Vector genome loss and epigenetic modifications mediate decline in transgene expression of AAV5 vectors produced in mammalian and insect cells.

Recombinant adeno-associated virus (rAAV) vectors are often produced in HEK293 or Spodoptera frugiperda (Sf)-based cell lines. We compared expression profiles of "oversized" (∼5,000 bp) and "standard-sized" (4,600 bp) rAAV5-human α1-antitrypsin (rAAV5-hA1AT) vectors manufactured in HEK293 or Sf cells and investigated molecular mechanisms mediating expression decline. C57BL/6 mice received 6 × 10 vg/kg of vector, and blood and liver samples were collected through week 57.

Application of the Pluripotent Stem Cells and Genomics in Cardiovascular Research-What We Have Learnt and Not Learnt until Now.

Personalized regenerative medicine and biomedical research have been galvanized and revolutionized by human pluripotent stem cells in combination with recent advances in genomics, artificial intelligence, and genome engineering. More recently, we have witnessed the unprecedented breakthrough life-saving translation of mRNA-based vaccines for COVID-19 to contain the global pandemic and the investment in billions of US dollars in space exploration projects and the blooming space-tourism industry fueled by the latest reusable space vessels. Now, it is time to examine where the translation of pluripotent stem cell research stands currently, which has been touted for more than the last two decades to cure and treat millions of patients with severe debilitating degenerative diseases and tissue injuries.

Transcriptional changes associated with advancing stages of heart failure underlie atrial and ventricular arrhythmogenesis.

Background: Heart failure (HF) is a leading cause of mortality and is associated with cardiac remodeling. Vulnerability to atrial fibrillation (AF) has been shown to be greater in the early stages of HF, whereas ventricular tachycardia/fibrillation develop during late stages. Here, we explore changes in gene expression that underlie the differential development of fibrosis and structural alterations that predispose to atrial and ventricular arrhythmias.

Current Challenges of iPSC-Based Disease Modeling and Therapeutic Implications.

Induced pluripotent stem cell (iPSC)-based disease modelling and the cell replacement therapy approach have proven to be very powerful and instrumental in biomedical research and personalized regenerative medicine as evidenced in the past decade by unraveling novel pathological mechanisms of a multitude of monogenic diseases at the cellular level and the ongoing and emerging clinical trials with iPSC-derived cell products. iPSC-based disease modelling has sparked widespread enthusiasm and has presented an unprecedented opportunity in high throughput drug discovery platforms and safety pharmacology in association with three-dimensional multicellular organoids such as personalized organs-on-chips, gene/base editing, artificial intelligence and high throughput "omics" methodologies. This critical review summarizes the progress made in the past decade with the advent of iPSC discovery in biomedical applications and regenerative medicine with case examples and the current major challenges that need to be addressed to unleash the full potential of iPSCs in clinical settings and pharmacology for more effective and safer regenerative therapy.

Overexpression of Map3k7 activates sinoatrial node-like differentiation in mouse ES-derived cardiomyocytes.

In vivo, cardiomyocytes comprise a heterogeneous population of contractile cells defined by unique electrophysiologies, molecular markers and morphologies. The mechanisms directing myocardial cells to specific sub-lineages remain poorly understood. Here we report that overexpression of TGFβ-Activated Kinase (TAK1/Map3k7) in mouse embryonic stem (ES) cells faithfully directs myocardial differentiation of embryoid body (EB)-derived cardiac cells toward the sinoatrial node (SAN) lineage.

Depletion of Mageb16 induces differentiation of pluripotent stem cells predominantly into mesodermal derivatives.

The Melanoma-associated Antigen gene family (MAGE) generally encodes for tumour antigens. We had identified that one of the MAGE gene members, Mageb16 was highly expressed in undifferentiated murine embryonic stem cells (ESCs). While the role of Mageb16 in stemness and differentiation of pluripotent stem cells is completely unknown, here, in our current study, we have demonstrated that Mageb16 (41 kDa) is distributed in cytosol and/or in surface membrane in undifferentiated ESCs.

STRIP2 Is Indispensable for the Onset of Embryonic Stem Cell Differentiation.

The role of striatin interacting protein 2 (Strip2) in differentiation of embryonic stem cells (ESCs) is still under debate. Strip2-silenced murine (KD) ESCs were differentiated for 4, 8, 12, and 16 days. We show that Strip2 is distributed in the perinucleus or nuclei of wild-type (WT) undifferentiated ESCs, but is localized in high-density nuclear bodies in differentiated cells.

Electrophysiological characteristics of embryonic stem cell-derived cardiomyocytes are cell line-dependent.

Background: Modelling of cardiac development, physiology and pharmacology by differentiation of embryonic stem cells (ESCs) requires comparability of cardiac differentiation between different ESC lines. To investigate whether the outcome of cardiac differentiation is consistent between different ESC lines, we compared electrophysiological properties of ESC-derived cardiomyocytes (ESC-CMs) of different murine ESC lines.

