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Cardiopoietic programming of embryonic stem cells for tumor-free heart repair.

Behfar A, Perez-Terzic C, Faustino RS, Arrell DK, Hodgson DM, Yamada S, Puceat M, Niederländer N, Alekseev AE, Zingman LV, Terzic A - J. Exp. Med. (2007)

Bottom Line: Here, the tumorigenic threat associated with embryonic stem cell transplantation was suppressed by cardiac-restricted transgenic expression of the reprogramming cytokine TNF-alpha, enhancing the cardiogenic competence of recipient heart.Characterized by a down-regulation of oncogenic markers, up-regulation, and nuclear translocation of cardiac transcription factors, this predetermined population yielded functional cardiomyocyte progeny.Thus, cardiopoietic programming establishes a strategy to hone stem cell pluripotency, offering a tumor-resistant approach for regeneration.

View Article: PubMed Central - PubMed

Affiliation: Marriott Heart Disease Research Program, Division of Cardiovascular Diseases, Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA.

ABSTRACT
Embryonic stem cells have the distinct potential for tissue regeneration, including cardiac repair. Their propensity for multilineage differentiation carries, however, the liability of neoplastic growth, impeding therapeutic application. Here, the tumorigenic threat associated with embryonic stem cell transplantation was suppressed by cardiac-restricted transgenic expression of the reprogramming cytokine TNF-alpha, enhancing the cardiogenic competence of recipient heart. The in vivo aptitude of TNF-alpha to promote cardiac differentiation was recapitulated in embryoid bodies in vitro. The procardiogenic action required an intact endoderm and was mediated by secreted cardio-inductive signals. Resolved TNF-alpha-induced endoderm-derived factors, combined in a cocktail, secured guided differentiation of embryonic stem cells in monolayers produce cardiac progenitors termed cardiopoietic cells. Characterized by a down-regulation of oncogenic markers, up-regulation, and nuclear translocation of cardiac transcription factors, this predetermined population yielded functional cardiomyocyte progeny. Recruited cardiopoietic cells delivered in infarcted hearts generated cardiomyocytes that proliferated into scar tissue, integrating with host myocardium for tumor-free repair. Thus, cardiopoietic programming establishes a strategy to hone stem cell pluripotency, offering a tumor-resistant approach for regeneration.

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Synergy of recombinant secretome components drives cardiogenesis of embryonic stem cells in monolayer, revealing a cardiopoietic population. (A) Untreated embryonic stem cells (D0, row 1) or cells treated for 2 d (D2, row 2) or 8 d (D8, row 3) with individual components (i.e., BMP-4, FGF-2, and TGF-β1) did not demonstrate cardiogenic specification as expression of cardiac transcription factors (Nkx2.5, columns 1–3; MEF-2C, column 4; GATA-4, column 5) was limited to the cytosol at D2 and diminished in expression by D8. (B) Combinatorial stimulation with identified secretome components resulted by day 2 in nuclear translocation of Nkx2.5 with strong cytosolic expression of MEF-2C (D2, row 1), followed by recruitment of the cardiopoietic phenotype with nuclear translocation of Nkx2.5, MEF-2C, and GATA-4 (D4, row 2) to yield cardiomyocytes with completed sarcomerogenesis by day 7 demonstrated by α-actinin (D7, row 3). Bars: (A) 20 μm; (B) 15 μm.
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fig6: Synergy of recombinant secretome components drives cardiogenesis of embryonic stem cells in monolayer, revealing a cardiopoietic population. (A) Untreated embryonic stem cells (D0, row 1) or cells treated for 2 d (D2, row 2) or 8 d (D8, row 3) with individual components (i.e., BMP-4, FGF-2, and TGF-β1) did not demonstrate cardiogenic specification as expression of cardiac transcription factors (Nkx2.5, columns 1–3; MEF-2C, column 4; GATA-4, column 5) was limited to the cytosol at D2 and diminished in expression by D8. (B) Combinatorial stimulation with identified secretome components resulted by day 2 in nuclear translocation of Nkx2.5 with strong cytosolic expression of MEF-2C (D2, row 1), followed by recruitment of the cardiopoietic phenotype with nuclear translocation of Nkx2.5, MEF-2C, and GATA-4 (D4, row 2) to yield cardiomyocytes with completed sarcomerogenesis by day 7 demonstrated by α-actinin (D7, row 3). Bars: (A) 20 μm; (B) 15 μm.

