A GPS for Heart Progenitor Cell Lineages
Authors: Jolanta Chmielowiec, Yuh-Shin Chang, Kenneth R Chien
Journal: Molecular Therapy (2011)

The good news about human embryonic stem cells (hESCs) is that they can potentially become any cell. The bad news is that hESCs can potentially become any cell. It is this very conundrum that demands the construction of a road map that will allow high-efficiency generation of a given differentiated daughter cell from pluripotent human stem cells, regardless of whether they are derived from ESCs or induced pluripotent stem cells (iPSCs). In work described in this issue of Molecular Therapy, Dixon and colleagues have devised and implemented a genetics-based approach toward the goal of identifying the key transcriptional factors that can drive the differentiation of hESCs into heart progenitors and their downstream lineages.1Dixon JE Dick E Rajamohan D Shakesheff KM Denning C Directed differentiation of human embryonic stem cells to interrogate the cardiac gene regulatory network.Mol Ther. 2011; 19: 1695-1703Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar The study employed a panel of lentiviral vectors that encompass a hit list of likely candidate factors and then applied them, both individually and in combination, to an engineered, stable hESC line that carries a fluorescent reporter driven by a cardiac-specific enhancer/promoter construct. Using fluorescence-activated cell-sorting analysis, the investigators defined four key transcription factors—GATA4, TBX5, NKX2.5, and the cardiac-specific transcriptional cofactor BAF60c—as being sufficient for the maximal generation of heart progenitors, as identified by the progenitor marker ISL1,2Bu L Jiang X Martin-Puig S Caron L Zhu S Shao Y et al.Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages.Nature. 2009; 460: 113-117Crossref PubMed Scopus (449) Google Scholar,3Laugwitz K-L Moretti A Lam J Gruber P Chen Y Woodard S et al.Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages.Nature. 2005; 433: 647-653Crossref PubMed Scopus (1129) Google Scholar,4Moretti A Caron L Nakano A Lam JT Bernshausen A Chen Y et al.Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification.Cell. 2006; 127: 1151-1165Abstract Full Text Full Text PDF PubMed Scopus (835) Google Scholar as well as of the desired downstream differentiated cardiac muscle cells. In essence, the study recapitulates approaches that Takahashi and Yamanaka5Takahashi K Yamanaka S Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (19291) Google Scholar utilized in the landmark series of experiments outlining the ability to reprogram skin fibroblasts directly into pluripotent stem cells, but in this case for the purpose of determining the minimal combinatorial transcriptional cocktail to drive cardiogenesis. The article by Dixon et al. comes on the heels of a series of studies6Domian IJ Chiravuri M van der Meer P Feinberg AW Wu SM Parker KK et al.Generation of functional ventricular heart muscle from mouse ventricular progenitor cells.Science. 2009; 326: 426-429Crossref PubMed Scopus (169) Google Scholar,7Efe JA Hilcove S Kim J Zhou H Ouyang K Wang G et al.Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy.Nat Cell Biol. 2011; 13: 215-222Crossref PubMed Scopus (525) Google Scholar,8Ieda M Fu J-D Delgado-Olguin P Vedantham V Hayashi Y Bruneau BG et al.Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.Cell. 2010; 142: 375-386Abstract Full Text Full Text PDF PubMed Scopus (1912) Google Scholar,9Qyang Y Martin-Puig S Chiravuri M Chen S Xu H Bu L et al.The renewal and differentiation of Isl1+ cardiovascular progenitors are controlled by a Wnt/beta-catenin pathway.Cell Stem Cell. 2007; 1: 165-179Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar,10Yang L Soonpaa MH Adler ED Roepke TK Kattman SJ Kennedy M et al.Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population.Nature. 2008; 453: 524-528Crossref PubMed Scopus (1162) Google Scholar that have approached the same core problem—generating a sufficient number of mature ventricular muscle cells—via a diverse set of approaches, including optimizing growth-factor cocktails, identifying heart progenitor renewal pathways, direct reprogramming of fibroblasts into cardiomyocyte lineages, and directing the differentiation of pluripotent stem cells into heart progenitor lineages by means of a combination of transcriptional and growth-factor cues (Figure 1). The challenge has its basis in the massive loss of viable cardiac muscle during a myocardial infarction, whereby more than a billion cells can easily be lost (for review, see ref. 11Hansson EM Lindsay ME Chien KR Regeneration next: toward heart stem cell therapeutics.Cell Stem Cell. 2009; 5: 364-377Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) and is compounded by the fact that results from current approaches to delivery, transplantation, and engrafting have suggested that fewer than 5% of the cells that are delivered to the heart will actually engraft long term. Dixon and colleagues’ study provides a genetic road map that will allow a more in-depth focus on the pathways that link these transcription factors with cardiogenesis, and their findings support the concept that cardiac specification is achieved in a combinatorial manner, as opposed to through a single master transcription factor that can specify a myogenic fate in a dominant fashion as is the case with the MyoD gene family in skeletal muscle. To the authors’ credit, and with a refreshing dose of intellectual honesty, they also note that the constitutive expression of the four factors in hESCs results in cardiomyocyte derivatives that are not completely normal, a concern that extends to all the approaches known to date for generating cardiomyocytes from pluripotent stem cell lines or via direct reprogramming of fibroblasts to cardiomyocytes. To document this, the authors used a conditional expression strategy for the four factors utilizing a doxycycline switch system, a commendable approach that has been lacking in some of the recently published studies. Transient expression of TBX5, NKX2.5, and BAF60c leads to activation of the cardiogenesis program in multiple pluripotent hESC lines and ultimately to a mixed population of mature atrial and ventricular cardiomyocytes, and the authors show that this process transitions through all intermediate stages (MESP1+ primitive mesoderm progenitors, multipotent ISL1+ heart progenitor cells, GATA4+/TBX5+/NKX2.5+ committed cardiac progenitor cells). The novelty of the system lies in the ingenious application of the chemotherapeutic agent Ara-C to enrich for cTnI/a-actinin double-positive cells up to 45%. It is unlikely that the constitutive overexpression of any cardiogenic gene will lead to the generation of fully normal mature and durable ventricular muscle cells over the long term. Until this goal can be reached, suggestions to plug any of these cells into large animals for attempts at in vivo cardiac repair may be misplaced. But clearly, as pointed out by Dixon and co-workers, once the ultimate navigation system is devised for human ventriculogenesis, it will probably show us the road to science-driven cardiac regenerative therapeutics.