able to be activated with light. ChR2 is stably transduced into undifferentiated hESC via a lentiviral vector. Via directed differentiation,hESCChR2-CM are produced and subjected to optical stimulation. hESCChR2-CM respond to traditional electrical stimulation and produce similar contractility features as their wild-type counterparts but only hESCChR2-CM can be activated by optical stimulation. Here it is shown that a light sensitive protein can enable in vitro optical control of hESC-CM and that this activation occurs optimally above specific light stimulation intensity and pulse width thresholds. For future therapy,in vivo optical stimulation along with optical inhibition could allow for acute synchronization of implanted hPSC-CM with patient cardiac rhythms. View Publication

Mitochondrial dynamics play an important role in numerous physiological and pathophysiological phenomena in the developing and adult human heart. Alterations in structural aspects of cellular mitochondrial composition as a function of changes in physiology can easily be visualized using fluorescence microscopy. Commonly,mitochondrial location,number,and morphology are reported qualitatively due to the lack of automated and user-friendly computer-based analysis tools. Mitochondrial Quantification using MATLAB (MQM) is a computer-based tool to quantitatively assess these parameters by analyzing fluorescently labeled mitochondria within the cell; in particular,MQM provides numerical information on the number,area,and location of mitochondria within a cell in a time-efficient,automated,and unbiased way. This chapter describes the use of MQM's capabilities to quantify mitochondrial changes during human pluripotent stem cell (hPSC) differentiation into spontaneously contracting cardiomyocytes (SC-CMs),which follows physiological pathways of human heart development. View Publication

Neurogenin3 (NEUROG3) is a basic helix-loop-helix transcription factor required for development of the endocrine pancreas in mice. In contrast,humans with NEUROG3 mutations are born with endocrine pancreas function,calling into question whether NEUROG3 is required for human endocrine pancreas development. To test this directly,we generated human embryonic stem cell (hESC) lines where both alleles of NEUROG3 were disrupted using CRISPR/Cas9-mediated gene targeting. NEUROG3(-/-) hESC lines efficiently formed pancreatic progenitors but lacked detectible NEUROG3 protein and did not form endocrine cells in vitro. Moreover,NEUROG3(-/-) hESC lines were unable to form mature pancreatic endocrine cells after engraftment of PDX1(+)/NKX6.1(+) pancreatic progenitors into mice. In contrast,a 75-90% knockdown of NEUROG3 caused a reduction,but not a loss,of pancreatic endocrine cell development. We conclude that NEUROG3 is essential for endocrine pancreas development in humans and that as little as 10% NEUROG3 is sufficient for formation of pancreatic endocrine cells. View Publication

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes,smooth muscle cells (SMC),and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs,differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result,numerous strategies have been developed to derive CPCs from ESCs. In this protocol,differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs,ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus,CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs. View Publication

UNLABELLED In the classical form of $\$1-antitrypsin deficiency (ATD),aberrant intracellular accumulation of misfolded mutant $\$1-antitrypsin Z (ATZ) in hepatocytes causes hepatic damage by a gain-of-function,proteotoxic" mechanism. Whereas some ATD patients develop severe liver disease (SLD) that necessitates liver transplantation� View Publication

Human induced pluripotent stem cells (hiPSCs) provide a platform for studying human disease in vitro,increase our understanding of human embryonic development,and provide clinically relevant cell types for transplantation,drug testing,and toxicology studies. Since their discovery,numerous advances have been made in order to eliminate issues such as vector integration into the host genome,low reprogramming efficiency,incomplete reprogramming and acquisition of genomic instabilities. One of the ways to achieve integration-free reprogramming is by using RNA-based Sendai virus. Here we describe a method to generate hiPSCs with Sendai virus in both feeder-free and feeder-dependent culture systems. Additionally,we illustrate methods by which to validate pluripotency of the resulting stem cell population. View Publication

BACKGROUND: Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators with important functions in development and disease. Here,we sought to identify and functionally characterize novel lncRNAs critical for vertebrate development. METHODS AND RESULTS: By relying on human pluripotent stem cell differentiation models,we investigated lncRNAs differentially regulated at key steps during human cardiovascular development with a special focus on vascular endothelial cells. RNA sequencing led to the generation of large data sets that serve as a gene expression roadmap highlighting gene expression changes during human pluripotent cell differentiation. Stage-specific analyses led to the identification of 3 previously uncharacterized lncRNAs,TERMINATOR,ALIEN,and PUNISHER,specifically expressed in undifferentiated pluripotent stem cells,cardiovascular progenitors,and differentiated endothelial cells,respectively. Functional characterization,including localization studies,dynamic expression analyses,epigenetic modification monitoring,and knockdown experiments in lower vertebrates,as well as murine embryos and human cells,confirmed a critical role for each lncRNA specific for each analyzed developmental stage. CONCLUSIONS: We have identified and functionally characterized 3 novel lncRNAs involved in vertebrate and human cardiovascular development,and we provide a comprehensive transcriptomic roadmap that sheds new light on the molecular mechanisms underlying human embryonic development,mesodermal commitment,and cardiovascular specification. View Publication

Amyotrophic lateral sclerosis is a progressive disease characterized by the loss of upper and lower motor neurons,leading to paralysis of voluntary muscles. About 10% of all ALS cases are familial (fALS),among which 15-20% are linked to Cu/Zn superoxide dismutase (SOD1) mutations,usually inherited in an autosomal dominant manner. To date only one FDA approved drug is available which increases survival moderately. Our understanding of ALS disease mechanisms is largely derived from rodent model studies,however due to the differences between rodents and humans,it is necessary to have humanized models for studies of disease pathogenesis as well as drug development. Therefore,we generated a comprehensive library of a total 22 of fALS patient-specific induced pluripotent stem cell (iPSC) lines. These cells were thoroughly characterized before being deposited into the library. The library of cells includes a variety of C9orf72 mutations,sod1 mutations,FUS,ANG and FIG4 mutations. Certain mutations are represented with more than one line,which allows for studies of variable genetic backgrounds. In addition,these iPSCs can be successfully differentiated to astroglia,a cell type known to play a critical role in ALS disease progression. This library represents a comprehensive resource that can be used for ALS disease modeling and the development of novel therapeutics. View Publication