Forward Programming of Cardiac Stem Cells by Homogeneous Transduction with <i>MYOCD</i> plus <i>TBX5</i>

<div><p>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 <i>GATA4</i>, <i>MEF2C</i>, and <i>TBX5</i> (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 <i>MYOCD</i> 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). <i>MYOCD</i> + <i>TBX</i> was the most effective pairwise combination in this system. In clonal derivatives homogenously expressing <i>MYOCD</i> + <i>TBX</i> 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 <i>MYOCD</i> induced cardiac protein expression, indicating a more complete cardiac differentiation program. (3) Homogeneous transduction with <i>MYOCD</i> + <i>TBX5</i> facilitated the identification of differentiating cells and the validation of this combinatorial reprogramming strategy. Together, these results highlight the pivotal importance of <i>MYOCD</i> in driving CSCs toward a cardiac muscle fate.</p></div

Cardiac gene induction in CSCs by homogeneous transduction with <i>MYOCD</i> and <i>TBX5</i>.

<p>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.</p

Cardiac gene induction in CSCs by single GMT factors ± <i>MYOCD</i>.

<p>QRT-PCR comparing <i>MYOCD</i> alone with <i>GATA4</i>, <i>MEF2C</i>, and <i>TBX5</i> in the absence or presence of <i>MYOCD</i>. Data are the mean ± SD of three independent experiments. *, p <0.05, versus the GFP control.</p

Dox-dependent expression of exogenous transcription factors in adult mouse CSCs.

<p>A: Timeline for the cardiogenic differentiation assays. CSCs were seeded in 6-well dishes (day 0), transduced with the rtTA-Puro^R lentiviral vector (day 1), and subjected to selection of successfully transduced cells in Puro (days 3–17). The selected transduced CSCs were re-seeded, transduced with Dox-inducible vectors for co-expression of transcription factors plus fluorescent reporters (day 18), and treated with Dox to induce the respective ectopic proteins (days 19–33). CSCs were then processed for RNA isolation and immunocytochemistry. B: Schematic representaiton of the induced (+Dox) and silent (-Dox) state. The system involves paired lentiviral vectors, rtTA-Puro^R constitutively expressing the reverse Tet transactivator (rtTA) and a Puromycin resistance gene (Puro^R), and a Dox-inducible series encoding the respective cardiac transcription factors and targeted reporter proteins via a Tet-responsive element (TRE). Co-expression in these bicistronic vectors is mediated by an internal ribosome entry site (IRES). C: Immunocytochemistry showing Dox-dependent expression of the ectopic transcription factors in transfected 293FT cells, which lack the corresponding endogenous factors, and appropriate intracellular targeting of all the respective reporters. D, E: Flow cytometry showing Induction of the transcription factor-reporter cassettes in transduced CSCs (MOI = 100). D: Representative dot plots. E: The percentage of fluorescent protein-expressing CSCs is shown as the mean ± SD for three samples, and is representative of additional experiments using one or more of the indicated factors. *, p < 0.05 for the presence or absence of Dox.</p

Species-specific detection of ectopic cardiac transcription factors in CSCs.

<p>A: Validation of specificity was confirmed using mouse versus human TaqMan Gene Expression Assays to detect the indicated endogenous factors in the E14 embryonic mouse heart (blue) and 12-week human fetal heart (red). Data are the mean ± SD of three independent experiments. B: Human factor expression in adult mouse CSCs. Transduced cells were treated with Dox for 14 days. Upper low, results comparing transduction of MT, GMT, and MyoGMT; lower row, results comparing single transduction of the GMT factors versus co-transduction with <i>MYOCD</i>. Expression was typically decreased as the number of co-transducing viruses increased. *, p < 0.05 for the treated CSCs versus human fetal heart, for reference.</p

Cardiac protein induction in CSCs by homogeneous transduction with <i>MYOCD</i> and <i>TBX5</i>.

<p>CSCs transduced homogenously with MyoT were cultured for 14 days ± Dox, then were analyzed by immunocytochemistry as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125384#pone.0125384.g004" target="_blank">Fig 4</a>. A: Representative images. Bar, 20 μm. B: Prevalence of cardiac protein expression in the MYOCD-IRES-GFP^nuc-positive, TBX5-IRES-dsRed^nuc-positive cells. Three samples were analysed for each protein (≥ 1600 cells). Data are the mean ± SD of three independent experiments (14d) or are single experiments for 2 and 7d. p < 0.01 for all proteins at 14 versus 0 days. Bar, 20 μm.</p

TaqMan QRT-PCR gene expression assays.

<p>All assays were carried out in TLDA format except for SRF, which was analysed individually.</p><p>TaqMan QRT-PCR gene expression assays.</p

Homogeneous transduction of CSCs with <i>MYOCD</i> and <i>TBX5</i>.

<p>A: Timeline of the protocol, highlighting the use of rtTA-Puro^r CSCs and secondary transduction with Dox-dependent <i>MYOCD</i> + TBX5, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125384#pone.0125384.g002" target="_blank">Fig 2A</a>. 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-GFP^nuc and TBX5-IRES-dsRed^nuc in clonal CSCs engineered by the method in panel A. Note, by comparison, the marked heterogeneity of transduction in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125384#pone.0125384.g004" target="_blank">Fig 4B</a>. D: QRT-PCR showing stringent Dox-dependent expression of <i>MYOCD</i> and <i>TBX5</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125384#pone.0125384.g007" target="_blank">7</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125384#pone.0125384.g008" target="_blank">8</a>. **, p < 0.01 for day 0 versus day 14.</p

Antibodies.

<p>IF, immunofluorescence; HRP, horseradish peroxidase; WB, Western blot. All secondary antibodies were F(ab')2 fragments excepting horse anti-mouse IgG, which was intact IgG.</p><p>Antibodies.</p

Cardiac gene and protein induction in CSCs by MyoGMT.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125384#pone.0125384.g005" target="_blank">Fig 5</a> 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 GFP^perox + GFP^nuc + dsRed^nuc (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 <i>MYOCD</i> (not shown). Similar results were observed in each of two independent experiments.</p