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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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Related in: MedlinePlus

Procardiogenic TNF-α effects are mediated through the endoderm. (A) During embryoid body differentiation of embryonic stem cells (ES), mRNA expression profiling of cardiac transcription factors (Nkx2.5, MEF-2C, GATA4) revealed a window (days D4–D7) of exponential up-regulation (CARDIOPOIETIC WINDOW) 3 d before the appearance of sarcomeric mRNA (βMHC). Arbitrary units were calculated relative to the expression of β-tubulin used as denominator. (B and C) TNF-α (30 ng/ml) doubled the expression of cardiac transcription factors in comparison to untreated controls during the cardiopoietic window (B) and accelerated the formation of beating embryoid bodies (C). (D and E) TNF-α (30 ng/ml) increased sarcomeric content in treated ((+)TNFα) versus untreated ((−)TNFα) embryoid bodies determined by α-actinin immunofluorescence at day 9 (D9) of differentiation (middle) without altering sarcomeric organization (right). Bars in D apply also to E. (F) The percentage of embryoid body area beating was dependent on the TNF-α concentration. (G) TNF-α treatment increased the yield of cardiomyocytes after isolation from dissociated embryoid bodies. (H and I) The cardiogenic effect of TNF-α (30 ng/ml) on embryoid bodies was inhibited by infliximab (150 ng/ml), a neutralizing TNF-α antibody, as determined by beating area on video microscopy (H) and α-actinin staining (I). (H, inset) TNF-α is effective only after the initiation of embryoid body differentiation (days 2–5) producing a 2.5-fold increase in cardiomyocyte yield compared with TNF-α–treated embryonic stem cells (day 0). (J) TNF-α required an intact endoderm to induce cardiogenesis, as endodermal disruption prevented cytokine action. Addition of conditioned medium from isolated ventral endoderm (Endo. Secretome) rescued cardiogenesis but did not restore the TNF-α effect. Addition of condition medium from TNF-α–treated endoderm (TNFα Rx Endo. Secr.) resulted in cardiogenesis at levels equal to that of TNF-α–treated embryoid bodies irrespective of the presence of TNF-α on the embryoid body itself. *, P < 0.05 versus untreated.
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fig3: Procardiogenic TNF-α effects are mediated through the endoderm. (A) During embryoid body differentiation of embryonic stem cells (ES), mRNA expression profiling of cardiac transcription factors (Nkx2.5, MEF-2C, GATA4) revealed a window (days D4–D7) of exponential up-regulation (CARDIOPOIETIC WINDOW) 3 d before the appearance of sarcomeric mRNA (βMHC). Arbitrary units were calculated relative to the expression of β-tubulin used as denominator. (B and C) TNF-α (30 ng/ml) doubled the expression of cardiac transcription factors in comparison to untreated controls during the cardiopoietic window (B) and accelerated the formation of beating embryoid bodies (C). (D and E) TNF-α (30 ng/ml) increased sarcomeric content in treated ((+)TNFα) versus untreated ((−)TNFα) embryoid bodies determined by α-actinin immunofluorescence at day 9 (D9) of differentiation (middle) without altering sarcomeric organization (right). Bars in D apply also to E. (F) The percentage of embryoid body area beating was dependent on the TNF-α concentration. (G) TNF-α treatment increased the yield of cardiomyocytes after isolation from dissociated embryoid bodies. (H and I) The cardiogenic effect of TNF-α (30 ng/ml) on embryoid bodies was inhibited by infliximab (150 ng/ml), a neutralizing TNF-α antibody, as determined by beating area on video microscopy (H) and α-actinin staining (I). (H, inset) TNF-α is effective only after the initiation of embryoid body differentiation (days 2–5) producing a 2.5-fold increase in cardiomyocyte yield compared with TNF-α–treated embryonic stem cells (day 0). (J) TNF-α required an intact endoderm to induce cardiogenesis, as endodermal disruption prevented cytokine action. Addition of conditioned medium from isolated ventral endoderm (Endo. Secretome) rescued cardiogenesis but did not restore the TNF-α effect. Addition of condition medium from TNF-α–treated endoderm (TNFα Rx Endo. Secr.) resulted in cardiogenesis at levels equal to that of TNF-α–treated embryoid bodies irrespective of the presence of TNF-α on the embryoid body itself. *, P < 0.05 versus untreated.