Mentions: The cardiogenic aptitude of individual candidates identified within the TNF-α–primed endodermal secretome was tested on embryonic stem cells in monolayer and found to increase the expression of early cardiac transcription factors Nkx2.5 and MEF-2C localized to the cytosol (Fig. 6 A). Induction with single recombinant factors, e.g., BMP-2 or -4, TGF-β1, IGF-1 or -2, and FGF-2 or -4, was, however, not sufficient to mediate nuclear import of cardiac transcription factors in differentiating stem cells, a critical step for definitive engagement into the cardiac program (Fig. 6 A; reference 47). Rather, the synergy of factors (TGF-β1, BMP-2 and -4, activin-A, VEGF-A, IL-6, FGF-2 and -4, IGF-1 and -2, and EGF) identified in the secretome, used as a recombinant cocktail regimen, induced nuclear translocation of Nkx2.5 by day 2, and of later cardiac transcription factors MEF-2C and GATA4 by day 4, indicative of definitive commitment to cardiac differentiation (Fig. 6 B). In this way, the cardiogenic cocktail demonstrated a capacity to recruit from embryonic stem cells a monolayer of cells en route to cardiac maturation. This intermediate cell phenotype—termed cardiopoietic progenitor cells—completed the cardiac differentiation program by day 7, demonstrating definitive expression of myofibrillar proteins (α-actinin) and sarcomeric organization (Fig. 6 B). Specifically, when day 4 cardiopoietic stem cells at 10,000 cells/ml were continuously cultured in the presence of the cardiogenic cocktail, sarcomeric differentiation was achieved by day 7 in ≥10% of cells, day 9 in ≥30% of cells, and day 12 in ≥65% of cells in culture (Fig. 7, A–C). Removal of the cardiogenic cocktail after 4 d of recombinant stimulation resulted in continued engagement of cardiopoietic cells in the cell cycle dividing every 36 h without differentiation into cardiomyocytes by day 9 (≤5% of cells), and withdrawal from cell cycle upon confluence (Fig. 7 D). Electron microscopy further established the transitional state of cardiopoietic cells relinquishing a phenotype of high nucleus-to-cytosol ratio typical of embryonic stem cells (48), while acquiring a progressively mature cardiac structure (Fig. 7 E). Thus, the identified cardiopoietic cell population demonstrated maintained mitotic activity, a remnant property of the embryonic source, and reproducibly acquired contact inhibition with execution of the cardiac program under the combinatorial guidance of the cardiogenic cocktail composed of factors identified in the TNF-α–primed endodermal secretome.


Cardiopoietic programming of embryonic stem cells for tumor-free heart repair.

Behfar A, Perez-Terzic C, Faustino RS, Arrell DK, Hodgson DM, Yamada S, Puceat M, Niederländer N, Alekseev AE, Zingman LV, Terzic A - J. Exp. Med. (2007)

Synergy of recombinant secretome components drives cardiogenesis of embryonic stem cells in monolayer, revealing a cardiopoietic population. (A) Untreated embryonic stem cells (D0, row 1) or cells treated for 2 d (D2, row 2) or 8 d (D8, row 3) with individual components (i.e., BMP-4, FGF-2, and TGF-β1) did not demonstrate cardiogenic specification as expression of cardiac transcription factors (Nkx2.5, columns 1–3; MEF-2C, column 4; GATA-4, column 5) was limited to the cytosol at D2 and diminished in expression by D8. (B) Combinatorial stimulation with identified secretome components resulted by day 2 in nuclear translocation of Nkx2.5 with strong cytosolic expression of MEF-2C (D2, row 1), followed by recruitment of the cardiopoietic phenotype with nuclear translocation of Nkx2.5, MEF-2C, and GATA-4 (D4, row 2) to yield cardiomyocytes with completed sarcomerogenesis by day 7 demonstrated by α-actinin (D7, row 3). Bars: (A) 20 μm; (B) 15 μm.
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Related In: Results  -  Collection

