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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

Proteomic and genomic dissection of TNF-α–primed endodermal secretome. (A) Two-dimensional gel electrophoresis (at pH 3–10 and 6–11) was used to resolve the endodermal secretome concentrated from 5-ml aliquots, with examples of TNF-α up-regulated proteins circled. Protein content was increased approximately twofold in TNF-α–primed versus naive secretome (inset). Triangles: examples of down-regulated or unchanged proteins. (B) Example LTQ tandem mass spectrum showing FKBP-1A peptide (amino acids 2–14). This approach was used to resolve protein identities by respective constituent peptide sequence signatures of individual peptide mass spectra, with relative abundance quantified (inset left) based on spot density (inset right). (C) Transcriptional profiling of total RNA from TNF-α–treated and untreated endoderm showing differential gene expression, including examples of genes encoding proteins identified by proteomics. (D) Volcano plots revealed that 64% of genes were up-regulated and 36% down-regulated in TNF-α–treated versus untreated endoderm. (E) Proteomic shotgun analysis of the TNF-α–treated endodermal secretome and ELISA revealed the identity of secreted proteins, including FGFs, leukemia inhibitory factor, VEGFs, TGF-βs, BMPs, CSF, IGFs, EGF, NGF, and ILs. Pathway analysis of identified proteins visualized the interactome triggered by cytokine stimulation and demonstrated secreted candidate cardiotrophic factors downstream of TNF-α integrated through p38-driven networks with individual proteins depicted in their corresponding cellular compartments. Blue nodes reflect detection by proteomic analysis, and yellow nodes reflect ELISA confirmation. (F) Example of LTQ-FT shotgun tandem mass spectrum showing IGF-2 peptide (amino acids 74–89). This approach was used to provide a more sensitive means of detecting the presence of secreted proteins. *, P < 0.05.
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fig4: Proteomic and genomic dissection of TNF-α–primed endodermal secretome. (A) Two-dimensional gel electrophoresis (at pH 3–10 and 6–11) was used to resolve the endodermal secretome concentrated from 5-ml aliquots, with examples of TNF-α up-regulated proteins circled. Protein content was increased approximately twofold in TNF-α–primed versus naive secretome (inset). Triangles: examples of down-regulated or unchanged proteins. (B) Example LTQ tandem mass spectrum showing FKBP-1A peptide (amino acids 2–14). This approach was used to resolve protein identities by respective constituent peptide sequence signatures of individual peptide mass spectra, with relative abundance quantified (inset left) based on spot density (inset right). (C) Transcriptional profiling of total RNA from TNF-α–treated and untreated endoderm showing differential gene expression, including examples of genes encoding proteins identified by proteomics. (D) Volcano plots revealed that 64% of genes were up-regulated and 36% down-regulated in TNF-α–treated versus untreated endoderm. (E) Proteomic shotgun analysis of the TNF-α–treated endodermal secretome and ELISA revealed the identity of secreted proteins, including FGFs, leukemia inhibitory factor, VEGFs, TGF-βs, BMPs, CSF, IGFs, EGF, NGF, and ILs. Pathway analysis of identified proteins visualized the interactome triggered by cytokine stimulation and demonstrated secreted candidate cardiotrophic factors downstream of TNF-α integrated through p38-driven networks with individual proteins depicted in their corresponding cellular compartments. Blue nodes reflect detection by proteomic analysis, and yellow nodes reflect ELISA confirmation. (F) Example of LTQ-FT shotgun tandem mass spectrum showing IGF-2 peptide (amino acids 74–89). This approach was used to provide a more sensitive means of detecting the presence of secreted proteins. *, P < 0.05.

Mentions: Proteomic two-dimensional gel analysis revealed that the TNF-α stimulation of isolated visceral endoderm-like cells induced secretion of proteins involved in sarcomerogenesis (profilin and cofilin), calcium signaling (calcyclin), myocardial reprogramming (nucleotide diphosphate kinase), and heart formation (FK506 binding protein FKBP12, cystatin, and ubiquitin) (39–42; Fig. 4 A). The identity of each protein was resolved by reconstruction of constitutive peptides using tandem mass spectrometry (Fig. 4 B) with the overall twofold increase in protein content after cytokine induction (Fig. 4 A, inset) confirmed at an individual protein level (Fig. 4 B, insets). Transcriptional profiling of total RNA revealed that 970 genes changed >1.5-fold, of which 616 (64%) were up-regulated and 354 (36%) down-regulated in TNF-α–stimulated compared with untreated visceral endoderm (Fig. 4, C and D), underlying the cytokine induction of protein synthesis. Multidimensional liquid chromatography tandem mass spectrometric shotgun analysis, along with pathway analysis, was used to dissect the intracellular network downstream of TNF-α (Fig. 4 E), with protein identities established from individual mass spectra (Fig. 4 F). Subtractive analysis of the genomic and proteomic data obtained from TNF-α–treated versus untreated endodermal secretome resolved several cytokine-induced secreted growth factors including TGF-β1, bone morphogenetic protein (BMP)-1, -2, and -4, vascular endothelial growth factor (VEGF)-A, IL-6, epidermal growth factor (EGF), fibroblast growth factor (FGF)-2 and -4, haploglobin, CSF-1, nerve growth factor (NGF)-β, and insulin-like growth factor (IGF)-1, and -2 (Fig. 4 E). Unbiased network analysis of the identified nodes in the endoderm secretome using the Ingenuity Pathway Knowledge Base ranked the “cardiovascular system development” function as the most overrepresented subnetwork following TNF-α induction, up from the twelfth rank in the untreated secretome and distanced by fivefold from the nearest function, identifying secreted factors as candidate cardiotrophs (Fig. 4 E).