Methods: Two wild-type (D3 and R1) and two transgenic ESC lines (D3/aPIG44 and CGR8/AMPIGX-7) were differentiated under identical culture conditions.

Unique metabolic features of stem cells, cardiomyocytes, and their progenitors.

Recently, growing attention has been directed toward stem cell metabolism, with the key observation that the plasticity of stem cells also reflects the plasticity of their energy substrate metabolism. There seems to be a clear link between the self-renewal state of stem cells, in which cells proliferate without differentiation, and the activity of specific metabolic pathways. Differentiation is accompanied by a shift from anaerobic glycolysis to mitochondrial respiration.

A temporal window of vulnerability for development of atrial fibrillation with advancing heart failure.

Aims: Heart failure (HF) is associated with development of AF and life-threatening ventricular tachycardia and fibrillation (VT/VF). Vulnerability to development of AF and VT/VF at different stages of HF and the underlying pathophysiological mechanisms are poorly defined. The present study was designed to determine the time-course of development of electrical and structural remodelling of the atria and ventricles, and their contribution to induction of AF and VT/VF in a canine model of HF.

Identification and characterization of a transient outward K+ current in human induced pluripotent stem cell-derived cardiomyocytes.

Background: The ability to recapitulate mature adult phenotypes is critical to the development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) as models of disease. The present study examines the characteristics of the transient outward current (Ito) and its contribution to the hiPSC-CM action potential (AP).

Method: Embryoid bodies were made from a hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2.

Maximum diastolic potential of human induced pluripotent stem cell-derived cardiomyocytes depends critically on I(Kr).

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold promise for therapeutic applications. To serve these functions, the hiPSC-CM must recapitulate the electrophysiologic properties of native adult cardiomyocytes. This study examines the electrophysiologic characteristics of hiPSC-CM between 11 and 121 days of maturity.

Gene expression signatures defining fundamental biological processes in pluripotent, early, and late differentiated embryonic stem cells.

Investigating the molecular mechanisms controlling the in vivo developmental program postembryogenesis is challenging and time consuming. However, the developmental program can be partly recapitulated in vitro by the use of cultured embryonic stem cells (ESCs). Similar to the totipotent cells of the inner cell mass, gene expression and morphological changes in cultured ESCs occur hierarchically during their differentiation, with epiblast cells developing first, followed by germ layers and finally somatic cells.

Identification of specific pluripotent stem cell death--inducing small molecules by chemical screening.

A potential application of embryonic and inducible pluripotent stem cells for the therapy of degenerative diseases involves pure somatic cells, free of tumorigenic undifferentiated embryonic and inducible pluripotent stem cells. In complex collections of chemicals with pharmacological potential we expect to find molecules able to induce specific pluripotent stem cell death, which could be used in some cell therapy settings to eliminate undifferentiated cells. Therefore, we have screened a chemical library of 1120 small chemicals to identify compounds that induce specifically apoptotic cell death in undifferentiated mouse embryonic stem cells (ESCs).

Specific gene signatures and pathways in mesodermal cells and their derivatives derived from embryonic stem cells.

The vertebrate early stage embryo is consisting of the three primary germ layers ectoderm, mesoderm and endoderm, from which all organ tissues are developed. During early embryonic development, mesodermal cells become sequentially determined to more precisely defined cell types including muscle, heart, vasculature, blood, kidney, gonads, dermis and cartilage. How the prospective mesodermal cells integrate the various signals they receive and how they resolve this information to regulate their morphogenetic behavior and cell fate decisions is largely unknown.

eXtraembryonic ENdoderm (XEN) stem cells produce factors that activate heart formation.

Background: Initial specification of cardiomyocytes in the mouse results from interactions between the extraembryonic anterior visceral endoderm (AVE) and the nascent mesoderm. However the mechanism by which AVE activates cardiogenesis is not well understood, and the identity of specific cardiogenic factors in the endoderm remains elusive. Most mammalian studies of the cardiogenic potential of the endoderm have relied on the use of cell lines that are similar to the heart-inducing AVE.

A comparative analysis of extra-embryonic endoderm cell lines.

Prior to gastrulation in the mouse, all endodermal cells arise from the primitive endoderm of the blastocyst stage embryo. Primitive endoderm and its derivatives are generally referred to as extra-embryonic endoderm (ExEn) because the majority of these cells contribute to extra-embryonic lineages encompassing the visceral endoderm (VE) and the parietal endoderm (PE). During gastrulation, the definitive endoderm (DE) forms by ingression of cells from the epiblast.

Isolation and functional characterization of alpha-smooth muscle actin expressing cardiomyocytes from embryonic stem cells.