Mentions: The procardiogenic action of TNF-α revealed through in vivo transgenesis was validated and dissected in vitro through differentiation of embryonic stem cells in a controlled model of cardiogenesis (37). Within developing embryoid bodies, early cardiogenesis was characterized by a time window of exponential rise in markers of cardiac specification (Fig. 3 A). Between day 4 and 7 of differentiation, genes encoding cardiac transcription factors (Nkx2.5, MEF-2C, and GATA4) were reproducibly up-regulated before expression of sarcomeric genes (β-MHC) indicative of terminal differentiation (n = 6 differentiation experiments with 20 embryoid bodies for each time point; Fig. 3 A). Within this time window of early differentiation, embryoid bodies treated with TNF-α (30 ng/ml) demonstrated significant up-regulation of Nkx2.5, MEF-2C, and GATA4 (n = 6 differentiation experiments; Fig. 3 B) compared with untreated controls (P < 0.05). TNF-α treatment translated into an accelerated cardiac differentiation determined by an earlier increase in the number of beating embryoid bodies sustained throughout differentiation (n = 80 embryoid bodies; Fig. 3 C). TNF-α, in a concentration-dependent manner, increased cardiac content within each embryoid body as determined at day 9 of differentiation by α-actinin staining of sarcomeres (n = 30 embryoid bodies; Fig. 3, D and E), areas of beating activity (n = 30 embryoid bodies; Fig. 3 F), and quantification of cardiomyocyte yield (n = 3 differentiation experiments with 30 embryoid bodies for each isolation; Fig. 3 G). The cardiogenic effect of TNF-α was prevented by cotreatment with the TNF-α antagonist, infliximab (150 ng/ml, n = 6; Fig. 3, H and I). The action of TNF-α required that embryonic stem cells undergo trigerminal differentiation into embryoid bodies as treatment of stem cells in the pluripotent state did not promote cardiogenesis (Fig. 3 I, inset). In fact, the cardiogenic effect of TNF-α was found to be dependent on the endoderm (Fig. 3 J). Although trigerminal embryoid bodies displayed a vigorous response to TNF-α that augmented the beating area by >2-fold above baseline (Fig. 3 J, first pair), embryoid bodies lacking endoderm after treatment with leukemia inhibitory factor (100 U/μl; 38) lost their cardiogenic response to TNF-α (Fig. 3 J, second pair). In endoderm-deficient embryoid bodies, rescue of baseline cardiogenesis was achieved by addition of conditioned medium derived from isolated visceral endoderm-like cells (Fig. 3 J, third pair). Endoderm-deficient embryoid bodies rescued by visceral endoderm-conditioned medium did not, however, demonstrate a cardiogenic response to TNF-α treatment, indicating that the cytokine must target the endodermal layer (Fig. 3 J, third pair). Rescue of TNF-α action was achieved through treatment of endoderm-deficient embryoid bodies with condition medium derived from TNF-α–primed visceral endoderm-like cells (Fig. 3 J, fourth pair). The boost in the cardiogenic propensity of endoderm-derived conditioned medium after TNF-α stimulation was reproduced on pluripotent stem cells differentiating in monolayer. Visceral endodermal-like cells treated with TNF-α had a twofold increased capacity to induce cardiogenic differentiation of embryonic stem cells, with the total yield of cells demonstrating nuclear translocation of Nkx2.5 and MEF-2C increased from 34 ± 4% in the untreated condition to 73 ± 6% in the TNF-α–treated condition (data not shown). Thus, the procardiogenic action of TNF-α observed in vivo can be recapitulated in vitro with the action of the cytokine dependent on the endoderm.


