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Forward Programming of Cardiac Stem Cells by Homogeneous Transduction with MYOCD plus TBX5.

Belian E, Noseda M, Abreu Paiva MS, Leja T, Sampson R, Schneider MD - PLoS ONE (2015)

Bottom Line: However, no protein was detected for the induced sarcomeric genes Actc1, Myh6, and Myl2.In clonal derivatives homogenously expressing MYOCD + TBX at high levels, 93% of cardiac transcripts were up-regulated and all five proteins tested were visualized. (1) GMT induced cardiac genes in CSCs, but not cardiac proteins under the conditions used. (2) Complementing GMT with MYOCD induced cardiac protein expression, indicating a more complete cardiac differentiation program. (3) Homogeneous transduction with MYOCD + TBX5 facilitated the identification of differentiating cells and the validation of this combinatorial reprogramming strategy.Together, these results highlight the pivotal importance of MYOCD in driving CSCs toward a cardiac muscle fate.

View Article: PubMed Central - PubMed

Affiliation: British Heart Foundation Centre of Research Excellence, National Heart and Lung Institute, Imperial College London, London W14 0NN, United Kingdom.

ABSTRACT

Unlabelled: Adult cardiac stem cells (CSCs) express many endogenous cardiogenic transcription factors including members of the Gata, Hand, Mef2, and T-box family. Unlike its DNA-binding targets, Myocardin (Myocd)-a co-activator not only for serum response factor, but also for Gata4 and Tbx5-is not expressed in CSCs. We hypothesised that its absence was a limiting factor for reprogramming. Here, we sought to investigate the susceptibility of adult mouse Sca1+ side population CSCs to reprogramming by supplementing the triad of GATA4, MEF2C, and TBX5 (GMT), and more specifically by testing the effect of the missing co-activator, Myocd. Exogenous factors were expressed via doxycycline-inducible lentiviral vectors in various combinations. High throughput quantitative RT-PCR was used to test expression of 29 cardiac lineage markers two weeks post-induction. GMT induced more than half the analysed cardiac transcripts. However, no protein was detected for the induced sarcomeric genes Actc1, Myh6, and Myl2. Adding MYOCD to GMT affected only slightly the breadth and level of gene induction, but, importantly, triggered expression of all three proteins examined (α-cardiac actin, atrial natriuretic peptide, sarcomeric myosin heavy chains). MYOCD + TBX was the most effective pairwise combination in this system. In clonal derivatives homogenously expressing MYOCD + TBX at high levels, 93% of cardiac transcripts were up-regulated and all five proteins tested were visualized.

In summary: (1) GMT induced cardiac genes in CSCs, but not cardiac proteins under the conditions used. (2) Complementing GMT with MYOCD induced cardiac protein expression, indicating a more complete cardiac differentiation program. (3) Homogeneous transduction with MYOCD + TBX5 facilitated the identification of differentiating cells and the validation of this combinatorial reprogramming strategy. Together, these results highlight the pivotal importance of MYOCD in driving CSCs toward a cardiac muscle fate.

No MeSH data available.


Related in: MedlinePlus

Cardiac gene and protein induction in CSCs by MyoGMT.A: QRT-PCR comparing MEF2C alone (the GMT factor absent from the CSCs), the GMT triad, and the 4-factor combination of MyoGMT. Data are the mean ± SD of three independent experiments. *, p <0.05, for factor-transduced versus GFP-transduced CSCs. Results here and in Fig 5 are taken from the identical three experiments, separated for clarity of the respective comparisons. B: Immunocytochemistry showing induction of the indicated cardiac proteins in CSCs by MyoGMT. Cells expressing the indicated proteins were identified using secondary antibodies conjugated with Alexa 647 (pseudocoloured yellow for enhanced visibility). Co-expression of GFPperox + GFPnuc + dsRednuc (expressed with the ectopic factors G, Myo / M and T, respectively), was assessed to identify the successfully co-transduced cells. No induction was detected in cells transduced with the GFP control vector (lower panels), or cells transduced with M and GMT in the absence of MYOCD (not shown). Similar results were observed in each of two independent experiments.
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pone.0125384.g004: Cardiac gene and protein induction in CSCs by MyoGMT.A: QRT-PCR comparing MEF2C alone (the GMT factor absent from the CSCs), the GMT triad, and the 4-factor combination of MyoGMT. Data are the mean ± SD of three independent experiments. *, p <0.05, for factor-transduced versus GFP-transduced CSCs. Results here and in Fig 5 are taken from the identical three experiments, separated for clarity of the respective comparisons. B: Immunocytochemistry showing induction of the indicated cardiac proteins in CSCs by MyoGMT. Cells expressing the indicated proteins were identified using secondary antibodies conjugated with Alexa 647 (pseudocoloured yellow for enhanced visibility). Co-expression of GFPperox + GFPnuc + dsRednuc (expressed with the ectopic factors G, Myo / M and T, respectively), was assessed to identify the successfully co-transduced cells. No induction was detected in cells transduced with the GFP control vector (lower panels), or cells transduced with M and GMT in the absence of MYOCD (not shown). Similar results were observed in each of two independent experiments.

