Transcriptional and Functional Profiling of Human Embryonic Stem Cell-Derived Cardiomyocytes

Department of Radiology, Stanford University School of Medicine, Stanford, California, United States of America.
PLoS ONE (Impact Factor: 3.23). 02/2008; 3(10):e3474. DOI: 10.1371/journal.pone.0003474
Source: PubMed

ABSTRACT Human embryonic stem cells (hESCs) can serve as a potentially limitless source of cells that may enable regeneration of diseased tissue and organs. Here we investigate the use of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in promoting recovery from cardiac ischemia reperfusion injury in a mouse model. Using microarrays, we have described the hESC-CM transcriptome within the spectrum of changes that occur between undifferentiated hESCs and fetal heart cells. The hESC-CMs expressed cardiomyocyte genes at levels similar to those found in 20-week fetal heart cells, making this population a good source of potential replacement cells in vivo. Echocardiographic studies showed significant improvement in heart function by 8 weeks after transplantation. Finally, we demonstrate long-term engraftment of hESC-CMs by using molecular imaging to track cellular localization, survival, and proliferation in vivo. Taken together, global gene expression profiling of hESC differentiation enables a systems-based analysis of the biological processes, networks, and genes that drive hESC fate decisions, and studies such as this will serve as the foundation for future clinical applications of stem cell therapies.

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Available from: Kitchener D Wilson, Sep 26, 2015
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    • "expressed gene in pluripotent hES cells vs. their differentiated counterparts (Cao et al., 2008). In addition, a global phosphoproteomic study of hES cells identified Lck as a signaling protein with more abundant phosphorylation site identifications in pluripotent hES cells compared to differentiated hES cell derivatives (Brill et al., 2009). "
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    ABSTRACT: Embryonic stem (ES) cells are characterized by pluripotency, defined as the developmental potential to generate cell lineages derived from all three primary germ layers. In the past decade, great progress has been made on the cell culture conditions, transcription factor programs and intracellular signaling pathways that control both murine and human ES cell fates. ES cells of mouse vs. human origin have distinct culture conditions, responding to some tyrosine kinase signaling pathways in opposite ways. Previous work has implicated the Src family of non-receptor protein-tyrosine kinases in mouse ES cell self-renewal and differentiation. Seven members of the Src kinase family are expressed in mouse ES cells, and individual family members appear to play distinct roles in regulating their developmental fate. Both Hck and c-Yes are important in self-renewal, while c-Src activity alone is sufficient to induce differentiation. While these findings implicate Src-family kinase signaling in mouse ES cell renewal and differentiation, the role of this kinase family in human ES cells is largely unknown. Here, we explored Src-family kinase expression patterns and signaling in human ES cells during self-renewal and differentiation. Of the eleven Src-related kinases in the human genome, Fyn, c-Yes, c-Src, Lyn, Lck and Hck were expressed in H1, H7 and H9 hES cells, while Fgr, Blk, Srm, Brk, and Frk transcripts were not detected. Of these, c-Yes, Lyn, and Hck transcript levels remained constant in self-renewing human ES cells vs. differentiated EBs, while c-Src and Fyn showed a modest increase in expression as a function of differentiation. In contrast, Lck expression levels dropped dramatically as a function of EB differentiation. To assess the role of overall Src-family kinase activity in human ES cell differentiation, cultures were treated with inhibitors specific for the Src kinase family. Remarkably, human ES cells maintained in the presence of the potent Src-family kinase inhibitor A-419259 retained the morphology of domed, pluripotent colonies and continued to express the self-renewal marker TRA-1-60 despite culture under differentiation conditions. Taken together, these observations support a role for Src-family kinase signaling in the regulation of human ES cell fate, and suggest that the activities of individual Src-family members are required for initiation of the differentiation program.
    Stem Cell Research 09/2014; 13(3). DOI:10.1016/j.scr.2014.09.007 · 3.69 Impact Factor
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    • "Therefore, it is of major importance to address this problem in order to improve the engraftment and enhance the therapeutic efficacy of transplanted cells. BL imaging using FLuc is a powerful method for the quantitative assessment of cell survival in vivo and has frequently been used for longitudinal monitoring of various types of intramyocardially transplanted cells in small animals including adult stem cells [16], [40]–[46], fibroblasts [16], embryonic cardiomyoblasts [26], [47], [48], cardiac stem cells [24], [49]–[51], undifferentiated ESC [52], [53], ESC-derived endothelial cells [54] and ES-CM [22], [23], [31], [55]. In this study we describe the generation of transgenic murine iPSC lines in which constitutively active FLuc and CM-specific PAC and EGFP expression allow for isolation, longitudinal in vivo bioluminescent tracking and in situ fluorescent detection of highly purified iPS-CM. "
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    ABSTRACT: Cell loss after transplantation is a major limitation for cell replacement approaches in regenerative medicine. To assess the survival kinetics of induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) we generated transgenic murine iPSC lines which, in addition to CM-specific expression of puromycin N-acetyl-transferase and enhanced green fluorescent protein (EGFP), also constitutively express firefly luciferase (FLuc) for bioluminescence (BL) in vivo imaging. While undifferentiated iPSC lines generated by random integration of the transgene into the genome retained stable FLuc activity over many passages, the BL signal intensity was strongly decreased in purified iPS-CM compared to undifferentiated iPSC. Targeted integration of FLuc-expression cassette into the ROSA26 genomic locus using zinc finger nuclease (ZFN) technology strongly reduced transgene silencing in iPS-CM, leading to a several-fold higher BL compared to iPS-CM expressing FLuc from random genomic loci. To investigate the survival kinetics of iPS-CM in vivo, purified CM obtained from iPSC lines expressing FLuc from a random or the ROSA26 locus were transplanted into cryoinfarcted hearts of syngeneic mice. Engraftment of viable cells was monitored by BL imaging over 4 weeks. Transplanted iPS-CM were poorly retained in the myocardium independently of the cell line used. However, up to 8% of cells survived for 28 days at the site of injection, which was confirmed by immunohistological detection of EGFP-positive iPS-CM in the host tissue. Transplantation of iPS-CM did not affect the scar formation or capillary density in the periinfarct region of host myocardium. This report is the first to determine the survival kinetics of drug-selected iPS-CM in the infarcted heart using BL imaging and demonstrates that transgene silencing in the course of iPSC differentiation can be greatly reduced by employing genome editing technology. FLuc-expressing iPS-CM generated in this study will enable further studies to reduce their loss, increase long-term survival and functional integration upon transplantation.
    PLoS ONE 09/2014; 9(9):e107363. DOI:10.1371/journal.pone.0107363 · 3.23 Impact Factor
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    • "Similarly, hESC-CMs are predominantly glycolytic, highlighting their immature and fetal like phenotypes [48], although oxidative metabolism [43], mainly in the form of lactate oxidation, is present [49] (Figure 3B). hESC-CMs express oxidative phosphorylation genes, although at low levels compared to fetal CMs [50]. "
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    ABSTRACT: Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells, are abundant sources of cardiomyocytes (CMs) for cell replacement therapy and other applications such as disease modeling, drug discovery and cardiotoxicity screening. However, hPSC-derived CMs display immature structural, electrophysiological, calcium-handling and metabolic properties. Here, we review various biological as well as physical and topographical cues that are known to associate with the development of native CMs in vivo to gain insights into the development of strategies for facilitated maturation of hPSC-CMs.
    Stem Cell Research & Therapy 01/2014; 5(1):17. DOI:10.1186/scrt406 · 3.37 Impact Factor
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