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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 induction in CSCs by homogeneous transduction with MYOCD and TBX5.A: QRT-PCR showing stable expression of exogenous cardiac transcription factors in CSCs transduced homogenously with MyoT. Levels of both factors were 2.5- to 4-fold higher than in human fetal heart. *, p < 0.05. Data are the mean ± SD of three independent experiments. B-D: QRT-PCR showing cardiac gene expression after Dox administration for 2, 7, and 14 days. Data are the mean ± SD of three independent experiments. *, p < 0.05 versus the GFP control.
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pone.0125384.g007: Cardiac gene induction in CSCs by homogeneous transduction with MYOCD and TBX5.A: QRT-PCR showing stable expression of exogenous cardiac transcription factors in CSCs transduced homogenously with MyoT. Levels of both factors were 2.5- to 4-fold higher than in human fetal heart. *, p < 0.05. Data are the mean ± SD of three independent experiments. B-D: QRT-PCR showing cardiac gene expression after Dox administration for 2, 7, and 14 days. Data are the mean ± SD of three independent experiments. *, p < 0.05 versus the GFP control.

Mentions: A: Timeline of the protocol, highlighting the use of rtTA-Puror CSCs and secondary transduction with Dox-dependent MYOCD + TBX5, as in Fig 2A. For homogeneous transduction, single-cell deposition was performed using a preparative cell sorter (day 1), clonal growth was permitted for four weeks, the single-cell derivatives were passaged (day 29), and the CSCs were then cultured ± Dox to confirm co-expression of the dual fluorescent reporters (days 30–32). Single-cell progeny expressing both were taken forward ± Dox for differentiation studies. B: Serial images of a representative single-cell clone at 6–21 days. C: Homogeneous co-expression of MYOCD-IRES-GFPnuc and TBX5-IRES-dsRednuc in clonal CSCs engineered by the method in panel A. Note, by comparison, the marked heterogeneity of transduction in Fig 4B. D: QRT-PCR showing stringent Dox-dependent expression of MYOCD and TBX5 in the homogeneously transduced cells. The two clones are designated MyoT high and MyoT low, respectively, in accordance with the elicited expression levels. *, p < 0.05, and **, p < 0.01 versus human fetal heart. E: Dose-dependent induction by MyoT at 14 days was confirmed for all four cardiac genes, tested as a prelude to full evaluation of the higher line in Figs 7 and 8. **, p < 0.01 for day 0 versus day 14.


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 induction in CSCs by homogeneous transduction with MYOCD and TBX5.A: QRT-PCR showing stable expression of exogenous cardiac transcription factors in CSCs transduced homogenously with MyoT. Levels of both factors were 2.5- to 4-fold higher than in human fetal heart. *, p < 0.05. Data are the mean ± SD of three independent experiments. B-D: QRT-PCR showing cardiac gene expression after Dox administration for 2, 7, and 14 days. Data are the mean ± SD of three independent experiments. *, p < 0.05 versus the GFP control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4457652&req=5

pone.0125384.g007: Cardiac gene induction in CSCs by homogeneous transduction with MYOCD and TBX5.A: QRT-PCR showing stable expression of exogenous cardiac transcription factors in CSCs transduced homogenously with MyoT. Levels of both factors were 2.5- to 4-fold higher than in human fetal heart. *, p < 0.05. Data are the mean ± SD of three independent experiments. B-D: QRT-PCR showing cardiac gene expression after Dox administration for 2, 7, and 14 days. Data are the mean ± SD of three independent experiments. *, p < 0.05 versus the GFP control.
Mentions: A: Timeline of the protocol, highlighting the use of rtTA-Puror CSCs and secondary transduction with Dox-dependent MYOCD + TBX5, as in Fig 2A. For homogeneous transduction, single-cell deposition was performed using a preparative cell sorter (day 1), clonal growth was permitted for four weeks, the single-cell derivatives were passaged (day 29), and the CSCs were then cultured ± Dox to confirm co-expression of the dual fluorescent reporters (days 30–32). Single-cell progeny expressing both were taken forward ± Dox for differentiation studies. B: Serial images of a representative single-cell clone at 6–21 days. C: Homogeneous co-expression of MYOCD-IRES-GFPnuc and TBX5-IRES-dsRednuc in clonal CSCs engineered by the method in panel A. Note, by comparison, the marked heterogeneity of transduction in Fig 4B. D: QRT-PCR showing stringent Dox-dependent expression of MYOCD and TBX5 in the homogeneously transduced cells. The two clones are designated MyoT high and MyoT low, respectively, in accordance with the elicited expression levels. *, p < 0.05, and **, p < 0.01 versus human fetal heart. E: Dose-dependent induction by MyoT at 14 days was confirmed for all four cardiac genes, tested as a prelude to full evaluation of the higher line in Figs 7 and 8. **, p < 0.01 for day 0 versus day 14.

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