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: 3.23). 01/2011; 6(1):e16004. DOI: 10.1371/journal.pone.0016004
Source: PubMed


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.

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Available from: Shirley Mihardja
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    • "To determine miRNAs that are differentially expressed during differentiation of hESCs into CMs, we performed miRNA expression profiling by using the previously described myocardial reporter hESC line [15]. We compared miRNA expression profiles of undifferentiated hESCs with CMs sorted at Days 8 and 14 after embryoid body (EB) formation [15]. "
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    ABSTRACT: Introduction Posttranscriptional control of mRNA by microRNA (miRNA) has been implicated in the regulation of diverse biologic processes from directed differentiation of stem cells through organism development. We describe a unique pathway by which miRNA regulates the specialized differentiation of cardiomyocyte (CM) subtypes. Methods We differentiated human embryonic stem cells (hESCs) to cardiac progenitor cells and functional CMs, and characterized the regulated expression of specific miRNAs that target transcriptional regulators of left/right ventricular-subtype specification. Results From >900 known human miRNAs in hESC-derived cardiac progenitor cells and functional CMs, a subset of differentially expressed cardiac miRNAs was identified, and in silico analysis predicted highly conserved binding sites in the 3′-untranslated regions (3′UTRs) of Hand-and-neural-crest-derivative-expressed (HAND) genes 1 and 2 that are involved in left and right ventricular development. We studied the temporal and spatial expression patterns of four miRNAs in differentiating hESCs, and found that expression of miRNA (miR)-363, miR-367, miR-181a, and miR-181c was specific for stage and site. Further analysis showed that miR-363 overexpression resulted in downregulation of HAND1 mRNA and protein levels. A dual luciferase reporter assay demonstrated functional interaction of miR-363 with the full-length 3′UTR of HAND1. Expression of anti-miR-363 in-vitro resulted in enrichment for HAND1-expressing CM subtype populations. We also showed that BMP4 treatment induced the expression of HAND2 with less effect on HAND1, whereas miR-363 overexpression selectively inhibited HAND1. Conclusions These data show that miR-363 negatively regulates the expression of HAND1 and suggest that suppression of miR-363 could provide a novel strategy for generating functional left-ventricular CMs.
    Full-text · Article · Jun 2014 · Stem Cell Research & Therapy
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    • "For example, the overexpression of specific transcription or growth factors aiming at the directed differentiation into a desired cell type may overcome poor differentiation efficiencies that exist for several somatic lineages (David et al., 2009; Hartung et al., 2013). On the other hand, lineage-restricted reporter gene expression could be used to enrich for specific progenies (Boecker et al., 2004; Huber et al., 2007; Xu et al., 2008b; Kita-Matsuo et al., 2009; Ritner et al., 2011). Another application is the cell typespecific or ubiquitous expression of marker genes to monitor cell engraftment in respective animal models posttransplantation (Dai et al., 2007; Sun et al., 2009; Mauritz et al., 2011). "
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    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.
    Full-text · Article · Feb 2014 · Human Gene Therapy Methods
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    • "Latest generation self-inactivated lentiviral vectors (SIN-LVs) express the transgene under internal promoters [13], [14] allowing the use of physiological or tissue-specific promoters to obtain regulated transgene expression [15]. Using this technology, several groups have developed cardiac-specific lentiviral vectors able to specifically mark cardiomyocites derived from hESCs [16], [17], [18]. However, no LVs capable of specifically marking blood cells differentiating from hESCs have been reported so far. "
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    ABSTRACT: Genetic manipulation of human embryonic stem cells (hESCs) is instrumental for tracing lineage commitment and to studying human development. Here we used hematopoietic-specific Wiskott-Aldrich syndrome gene (WAS)-promoter driven lentiviral vectors (LVs) to achieve highly specific gene expression in hESCs-derived hematopoietic cells. We first demonstrated that endogenous WAS gene was not expressed in undifferentiated hESCs but was evident in hemogenic progenitors (CD45(-)CD31(+)CD34(+)) and hematopoietic cells (CD45(+)). Accordingly, WAS-promoter driven LVs were unable to express the eGFP transgene in undifferentiated hESCs. eGFP(+) cells only appeared after embryoid body (EB) hematopoietic differentiation. The phenotypic analysis of the eGFP(+) cells showed marking of different subpopulations at different days of differentiation. At days 10-15, AWE LVs tag hemogenic and hematopoietic progenitors cells (CD45(-)CD31(+)CD34(dim) and CD45(+)CD31(+)CD34(dim)) emerging from hESCs and at day 22 its expression became restricted to mature hematopoietic cells (CD45(+)CD33(+)). Surprisingly, at day 10 of differentiation, the AWE vector also marked CD45(-)CD31(low/-)CD34(-) cells, a population that disappeared at later stages of differentiation. We showed that the eGFP(+)CD45(-)CD31(+) population generate 5 times more CD45(+) cells than the eGFP(-)CD45(-)CD31(+) indicating that the AWE vector was identifying a subpopulation inside the CD45(-)CD31(+) cells with higher hemogenic capacity. We also showed generation of CD45(+) cells from the eGFP(+)CD45(-)CD31(low/-)CD34(-) population but not from the eGFP(-)CD45(-)CD31(low/-)CD34(-) cells. This is, to our knowledge, the first report of a gene transfer vector which specifically labels hemogenic progenitors and hematopoietic cells emerging from hESCs. We propose the use of WAS-promoter driven LVs as a novel tool to studying human hematopoietic development.
    Full-text · Article · Jun 2012 · PLoS ONE
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