Mentions: We postulated that the absence of Mef2c, Myocd or both might be a mechanism by which adult CSCs are stably maintained in a progenitor state whose gene expression profile otherwise largely resembles the cardiac mesoderm seen transiently in native embryos and differentiating pluripotent cells. Alternatively, or in addition, one or more of the expressed factors might be insufficient, at its native level of expression. To investigate these hypotheses, we overexpressed key cardiac transcription factors or their co-factor Myocd in Sca1+ SP+ CSCs using a doxycycline (Dox)-inducible lentiviral system (Fig 2A) enabling temporally controlled induction and repression of the exogenous cardiac transcription factors [42,43]. The system consists of a vector encoding the reverse tetracycline transactivator (rtTA) [42] and a series of vectors encoding the inducible cardiac transcription factor genes downstream of the tetracycline-responsive element (TRE) (Fig 2B). The rtTA-encoding vector also encodes a puromycin resistance cassette to enable selection of successfully transduced cells. In the absence of Dox, rtTA is unable to bind the TRE and the cardiac transcription factor is not transcribed. However, when Dox is added it interacts with the rtTA, which consequently binds to the TRE and induces the transgene. Four lentiviral constructs were tested, encoding the triad of GATA4, MEF2C and TBX5 plus the co-activator MYOCD (Fig 2B). A further feature of the lentiviral backbone utilised is co-expression of the cardiogenic gene plus a nuclear- or peroxisomal-localised fluorescent protein via an internal ribosome entry site (IRES), simplifying the detection of Dox-dependent transgene induction. Dox-dependent induction of the cardiogenic proteins was confirmed by immunostaining in 293FT cells and was concordant with the respective reporter proteins (Fig 2C; see also S1 Fig). The prevalence of transduction was maximal at a multiplicity of infection (MOI) of 100, and the concentration of Dox saturating at 500–1000 ng/ml (S2 Fig). Dox-dependent control was then confirmed in CSCs themselves, using flow cytometry (Fig 2D). Here and in Figs 3 and 4, Sca1+ SP+ CSCs were isolated from adult mouse hearts, purified by preparative flow sorting (roughly 10,000 per heart), expanded for five passages in growth medium, transduced with lentiviral constructs, and then treated with Dox for 14 days to activate the exogenous factors.


Forward Programming of Cardiac Stem Cells by Homogeneous Transduction with MYOCD plus TBX5.

Belian E, Noseda M, Abreu Paiva MS, Leja T, Sampson R, Schneider MD - PLoS ONE (2015)

Cardiac gene and protein induction in CSCs by MyoGMT.A: QRT-PCR comparing MEF2C alone (the GMT factor absent from the CSCs), the GMT triad, and the 4-factor combination of MyoGMT. Data are the mean ± SD of three independent experiments. *, p <0.05, for factor-transduced versus GFP-transduced CSCs. Results here and in Fig 5 are taken from the identical three experiments, separated for clarity of the respective comparisons. B: Immunocytochemistry showing induction of the indicated cardiac proteins in CSCs by MyoGMT. Cells expressing the indicated proteins were identified using secondary antibodies conjugated with Alexa 647 (pseudocoloured yellow for enhanced visibility). Co-expression of GFPperox + GFPnuc + dsRednuc (expressed with the ectopic factors G, Myo / M and T, respectively), was assessed to identify the successfully co-transduced cells. No induction was detected in cells transduced with the GFP control vector (lower panels), or cells transduced with M and GMT in the absence of MYOCD (not shown). Similar results were observed in each of two independent experiments.
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Related In: Results  -  Collection