Early mammalian heart development is characterized by transient expression of alpha-smooth muscle actin (Acta2). To date, cardiomyocytes expressing Acta2 in the early stages of in vivo development have not been characterized. To functionally characterize Acta2-expressing cardiomyocytes, we used a transgenic ES cell line expressing both the puromycin acetyl transferase (Pac) and enhanced green fluorescent protein (EGFP) cassettes under the control of the Acta2 promoter.

Global transcriptomic analysis of murine embryonic stem cell-derived brachyury(+) (T) cells.

Brachyury(+) mesodermal cell population with purity over 79% was obtained from differentiating brachyury embryonic stem cells (ESC) generated with brachyury promoter driven enhanced green fluorescent protein and puromycin-N-acetyltransferase. A comprehensive transcriptomic analysis of brachyury(+) cells enriched with puromycin application from 6-day-old embryoid bodies (EBs), 6-day-old control EBs and undifferentiated ESCs led to identification of 1573 uniquely up-regulated and 1549 uniquely down-regulated transcripts in brachyury(+) cells. Furthermore, transcripts up-regulated in brachyury(+) cells have overrepresented the Gene Ontology annotations (cell differentiation, blood vessel morphogenesis, striated muscle development, placenta development and cell motility) and Kyoto Encyclopedia of Genes and Genomes pathway annotations (mitogen-activated protein kinase signaling and transforming growth factor beta signaling).

The FunGenES database: a genomics resource for mouse embryonic stem cell differentiation.

Embryonic stem (ES) cells have high self-renewal capacity and the potential to differentiate into a large variety of cell types. To investigate gene networks operating in pluripotent ES cells and their derivatives, the "Functional Genomics in Embryonic Stem Cells" consortium (FunGenES) has analyzed the transcriptome of mouse ES cells in eleven diverse settings representing sixty-seven experimental conditions. To better illustrate gene expression profiles in mouse ES cells, we have organized the results in an interactive database with a number of features and tools.

A genome-scale RNAi screen for Oct4 modulators defines a role of the Paf1 complex for embryonic stem cell identity.

Pluripotent embryonic stem cells (ESCs) maintain self-renewal while ensuring a rapid response to differentiation cues. The identification of genes maintaining ESC identity is important to develop these cells for their potential therapeutic use. Here we report a genome-scale RNAi screen for a global survey of genes affecting ESC identity via alteration of Oct4 expression.

Three LIF-dependent signatures and gene clusters with atypical expression profiles, identified by transcriptome studies in mouse ES cells and early derivatives.

Background: Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of the cytokine Leukaemia Inhibitory Factor (LIF). Identification of LIF targets and of genes regulating the transition between pluripotent and early differentiated cells is a critical step for understanding the control of ES cell pluripotency.

Results: By gene profiling studies carried out with mRNAs from ES cells and their early derivatives treated or not with LIF, we have identified i) LIF-dependent genes, highly expressed in pluripotent cells, whose expression level decreases sharply upon LIF withdrawal [Pluri genes], ii) LIF induced genes [Lifind genes] whose expression is differentially regulated depending upon cell context and iii) genes specific to the reversible or irreversible committed states.

Human ES cell derived cardiomyocytes for cell replacement therapy: a current update.

Cardiovascular diseases are the leading cause of death globally. The pluripotency and indefinite proliferative capacity of embryonic stem (ES) cells make them a promising candidate for the cell replacement therapy where the damaged cells are replaced by the functional cells derived from stem cells in vitro. Emerging results with human ES cells for the myocardial repair are encouraging, but this approach is still in its infancy and is under extensive investigation.

Entrapment of embryonic stem cells-derived cardiomyocytes in macroporous biodegradable microspheres: preparation and characterization.

Embryonic Stem (ES) cells-derived cardiomyocytes can possibly be applied for cell therapy of diseases such as heart failure. Biodegradable scaffolds will significantly improve the expansion of sufficient functional ES cell-derived cardiomyocytes and may also increase the survival rate of cardiomyocytes after their transplantation. In the present study, we cultivated cardiomyocytes isolated from a transgenic a-myosin heavy chain (alpha-MHC) ES cell lineage expressing both puromycin resistance and enhanced green fluorescent protein (EGFP) under the control of the alpha-MHC promoter in macroporous gelatine microspheres using small-scale bioreactors and proved that cardiomyocytes function after their cultivation in micropsperes.

Functional characterization and transcriptome analysis of embryonic stem cell-derived contractile smooth muscle cells.

Complete transcriptome profiling of contractile smooth muscle cells (SMCs) differentiated from embryonic stem cells is crucial for the characterization of smooth muscle gene expression signatures and will contribute to defining biological and physiological processes in these cells. We have generated a transgenic embryonic stem cell line expressing both the puromycin acetyl transferase and enhanced green fluorescent protein cassettes under the control of the Acta2 promoter. Applying a specific monolayer culture protocol using retinoic acid, a puromycin-resistant and enhanced green fluorescent protein-positive Acta2(+) SMC population of 95% purity was isolated.