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)

Procardiogenic TNF-α effects are mediated through the endoderm. (A) During embryoid body differentiation of embryonic stem cells (ES), mRNA expression profiling of cardiac transcription factors (Nkx2.5, MEF-2C, GATA4) revealed a window (days D4–D7) of exponential up-regulation (CARDIOPOIETIC WINDOW) 3 d before the appearance of sarcomeric mRNA (βMHC). Arbitrary units were calculated relative to the expression of β-tubulin used as denominator. (B and C) TNF-α (30 ng/ml) doubled the expression of cardiac transcription factors in comparison to untreated controls during the cardiopoietic window (B) and accelerated the formation of beating embryoid bodies (C). (D and E) TNF-α (30 ng/ml) increased sarcomeric content in treated ((+)TNFα) versus untreated ((−)TNFα) embryoid bodies determined by α-actinin immunofluorescence at day 9 (D9) of differentiation (middle) without altering sarcomeric organization (right). Bars in D apply also to E. (F) The percentage of embryoid body area beating was dependent on the TNF-α concentration. (G) TNF-α treatment increased the yield of cardiomyocytes after isolation from dissociated embryoid bodies. (H and I) The cardiogenic effect of TNF-α (30 ng/ml) on embryoid bodies was inhibited by infliximab (150 ng/ml), a neutralizing TNF-α antibody, as determined by beating area on video microscopy (H) and α-actinin staining (I). (H, inset) TNF-α is effective only after the initiation of embryoid body differentiation (days 2–5) producing a 2.5-fold increase in cardiomyocyte yield compared with TNF-α–treated embryonic stem cells (day 0). (J) TNF-α required an intact endoderm to induce cardiogenesis, as endodermal disruption prevented cytokine action. Addition of conditioned medium from isolated ventral endoderm (Endo. Secretome) rescued cardiogenesis but did not restore the TNF-α effect. Addition of condition medium from TNF-α–treated endoderm (TNFα Rx Endo. Secr.) resulted in cardiogenesis at levels equal to that of TNF-α–treated embryoid bodies irrespective of the presence of TNF-α on the embryoid body itself. *, P < 0.05 versus untreated.
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Related In: Results  -  Collection

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fig3: Procardiogenic TNF-α effects are mediated through the endoderm. (A) During embryoid body differentiation of embryonic stem cells (ES), mRNA expression profiling of cardiac transcription factors (Nkx2.5, MEF-2C, GATA4) revealed a window (days D4–D7) of exponential up-regulation (CARDIOPOIETIC WINDOW) 3 d before the appearance of sarcomeric mRNA (βMHC). Arbitrary units were calculated relative to the expression of β-tubulin used as denominator. (B and C) TNF-α (30 ng/ml) doubled the expression of cardiac transcription factors in comparison to untreated controls during the cardiopoietic window (B) and accelerated the formation of beating embryoid bodies (C). (D and E) TNF-α (30 ng/ml) increased sarcomeric content in treated ((+)TNFα) versus untreated ((−)TNFα) embryoid bodies determined by α-actinin immunofluorescence at day 9 (D9) of differentiation (middle) without altering sarcomeric organization (right). Bars in D apply also to E. (F) The percentage of embryoid body area beating was dependent on the TNF-α concentration. (G) TNF-α treatment increased the yield of cardiomyocytes after isolation from dissociated embryoid bodies. (H and I) The cardiogenic effect of TNF-α (30 ng/ml) on embryoid bodies was inhibited by infliximab (150 ng/ml), a neutralizing TNF-α antibody, as determined by beating area on video microscopy (H) and α-actinin staining (I). (H, inset) TNF-α is effective only after the initiation of embryoid body differentiation (days 2–5) producing a 2.5-fold increase in cardiomyocyte yield compared with TNF-α–treated embryonic stem cells (day 0). (J) TNF-α required an intact endoderm to induce cardiogenesis, as endodermal disruption prevented cytokine action. Addition of conditioned medium from isolated ventral endoderm (Endo. Secretome) rescued cardiogenesis but did not restore the TNF-α effect. Addition of condition medium from TNF-α–treated endoderm (TNFα Rx Endo. Secr.) resulted in cardiogenesis at levels equal to that of TNF-α–treated embryoid bodies irrespective of the presence of TNF-α on the embryoid body itself. *, P < 0.05 versus untreated.
