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. 2005 Feb 10;433(7026):647-53.
doi: 10.1038/nature03215.

Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages

Karl-Ludwig Laugwitz  1 , Alessandra Moretti, Jason Lam, Peter Gruber, Yinhong Chen, Sarah Woodard, Li-Zhu Lin, Chen-Leng Cai, Min Min Lu, Michael Reth, Oleksandr Platoshyn, Jason X-J Yuan, Sylvia Evans, Kenneth R Chien
Affiliations

Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages

Karl-Ludwig Laugwitz et al. Nature. .

Erratum in

  • Nature. 2007 Apr 19;446(7138):934

Abstract

The purification, renewal and differentiation of native cardiac progenitors would form a mechanistic underpinning for unravelling steps for cardiac cell lineage formation, and their links to forms of congenital and adult cardiac diseases. Until now there has been little evidence for native cardiac precursor cells in the postnatal heart. Herein, we report the identification of isl1+ cardiac progenitors in postnatal rat, mouse and human myocardium. A cardiac mesenchymal feeder layer allows renewal of the isolated progenitor cells with maintenance of their capability to adopt a fully differentiated cardiomyocyte phenotype. Tamoxifen-inducible Cre/lox technology enables selective marking of this progenitor cell population including its progeny, at a defined time, and purification to relative homogeneity. Co-culture studies with neonatal myocytes indicate that isl1+ cells represent authentic, endogenous cardiac progenitors (cardioblasts) that display highly efficient conversion to a mature cardiac phenotype with stable expression of myocytic markers (25%) in the absence of cell fusion, intact Ca2+-cycling, and the generation of action potentials. The discovery of native cardioblasts represents a genetically based system to identify steps in cardiac cell lineage formation and maturation in development and disease.

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Conflict of interest statement

Competing interests statement

The authors declare that they have no competing financial interests.