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)

Proteomic and genomic dissection of TNF-α–primed endodermal secretome. (A) Two-dimensional gel electrophoresis (at pH 3–10 and 6–11) was used to resolve the endodermal secretome concentrated from 5-ml aliquots, with examples of TNF-α up-regulated proteins circled. Protein content was increased approximately twofold in TNF-α–primed versus naive secretome (inset). Triangles: examples of down-regulated or unchanged proteins. (B) Example LTQ tandem mass spectrum showing FKBP-1A peptide (amino acids 2–14). This approach was used to resolve protein identities by respective constituent peptide sequence signatures of individual peptide mass spectra, with relative abundance quantified (inset left) based on spot density (inset right). (C) Transcriptional profiling of total RNA from TNF-α–treated and untreated endoderm showing differential gene expression, including examples of genes encoding proteins identified by proteomics. (D) Volcano plots revealed that 64% of genes were up-regulated and 36% down-regulated in TNF-α–treated versus untreated endoderm. (E) Proteomic shotgun analysis of the TNF-α–treated endodermal secretome and ELISA revealed the identity of secreted proteins, including FGFs, leukemia inhibitory factor, VEGFs, TGF-βs, BMPs, CSF, IGFs, EGF, NGF, and ILs. Pathway analysis of identified proteins visualized the interactome triggered by cytokine stimulation and demonstrated secreted candidate cardiotrophic factors downstream of TNF-α integrated through p38-driven networks with individual proteins depicted in their corresponding cellular compartments. Blue nodes reflect detection by proteomic analysis, and yellow nodes reflect ELISA confirmation. (F) Example of LTQ-FT shotgun tandem mass spectrum showing IGF-2 peptide (amino acids 74–89). This approach was used to provide a more sensitive means of detecting the presence of secreted proteins. *, P < 0.05.
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Related In: Results  -  Collection

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fig4: Proteomic and genomic dissection of TNF-α–primed endodermal secretome. (A) Two-dimensional gel electrophoresis (at pH 3–10 and 6–11) was used to resolve the endodermal secretome concentrated from 5-ml aliquots, with examples of TNF-α up-regulated proteins circled. Protein content was increased approximately twofold in TNF-α–primed versus naive secretome (inset). Triangles: examples of down-regulated or unchanged proteins. (B) Example LTQ tandem mass spectrum showing FKBP-1A peptide (amino acids 2–14). This approach was used to resolve protein identities by respective constituent peptide sequence signatures of individual peptide mass spectra, with relative abundance quantified (inset left) based on spot density (inset right). (C) Transcriptional profiling of total RNA from TNF-α–treated and untreated endoderm showing differential gene expression, including examples of genes encoding proteins identified by proteomics. (D) Volcano plots revealed that 64% of genes were up-regulated and 36% down-regulated in TNF-α–treated versus untreated endoderm. (E) Proteomic shotgun analysis of the TNF-α–treated endodermal secretome and ELISA revealed the identity of secreted proteins, including FGFs, leukemia inhibitory factor, VEGFs, TGF-βs, BMPs, CSF, IGFs, EGF, NGF, and ILs. Pathway analysis of identified proteins visualized the interactome triggered by cytokine stimulation and demonstrated secreted candidate cardiotrophic factors downstream of TNF-α integrated through p38-driven networks with individual proteins depicted in their corresponding cellular compartments. Blue nodes reflect detection by proteomic analysis, and yellow nodes reflect ELISA confirmation. (F) Example of LTQ-FT shotgun tandem mass spectrum showing IGF-2 peptide (amino acids 74–89). This approach was used to provide a more sensitive means of detecting the presence of secreted proteins. *, P < 0.05.
Mentions: Proteomic two-dimensional gel analysis revealed that the TNF-α stimulation of isolated visceral endoderm-like cells induced secretion of proteins involved in sarcomerogenesis (profilin and cofilin), calcium signaling (calcyclin), myocardial reprogramming (nucleotide diphosphate kinase), and heart formation (FK506 binding protein FKBP12, cystatin, and ubiquitin) (39–42; Fig. 4 A). The identity of each protein was resolved by reconstruction of constitutive peptides using tandem mass spectrometry (Fig. 4 B) with the overall twofold increase in protein content after cytokine induction (Fig. 4 A, inset) confirmed at an individual protein level (Fig. 4 B, insets). Transcriptional profiling of total RNA revealed that 970 genes changed >1.5-fold, of which 616 (64%) were up-regulated and 354 (36%) down-regulated in TNF-α–stimulated compared with untreated visceral endoderm (Fig. 4, C and D), underlying the cytokine induction of protein synthesis. Multidimensional liquid chromatography tandem mass spectrometric shotgun analysis, along with pathway analysis, was used to dissect the intracellular network downstream of TNF-α (Fig. 4 E), with protein identities established from individual mass spectra (Fig. 4 F). Subtractive analysis of the genomic and proteomic data obtained from TNF-α–treated versus untreated endodermal secretome resolved several cytokine-induced secreted growth factors including TGF-β1, bone morphogenetic protein (BMP)-1, -2, and -4, vascular endothelial growth factor (VEGF)-A, IL-6, epidermal growth factor (EGF), fibroblast growth factor (FGF)-2 and -4, haploglobin, CSF-1, nerve growth factor (NGF)-β, and insulin-like growth factor (IGF)-1, and -2 (Fig. 4 E). Unbiased network analysis of the identified nodes in the endoderm secretome using the Ingenuity Pathway Knowledge Base ranked the “cardiovascular system development” function as the most overrepresented subnetwork following TNF-α induction, up from the twelfth rank in the untreated secretome and distanced by fivefold from the nearest function, identifying secreted factors as candidate cardiotrophs (Fig. 4 E).

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