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Adult murine skeletal muscle contains cells that can differentiate into beating cardiomyocytes in vitro.

Winitsky SO, Gopal TV, Hassanzadeh S, Takahashi H, Gryder D, Rogawski MA, Takeda K, Yu ZX, Xu YH, Epstein ND - PLoS Biol. (2005)

Bottom Line: Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion.Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials.By 6 d they begin to differentiate without fusing to recipient cardiac cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Physiology Section, Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA.

ABSTRACT
It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.

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Related in: MedlinePlus

EMs of Day 0 Spoc CellsElectron micrograph of a freshly isolated Spoc cell (A) shows its small size relative to the typical biconcave profile of the red blood cell above it (arrow). (B) shows a typical Spoc cell with a large number of vesicular structures, lamellipodia, and filopodia. (C) displays three Spoc cells so tightly clumped that their borders are difficult to distinguish. This typical clumping makes fluorescence-activated cell sorting (FACS) analysis difficult.
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pbio-0030087-g001: EMs of Day 0 Spoc CellsElectron micrograph of a freshly isolated Spoc cell (A) shows its small size relative to the typical biconcave profile of the red blood cell above it (arrow). (B) shows a typical Spoc cell with a large number of vesicular structures, lamellipodia, and filopodia. (C) displays three Spoc cells so tightly clumped that their borders are difficult to distinguish. This typical clumping makes fluorescence-activated cell sorting (FACS) analysis difficult.

Mentions: At initial isolation, Spoc cells are CD34− and CD45− (data not shown). Dissociation of 10 g of leg muscle yields approximately 2 × 106 Spoc cells. C-kit+ cells, comprising less than 1% of Spoc cells, are present after isolation but can be removed by affinity purification without any change in the subsequent results (data not shown). Electron microscopy (EM) shows that on the day of isolation, Spoc cells are 4–8 μm in diameter, often smaller than a red blood cell, and display copious vesicles suggestive of active transport. Many lamellipodia and filopodia are present, which may in part explain the typical Velcro-like clumping of the cells that makes chemical and physical dispersion more difficult (Figure 1). The CD34−/CD45−/c-kit− phenotype differentiates Spoc cells from other nonadherent cells previously described as derived from skeletal muscle [5]. Spoc cells are distinguished from satellite cells by the following criteria: (1) Spoc cells do not express Pax-7 [21] or the surface marker c-met [22]; (2) approximately the same number of Spoc cells are isolated from both young (less than 4-wk-old) and older (12- to 16-wk-old) mice, whereas satellite cells are difficult to isolate from normal mouse muscle after 8 wk of age without first inducing muscle injury [21]; (3) at isolation, Spoc cells remain round floating cells approximately 4 μm in diameter, whereas isolated satellite cells soon become adherent; (4) Spoc cells are CD34− and Myf-5−, excluding them from the class of quiescent satellite cells [23]; and (5) the three skeletal muscle super-regulatory genes myf5, myoD, and myogenin are known to be present at some time in satellite cells [22], but in Spoc cells, all three markers remain negative from the day of isolation throughout long-term culture (data not shown). Thus, Spoc cells are neither satellite cells nor further-differentiated skeletal muscle cells.


Adult murine skeletal muscle contains cells that can differentiate into beating cardiomyocytes in vitro.

Winitsky SO, Gopal TV, Hassanzadeh S, Takahashi H, Gryder D, Rogawski MA, Takeda K, Yu ZX, Xu YH, Epstein ND - PLoS Biol. (2005)

EMs of Day 0 Spoc CellsElectron micrograph of a freshly isolated Spoc cell (A) shows its small size relative to the typical biconcave profile of the red blood cell above it (arrow). (B) shows a typical Spoc cell with a large number of vesicular structures, lamellipodia, and filopodia. (C) displays three Spoc cells so tightly clumped that their borders are difficult to distinguish. This typical clumping makes fluorescence-activated cell sorting (FACS) analysis difficult.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1064849&req=5

pbio-0030087-g001: EMs of Day 0 Spoc CellsElectron micrograph of a freshly isolated Spoc cell (A) shows its small size relative to the typical biconcave profile of the red blood cell above it (arrow). (B) shows a typical Spoc cell with a large number of vesicular structures, lamellipodia, and filopodia. (C) displays three Spoc cells so tightly clumped that their borders are difficult to distinguish. This typical clumping makes fluorescence-activated cell sorting (FACS) analysis difficult.
Mentions: At initial isolation, Spoc cells are CD34− and CD45− (data not shown). Dissociation of 10 g of leg muscle yields approximately 2 × 106 Spoc cells. C-kit+ cells, comprising less than 1% of Spoc cells, are present after isolation but can be removed by affinity purification without any change in the subsequent results (data not shown). Electron microscopy (EM) shows that on the day of isolation, Spoc cells are 4–8 μm in diameter, often smaller than a red blood cell, and display copious vesicles suggestive of active transport. Many lamellipodia and filopodia are present, which may in part explain the typical Velcro-like clumping of the cells that makes chemical and physical dispersion more difficult (Figure 1). The CD34−/CD45−/c-kit− phenotype differentiates Spoc cells from other nonadherent cells previously described as derived from skeletal muscle [5]. Spoc cells are distinguished from satellite cells by the following criteria: (1) Spoc cells do not express Pax-7 [21] or the surface marker c-met [22]; (2) approximately the same number of Spoc cells are isolated from both young (less than 4-wk-old) and older (12- to 16-wk-old) mice, whereas satellite cells are difficult to isolate from normal mouse muscle after 8 wk of age without first inducing muscle injury [21]; (3) at isolation, Spoc cells remain round floating cells approximately 4 μm in diameter, whereas isolated satellite cells soon become adherent; (4) Spoc cells are CD34− and Myf-5−, excluding them from the class of quiescent satellite cells [23]; and (5) the three skeletal muscle super-regulatory genes myf5, myoD, and myogenin are known to be present at some time in satellite cells [22], but in Spoc cells, all three markers remain negative from the day of isolation throughout long-term culture (data not shown). Thus, Spoc cells are neither satellite cells nor further-differentiated skeletal muscle cells.

Bottom Line: Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion.Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials.By 6 d they begin to differentiate without fusing to recipient cardiac cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Physiology Section, Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA.

ABSTRACT
It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.

Show MeSH
Related in: MedlinePlus