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

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 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

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

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

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

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

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

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 transcription factor, co-activator and differentiation marker expression in adult mouse CSCs compared to the embryonic heart.

<p>Here and throughout, Lin^- / Sca1⁺ / SP⁺ CSCs were isolated from adult mouse myocardium and expanded for five passages before use. A: QRT-PCR. mRNA levels in CSCs (red) are expressed relative to the E14.5 mouse embryonic heart and are the mean ± SD of three independent experiments. *, p < 0.05 for CSCs versus embryonic heart. B, C: Immunocytocvhemistry. B: CSCs were cultured to subconfluent density and fixed for immunostaining. The transcription factors Gata4, Hand2, Mef2a, Nkx2-5, Tbx5, Tbx20 and chromatin remodeling protein Baf60c were present as nuclear-localized proteins, whereas neither Mef2c nor Myocd was detected. Factor-specific primary antibody binding was visualized using species-specific secondary antibodies conjugated with Alexa 488. Isotype control primary anitibodies were used as the control. Nuclei were counter-stained with DAPI. Bar, 20 μm. C: Quantitation of immunostaining by automated high-content image analysis. Data are the mean ± SD of three wells (≥ 2500 cells counted per well for each protein) and were concordant with the QRT-PCR profile for all nine factors tested by both methods.</p