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fig6: Synergy of recombinant secretome components drives cardiogenesis of embryonic stem cells in monolayer, revealing a cardiopoietic population. (A) Untreated embryonic stem cells (D0, row 1) or cells treated for 2 d (D2, row 2) or 8 d (D8, row 3) with individual components (i.e., BMP-4, FGF-2, and TGF-β1) did not demonstrate cardiogenic specification as expression of cardiac transcription factors (Nkx2.5, columns 1–3; MEF-2C, column 4; GATA-4, column 5) was limited to the cytosol at D2 and diminished in expression by D8. (B) Combinatorial stimulation with identified secretome components resulted by day 2 in nuclear translocation of Nkx2.5 with strong cytosolic expression of MEF-2C (D2, row 1), followed by recruitment of the cardiopoietic phenotype with nuclear translocation of Nkx2.5, MEF-2C, and GATA-4 (D4, row 2) to yield cardiomyocytes with completed sarcomerogenesis by day 7 demonstrated by α-actinin (D7, row 3). Bars: (A) 20 μm; (B) 15 μm.
Mentions: The cardiogenic aptitude of individual candidates identified within the TNF-α–primed endodermal secretome was tested on embryonic stem cells in monolayer and found to increase the expression of early cardiac transcription factors Nkx2.5 and MEF-2C localized to the cytosol (Fig. 6 A). Induction with single recombinant factors, e.g., BMP-2 or -4, TGF-β1, IGF-1 or -2, and FGF-2 or -4, was, however, not sufficient to mediate nuclear import of cardiac transcription factors in differentiating stem cells, a critical step for definitive engagement into the cardiac program (Fig. 6 A; reference 47). Rather, the synergy of factors (TGF-β1, BMP-2 and -4, activin-A, VEGF-A, IL-6, FGF-2 and -4, IGF-1 and -2, and EGF) identified in the secretome, used as a recombinant cocktail regimen, induced nuclear translocation of Nkx2.5 by day 2, and of later cardiac transcription factors MEF-2C and GATA4 by day 4, indicative of definitive commitment to cardiac differentiation (Fig. 6 B). In this way, the cardiogenic cocktail demonstrated a capacity to recruit from embryonic stem cells a monolayer of cells en route to cardiac maturation. This intermediate cell phenotype—termed cardiopoietic progenitor cells—completed the cardiac differentiation program by day 7, demonstrating definitive expression of myofibrillar proteins (α-actinin) and sarcomeric organization (Fig. 6 B). Specifically, when day 4 cardiopoietic stem cells at 10,000 cells/ml were continuously cultured in the presence of the cardiogenic cocktail, sarcomeric differentiation was achieved by day 7 in ≥10% of cells, day 9 in ≥30% of cells, and day 12 in ≥65% of cells in culture (Fig. 7, A–C). Removal of the cardiogenic cocktail after 4 d of recombinant stimulation resulted in continued engagement of cardiopoietic cells in the cell cycle dividing every 36 h without differentiation into cardiomyocytes by day 9 (≤5% of cells), and withdrawal from cell cycle upon confluence (Fig. 7 D). Electron microscopy further established the transitional state of cardiopoietic cells relinquishing a phenotype of high nucleus-to-cytosol ratio typical of embryonic stem cells (48), while acquiring a progressively mature cardiac structure (Fig. 7 E). Thus, the identified cardiopoietic cell population demonstrated maintained mitotic activity, a remnant property of the embryonic source, and reproducibly acquired contact inhibition with execution of the cardiac program under the combinatorial guidance of the cardiogenic cocktail composed of factors identified in the TNF-α–primed endodermal secretome.

Bottom Line: Here, the tumorigenic threat associated with embryonic stem cell transplantation was suppressed by cardiac-restricted transgenic expression of the reprogramming cytokine TNF-alpha, enhancing the cardiogenic competence of recipient heart.Characterized by a down-regulation of oncogenic markers, up-regulation, and nuclear translocation of cardiac transcription factors, this predetermined population yielded functional cardiomyocyte progeny.Thus, cardiopoietic programming establishes a strategy to hone stem cell pluripotency, offering a tumor-resistant approach for regeneration.

View Article: PubMed Central - PubMed

Affiliation: Marriott Heart Disease Research Program, Division of Cardiovascular Diseases, Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA.

ABSTRACT
Embryonic stem cells have the distinct potential for tissue regeneration, including cardiac repair. Their propensity for multilineage differentiation carries, however, the liability of neoplastic growth, impeding therapeutic application. Here, the tumorigenic threat associated with embryonic stem cell transplantation was suppressed by cardiac-restricted transgenic expression of the reprogramming cytokine TNF-alpha, enhancing the cardiogenic competence of recipient heart. The in vivo aptitude of TNF-alpha to promote cardiac differentiation was recapitulated in embryoid bodies in vitro. The procardiogenic action required an intact endoderm and was mediated by secreted cardio-inductive signals. Resolved TNF-alpha-induced endoderm-derived factors, combined in a cocktail, secured guided differentiation of embryonic stem cells in monolayers produce cardiac progenitors termed cardiopoietic cells. Characterized by a down-regulation of oncogenic markers, up-regulation, and nuclear translocation of cardiac transcription factors, this predetermined population yielded functional cardiomyocyte progeny. Recruited cardiopoietic cells delivered in infarcted hearts generated cardiomyocytes that proliferated into scar tissue, integrating with host myocardium for tumor-free repair. Thus, cardiopoietic programming establishes a strategy to hone stem cell pluripotency, offering a tumor-resistant approach for regeneration.

Show MeSH
Related in: MedlinePlus