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Show All Figures
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pone.0125384.g004: Cardiac gene and protein induction in CSCs by MyoGMT.A: QRT-PCR comparing MEF2C alone (the GMT factor absent from the CSCs), the GMT triad, and the 4-factor combination of MyoGMT. Data are the mean ± SD of three independent experiments. *, p <0.05, for factor-transduced versus GFP-transduced CSCs. Results here and in Fig 5 are taken from the identical three experiments, separated for clarity of the respective comparisons. B: Immunocytochemistry showing induction of the indicated cardiac proteins in CSCs by MyoGMT. Cells expressing the indicated proteins were identified using secondary antibodies conjugated with Alexa 647 (pseudocoloured yellow for enhanced visibility). Co-expression of GFPperox + GFPnuc + dsRednuc (expressed with the ectopic factors G, Myo / M and T, respectively), was assessed to identify the successfully co-transduced cells. No induction was detected in cells transduced with the GFP control vector (lower panels), or cells transduced with M and GMT in the absence of MYOCD (not shown). Similar results were observed in each of two independent experiments.
Mentions: We postulated that the absence of Mef2c, Myocd or both might be a mechanism by which adult CSCs are stably maintained in a progenitor state whose gene expression profile otherwise largely resembles the cardiac mesoderm seen transiently in native embryos and differentiating pluripotent cells. Alternatively, or in addition, one or more of the expressed factors might be insufficient, at its native level of expression. To investigate these hypotheses, we overexpressed key cardiac transcription factors or their co-factor Myocd in Sca1+ SP+ CSCs using a doxycycline (Dox)-inducible lentiviral system (Fig 2A) enabling temporally controlled induction and repression of the exogenous cardiac transcription factors [42,43]. The system consists of a vector encoding the reverse tetracycline transactivator (rtTA) [42] and a series of vectors encoding the inducible cardiac transcription factor genes downstream of the tetracycline-responsive element (TRE) (Fig 2B). The rtTA-encoding vector also encodes a puromycin resistance cassette to enable selection of successfully transduced cells. In the absence of Dox, rtTA is unable to bind the TRE and the cardiac transcription factor is not transcribed. However, when Dox is added it interacts with the rtTA, which consequently binds to the TRE and induces the transgene. Four lentiviral constructs were tested, encoding the triad of GATA4, MEF2C and TBX5 plus the co-activator MYOCD (Fig 2B). A further feature of the lentiviral backbone utilised is co-expression of the cardiogenic gene plus a nuclear- or peroxisomal-localised fluorescent protein via an internal ribosome entry site (IRES), simplifying the detection of Dox-dependent transgene induction. Dox-dependent induction of the cardiogenic proteins was confirmed by immunostaining in 293FT cells and was concordant with the respective reporter proteins (Fig 2C; see also S1 Fig). The prevalence of transduction was maximal at a multiplicity of infection (MOI) of 100, and the concentration of Dox saturating at 500–1000 ng/ml (S2 Fig). Dox-dependent control was then confirmed in CSCs themselves, using flow cytometry (Fig 2D). Here and in Figs 3 and 4, Sca1+ SP+ CSCs were isolated from adult mouse hearts, purified by preparative flow sorting (roughly 10,000 per heart), expanded for five passages in growth medium, transduced with lentiviral constructs, and then treated with Dox for 14 days to activate the exogenous factors.

Bottom Line: However, no protein was detected for the induced sarcomeric genes Actc1, Myh6, and Myl2.In clonal derivatives homogenously expressing MYOCD + TBX at high levels, 93% of cardiac transcripts were up-regulated and all five proteins tested were visualized. (1) GMT induced cardiac genes in CSCs, but not cardiac proteins under the conditions used. (2) Complementing GMT with MYOCD induced cardiac protein expression, indicating a more complete cardiac differentiation program. (3) Homogeneous transduction with MYOCD + TBX5 facilitated the identification of differentiating cells and the validation of this combinatorial reprogramming strategy.Together, these results highlight the pivotal importance of MYOCD in driving CSCs toward a cardiac muscle fate.

View Article: PubMed Central - PubMed

Affiliation: British Heart Foundation Centre of Research Excellence, National Heart and Lung Institute, Imperial College London, London W14 0NN, United Kingdom.

ABSTRACT

Unlabelled: Adult cardiac stem cells (CSCs) express many endogenous cardiogenic transcription factors including members of the Gata, Hand, Mef2, and T-box family. Unlike its DNA-binding targets, Myocardin (Myocd)-a co-activator not only for serum response factor, but also for Gata4 and Tbx5-is not expressed in CSCs. We hypothesised that its absence was a limiting factor for reprogramming. Here, we sought to investigate the susceptibility of adult mouse Sca1+ side population CSCs to reprogramming by supplementing the triad of GATA4, MEF2C, and TBX5 (GMT), and more specifically by testing the effect of the missing co-activator, Myocd. Exogenous factors were expressed via doxycycline-inducible lentiviral vectors in various combinations. High throughput quantitative RT-PCR was used to test expression of 29 cardiac lineage markers two weeks post-induction. GMT induced more than half the analysed cardiac transcripts. However, no protein was detected for the induced sarcomeric genes Actc1, Myh6, and Myl2. Adding MYOCD to GMT affected only slightly the breadth and level of gene induction, but, importantly, triggered expression of all three proteins examined (α-cardiac actin, atrial natriuretic peptide, sarcomeric myosin heavy chains). MYOCD + TBX was the most effective pairwise combination in this system. In clonal derivatives homogenously expressing MYOCD + TBX at high levels, 93% of cardiac transcripts were up-regulated and all five proteins tested were visualized.

In summary: (1) GMT induced cardiac genes in CSCs, but not cardiac proteins under the conditions used. (2) Complementing GMT with MYOCD induced cardiac protein expression, indicating a more complete cardiac differentiation program. (3) Homogeneous transduction with MYOCD + TBX5 facilitated the identification of differentiating cells and the validation of this combinatorial reprogramming strategy. Together, these results highlight the pivotal importance of MYOCD in driving CSCs toward a cardiac muscle fate.

No MeSH data available.


Related in: MedlinePlus