Figures

Figure 1
Figure 1
Isl1+ progenitors in the late embryonic and postnatal heart. a–d, Isl1+ progenitors in mouse sections at ED12.5 (a, atrial septum; c, ventricular tissue) and 18.5 (b, free atrial wall; d, ventricular arterial region). Scale bar, 20 μm (a, b, d), 50 μm (c). EV, eustachian valve; RA, right atrium; Ao, aorta; LMCA, left main coronary artery; Pa, pulmonary artery. e, f, Cluster of isl1+ cardiac precursors embedded in right atrial tissue and in the right ventricle of a postnatal day 1 rat heart. Scale bars, 10 μm (e), 15 μm ( f). g, h, Isl1+ progenitors in human right atrial tissue from an 8-day-old patient and intra-atrial septum from a 2-day-old patient. Scale bars, 75 μm (g), 150 μm (h). i, Quantification and localization of isl1+ cells in postnatal day 1 rat hearts. Mean values ±s.e.m. from three hearts. Arrows designate isl1+ cells in different species and insets represent a magnification of the areas of interest.
Figure 2
Figure 2
Genetic marking of isl1+ progenitors and myocytic cell fate. a, Mice carry one isl1-IRES-Cre allele and one R26R reporter gene. Cre expression catalyses excision of the stop cassette, resulting in selective lacZ expression and genetic marking of isl1-expressing cells and their differentiated progeny. RTV, retroviral integration sequence. Shown are β-gal+ cardiomyocytes in the right ventricle (middle panel; inset demonstrates α-actinin expression; scale bar, 180 μm) and after cell isolation (right panel; scale bar, 20 μm) from double heterozygous hearts of 4-month- or 1-day-old animals, respectively. b, Immunocytochemistry for β-gal and sarcomeric α-actinin in isolated cardiac myocytes from animals carrying both alleles. Scale bar, 15 μm.
Figure 3
Figure 3
Specificity of recombination and distribution of β-gal+ cells in isl1-mER-Cre-mER/R26R hearts. a, Tamoxifen injection of isl1-mER-Cre-mER/R26R double heterozygous mice or administration of 4-OH-TM in culture results in heritable expression of lacZ. Labelling with C12FDG allows FACS purification of isl1+ cells. b, X-gal stain in whole-mount double heterozygous embryos with or without tamoxifen injection into pregnant mothers at ED7.5. c, Cre localization in isl1+ cells from isl1-mER-Cre-mER/R26R embryos is cytosolic without tamoxifen and nuclear with tamoxifen injection. Scale bar, 25 μm. d, Downregulation of isl1 and Cre in sequential sections of right ventricular and atrial tissue (left panels; scale bar, 80 μm) but not of dorsal mesocardium (right panels; scale bar, 30 μm). e, Sections from hearts of 1-day-old double heterozygous mice injected with tamoxifen at ED17, after X-gal stain and haematoxylin counterstain. LMCA, left main coronary artery; Ao, aorta; RA, right atrium; RV, right ventricle. Scale bar, 150 μm. Arrows designate β-gal+ cells and insets represent a magnification of the areas of interest.
Figure 4
Figure 4
Amplification, characterization and myocytic differentiation of isl1+ cardioblasts in vitro. a, b, Mesenchymal cell fractions from isl1-mER-Cre-mER/R26R hearts at 10 days in culture after 4-OH-TM treatment. Arrows point to β-gal+ cardioblasts detected by X-gal stain (a) and isl1 expression (b). Scale bars, 15 μm. c, d, Histograms of FACS-sorted β-gal+-C12FDG-labelled cells in cardiac mesenchymal fractions after 5 days (c) and 14 days (d) in culture. e, RT–PCR analysis for myocytic and progenitor markers in FACS-sorted progenitors (P) and neonatal myocytes (M). f–h, Flow cytometry profile of Hoechst 33342 dye efflux in cardiac mesenchymal cell fractions from double heterozygous animals after C12FDG and sca-1 labelling. i, j, Neonatal myocytes isolated from isl1-mER-Cre-mER/R26R mice at 4 days in culture. Arrows designate β-gal+ cells coexpressing troponin T and inset shows the same cells before immunocytochemistry. Scale bar, 15 μm (i). Frequency of β-gal+ myocytes and non-myocytes over time ( j, mean value ±s.e.m., n = 3). Exposure to 1 μM 4-OH-TM was performed at the first day in culture. k, Images of differentiated β-gal+ progenitors in co-culture with wild-type neonatal myocytes. Arrows mark progenitor-derived β-gal+ myocytes. Scale bar, 15 μm. l, Quantification of differentiation events over time in co-culture of β-gal+ progenitors FACS-sorted at day 10–14 (left panel) and comparison at 5 days co-culture between β-gal+ progenitors expanded for 5 days (early) or 14 days (late) in culture (right panel). Mean values ±s.e.m. from six experiments (n = 1,200 cells per group). m, Cell fusion independent cardioblast-myocyte conversion. Propidium-iodide stain in pre-fixed myocytes (scale bar, 25 μm). Arrows point to QD655-labelled precursors expressing α-actinin in β-gal+, but not in β-gal− cells during co-culture with pre-fixed myocytes. Scale bar, 15 μm. The diagram represents a quantification of fusion-independent myocytic transition. Grey columns indicate the percentage of β-gal+ cells expressing α-actinin in absence of pre-fixed myocytes. Mean values ±s.e.m. (n = 3).
Figure 5
Figure 5
Real time [Ca2+]i transients and action potentials in FACS-sorted β-gal+ precursors after myocytic differentiation in co-culture. a, b, Identification of β-gal+ cells by C12FDG labelling, before cell loading with the Ca2+ indicator fluo-4. Pseudo-colour images show minimal ( Camin2+) and maximal ( Camax2+) fluo-4 fluorescence intensity and circles indicate the Ca2+ measuring areas as outlined in the fluo-4 intensity traces in b. Eighteen measured cells: n = 11 differentiated progenitors, n = 7 neonatal myocytes. c, Increase in [Ca2+]i transient amplitude of a β-gal+ cell under isoproterenol (10−7 M) stimulation. d, Calcium transients of differentiated C12FDG+ precursor in response to electrical pacing at different frequencies after cell loading with the Ca2+ indicator fura-2. Six measured C12FDG+ cells. e, QD655-marked differentiated progenitors stained for α-actinin (left panel; scale bar, 20 μm) and impaled by an intracellular electrode to measure electrical activity (right panel). Inset represents the QD655+ cell. f, Action potentials in a differentiated progenitor and a neonatal cardiomyocyte. Summarized data (means ± s.e.m.) showing dV/dt max (the maximum rate of rise of the action potential stroke), APA, APD90 (action potential duration at 90% of repolarization) and MDP (the maximum diastolic potential). We analysed 160 action potentials from four different cells for each cell type. g, Working model of isl1+ cardiac progenitors for self-renewal and cardiomyocytic differentiation.
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