An Engineered Cardiac Reporter Cell Line Identifies Human Embryonic Stem Cell-Derived Myocardial Precursors

Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, United States of America.
PLoS ONE (Impact Factor: 3.53). 01/2011; 6(1):e16004. DOI: 10.1371/journal.pone.0016004
Source: PubMed

ABSTRACT Unlike some organs, the heart is unable to repair itself after injury. Human embryonic stem cells (hESCs) grow and divide indefinitely while maintaining the potential to develop into many tissues of the body. As such, they provide an unprecedented opportunity to treat human diseases characterized by tissue loss. We have identified early myocardial precursors derived from hESCs (hMPs) using an α-myosin heavy chain (αMHC)-GFP reporter line. We have demonstrated by immunocytochemistry and quantitative real-time PCR (qPCR) that reporter activation is restricted to hESC-derived cardiomyocytes (CMs) differentiated in vitro, and that hMPs give rise exclusively to muscle in an in vivo teratoma formation assay. We also demonstrate that the reporter does not interfere with hESC genomic stability. Importantly, we show that hMPs give rise to atrial, ventricular and specialized conduction CM subtypes by qPCR and microelectrode array analysis. Expression profiling of hMPs over the course of differentiation implicate Wnt and transforming growth factor-β signaling pathways in CM development. The identification of hMPs using this αMHC-GFP reporter line will provide important insight into the pathways regulating human myocardial development, and may provide a novel therapeutic reagent for the treatment of cardiac disease.

Download full-text


Available from: Shirley Mihardja, Jun 30, 2015
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Human pluripotent stem cells (hPSCs) are a prime cell source for regenerative therapies due to their extensive expansion potential and the ability to differentiate into essentially all somatic lineages in vitro. The introduction of transgenes into hPSCs will facilitate their pre-clinical testing and other applications such as the purification of desired cell lineages during differentiation and in vivo monitoring of transplanted progenies in relevant animal models as well. To date, several limits regarding the efficient generation of transgenic cell lines exist. This includes low transfection rates via non-viral methods, inefficient recovery of engineered clones, and silencing of transgene expression. Here we describe a fast and highly efficient method for the generation of multi-transgenic hPSC lines by overcoming the need for any pre-adaption to feeder-free culture conditions before genetic manipulation. Selection for a single antibiotic resistance gene encoded on one plasmid allowed for the stable genomic integration of several independent plasmid constructs thereby generating valuable multi-transgenic cell lines.
    Human Gene Therapy Methods 02/2014; DOI:10.1089/hgtb.2012.248 · 1.64 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: While the pathologies associated with in utero smoke exposure are well established, their underlying molecular mechanisms are incompletely understood. We differentiated human embryonic stem cells in the presence of physiological concentrations of tobacco smoke and nicotine. Using post hoc microarray analysis, quantitative PCR, and immunoblot analysis, we demonstrated that tobacco smoke has lineage- and stage-specific effects on human embryonic stem cell differentiation, through both nicotine-dependent and -independent pathways. We show that three major stem cell pluripotency/differentiation pathways, Notch, canonical Wnt, and transforming growth factor-β, are affected by smoke exposure, and that Nodal signaling through SMAD2 is specifically impacted by effects on Lefty1, Nodal, and FoxH1. These events are associated with upregulation of microRNA-302a, a post-transcriptional silencer of Lefty1. The described studies provide insight into the mechanisms by which tobacco smoke influences fetal development at the cellular level, and identify specific transcriptional, post-transcriptional, and signaling pathways by which this likely occurs.
    Differentiation 02/2012; 83(4):169-78. DOI:10.1016/j.diff.2011.12.005 · 2.84 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The liver is one of the largest and most complex organs in the human body. It is a vital organ weighing about 1,500 g and it continuously performs over 500 different functions. As a result, the liver is a vital organ and liver failure in the absence of liver transplantation often results in death to the patient. The shortage of donor livers for transplantation, the demand from industry to develop new drugs and new systems to test their safety, along with the need to better understand the many metabolic pathways of the liver, have been the major driving forces behind liver tissue engineering and advances to create livers synthetically, either in culture or ex vivo or by using animals as in vivo incubators. The liver is also a privileged organ in its ability to regenerate spontaneously in response to acute injury. For these reasons, it has been a major focus of research in tissue engineering and regenerative medicine. However, the ideal source of liver cells (hepatocytes) for synthetic livers has not yet been identified, and numerous research efforts are underway. The ultimate goal of these efforts is to produce an abundant, high quality and readily available source of primary human hepatocytes or synthetic liver tissue constructs for discovery, therapeutic, and diagnostic applications. This chapter explores many recent advances in the field of liver regeneration and liver tissue engineering, and new areas of research and future development.
    12/2010: pages 315-332;