Mentions: The procardiogenic action of TNF-α revealed through in vivo transgenesis was validated and dissected in vitro through differentiation of embryonic stem cells in a controlled model of cardiogenesis (37). Within developing embryoid bodies, early cardiogenesis was characterized by a time window of exponential rise in markers of cardiac specification (Fig. 3 A). Between day 4 and 7 of differentiation, genes encoding cardiac transcription factors (Nkx2.5, MEF-2C, and GATA4) were reproducibly up-regulated before expression of sarcomeric genes (β-MHC) indicative of terminal differentiation (n = 6 differentiation experiments with 20 embryoid bodies for each time point; Fig. 3 A). Within this time window of early differentiation, embryoid bodies treated with TNF-α (30 ng/ml) demonstrated significant up-regulation of Nkx2.5, MEF-2C, and GATA4 (n = 6 differentiation experiments; Fig. 3 B) compared with untreated controls (P < 0.05). TNF-α treatment translated into an accelerated cardiac differentiation determined by an earlier increase in the number of beating embryoid bodies sustained throughout differentiation (n = 80 embryoid bodies; Fig. 3 C). TNF-α, in a concentration-dependent manner, increased cardiac content within each embryoid body as determined at day 9 of differentiation by α-actinin staining of sarcomeres (n = 30 embryoid bodies; Fig. 3, D and E), areas of beating activity (n = 30 embryoid bodies; Fig. 3 F), and quantification of cardiomyocyte yield (n = 3 differentiation experiments with 30 embryoid bodies for each isolation; Fig. 3 G). The cardiogenic effect of TNF-α was prevented by cotreatment with the TNF-α antagonist, infliximab (150 ng/ml, n = 6; Fig. 3, H and I). The action of TNF-α required that embryonic stem cells undergo trigerminal differentiation into embryoid bodies as treatment of stem cells in the pluripotent state did not promote cardiogenesis (Fig. 3 I, inset). In fact, the cardiogenic effect of TNF-α was found to be dependent on the endoderm (Fig. 3 J). Although trigerminal embryoid bodies displayed a vigorous response to TNF-α that augmented the beating area by >2-fold above baseline (Fig. 3 J, first pair), embryoid bodies lacking endoderm after treatment with leukemia inhibitory factor (100 U/μl; 38) lost their cardiogenic response to TNF-α (Fig. 3 J, second pair). In endoderm-deficient embryoid bodies, rescue of baseline cardiogenesis was achieved by addition of conditioned medium derived from isolated visceral endoderm-like cells (Fig. 3 J, third pair). Endoderm-deficient embryoid bodies rescued by visceral endoderm-conditioned medium did not, however, demonstrate a cardiogenic response to TNF-α treatment, indicating that the cytokine must target the endodermal layer (Fig. 3 J, third pair). Rescue of TNF-α action was achieved through treatment of endoderm-deficient embryoid bodies with condition medium derived from TNF-α–primed visceral endoderm-like cells (Fig. 3 J, fourth pair). The boost in the cardiogenic propensity of endoderm-derived conditioned medium after TNF-α stimulation was reproduced on pluripotent stem cells differentiating in monolayer. Visceral endodermal-like cells treated with TNF-α had a twofold increased capacity to induce cardiogenic differentiation of embryonic stem cells, with the total yield of cells demonstrating nuclear translocation of Nkx2.5 and MEF-2C increased from 34 ± 4% in the untreated condition to 73 ± 6% in the TNF-α–treated condition (data not shown). Thus, the procardiogenic action of TNF-α observed in vivo can be recapitulated in vitro with the action of the cytokine dependent on the endoderm.

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