Issuu on Google+

Cardiovascular Research (2009) 84, 263–272 doi:10.1093/cvr/cvp211

Forward programming of pluripotent stem cells towards distinct cardiovascular cell types Robert David1, Juliane Stieber2, Evelyn Fischer1, Stefan Brunner1, Christoph Brenner1, Susanne Pfeiler3, Florian Schwarz1, and Wolfgang-Michael Franz1* 1

¨nchen, Germany; Medizinische Klinik und Poliklinik I, Klinikum Großhadern der LMU, Marchioninistraße 15, D-81377 Mu ¨t Erlangen, D-91054 Erlangen, Germany; and 3Institut fu ¨r Pharmakologie und Toxikologie der Universita ¨r Lehrstuhl fu ¨nchen, D-81377 Mu ¨nchen, Germany Klinische Chemie der LMU Mu 2

Received 8 December 2008; revised 18 June 2009; accepted 18 June 2009; online publish-ahead-of-print 29 June 2009 Time for primary review: 40 days

KEYWORDS

Aims The proliferative potential of pluripotent stem cell-derived cardiomyocytes is limited, and reasonable yields for novel therapeutic options have yet to be achieved. In addition, various clinical applications will require the generation of specific cardiac cell types. Whereas early cardiovascular precursors appear to be important for novel approaches such as reseeding decellularized hearts, direct cell transplantation may require ventricular cells. Our recent work demonstrated that MesP1 represents a master regulator sufficient to induce cardiovasculogenesis in pluripotent cells. This led to our hypothesis that ‘forward programming’ towards specific subtypes may be feasible via overexpression of distinct early cardiovascular transcription factors. Methods and results Here we demonstrate that forced expression of Nkx2.5 similar to MesP1 is sufficient to enhance cardiogenesis in murine embryonic stem cells (mES). In comparison to control transfected mES cells, a five-fold increased appearance of beating foci was observed as well as upregulated mRNA and protein expression levels. In contrast to MesP1, no increase of the endothelial lineage within the cardiovasculogenic mesoderm was observed. Likewise, Flk-1, the earliest known cardiovascular surface marker, was not induced via Nkx2.5 as opposed to MesP1. Detailed patch clamping analyses showed electrophysiological characteristics corresponding to all subtypes of cardiac ES cell differentiation in Nkx2.5 as well as MesP1 programmed embryoid bodies, but fractions of cardiomyocytes had distinct characteristics: MesP1 forced the appearance of early/intermediate type cardiomyocytes in comparison to control transfected ES cells whereas Nkx2.5 led to preferentially differentiated ventricular cells. Conclusion Our findings show proof of principle for cardiovascular subtype-specific programming of pluripotent stem cells and confirm the molecular hierarchy for cardiovascular specification initiated via MesP1 with differentiation factors such as Nkx2.5 further downstream.

1. Introduction Current therapeutic modalities for degenerative cardiovascular diseases are limited. They include medical therapy, mechanical left ventricular assist devices, and cardiac transplantation. Embryonic stem (ES) cells or pluripotent stem cells from recently described novel sources,1–11 which can differentiate into functional cardiovascular cells, may enable cardiovascular cell transplantation.12,13 However, the proliferative potential of stem cell-derived cardiomyocytes is limited and reasonable yields to repair a human infarcted heart (.1010 cardiomyocytes) have yet to be achieved.14 Therefore, it is crucial to decipher the underlying biological processes in order to pre-program

* Corresponding author. Tel: þ49 89 7095 3094; fax: þ49 89 7095 6094. E-mail address: wolfgang.franz@med.uni-muenchen.de

pluripotent stem cells towards a cardiovascular fate for cell therapy and cardiovascular tissue engineering. This knowledge may even provide a tool to elicit cardiac transdifferentiation in native human adult stem cells. In a first attempt of such ‘cardiovascular forward programming’ using pluripotent stem cells, we have recently shown that MesP1 is a key factor sufficient to induce cardiovasculogenesis.15 In ES cells, MesP1 overexpression resulted in significantly increased numbers of beating cardiomyocytes and of endothelial cells. Our experiments revealed a prominent function of MesP1 within a gene regulatory cascade causing Dkk-1 mediated blockage of canonical Wnt-signalling. We thereby defined the Dkk-1 promoter as a direct target, activated by MesP1 protein. We have also demonstrated that MesP1-induced ES cell based cardiomyogenesis requires the initial presence of general mesoderm inducing factors

Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2009. For permissions please email: journals.permissions@oxfordjournals.org.

Downloaded from cardiovascres.oxfordjournals.org at Islamic Azad University on August 28, 2011

Cardiovascular forward programming; Pluripotent stem cells; MesP1; Nkx2.5; Cardiac cell therapy; Cardiac tissue engineering


264

2. Methods 2.1 Plasmid construction and ES cell culture The human Nkx2.5 cDNA was cloned into pIRES-EGFP-2 and used for the generation of stably overexpressing ES cell clones as described.15 For details please refer to Supplementary material online.

2.2 Western blotting A polyclonal antibody specific for human Nkx2.5/Csx obtained from R&D systems was used for western blotting according to standard protocols.15

2.3 Flow cytometry FACS analyses were performed as described.15 For details please refer to Supplementary material online.

2.4 Confocal analyses Immunostaining was performed according to standard protocols as described.15 For details please refer to Supplementary material online.

2.5 Electrophysiological analyses Isolation and electrophysiological analysis of spontaneously beating cardiac cells from embryoid bodies (EBs) was done as described.15 For details please refer to Supplementary material online.

2.6 Real-time PCR Quantitative real-time PCR was performed as described.15 For details please refer to Supplementary material online.

3. Results 3.1 Generation of stably Nkx2.5 and Mesp1 overexpressing ES cell lines Relying on the high conservation of vertebrate Nkx2.5 proteins, we used the human Nkx2.5 in murine ES cells due to the specific traceability. We inserted the human Nkx2.5 cDNA in pIRES-EGFP-2 (Clontech) for overexpression in ES cells (Supplementary material online, Figure S1A) simultaneously allowing visualization of ES cell clones via EGFP (Figure 1A and B). Nkx2.5 overexpression was confirmed on the protein level (Supplementary material online, Figure S1B). The generation of MesP1 overexpressing ES cell lines has been described in detail.15 As expected qRT– PCRs showed no significant influence of Nkx2.5 overexpression on Oct4, Nanog and Rex-1 mRNA levels compared with control transfected cells. This corresponds with normal undifferentiated colony growth in leukemia inhibitory factor (LIF) containing medium (Figure 1A and B) and is in agreement with our previous findings for MesP1 overexpression.15 We concluded that similar to MesP1 likewise Nkx2.5 alone is not sufficient to induce cardiac differentiation of ES cells. However, in FACS analyses for Flk-1 (VEGFR-2, KDR), the earliest surface marker for the lateral plate mesoderm,16 we did not observe a shift of nascent mesoderm towards a cardiovascular fate. This is reflected in unaltered numbers of Flk-1 expressing cells after Nkx2.5 overexpression as opposed to MesP1 (Figure 2).

3.2 Effect of Nkx2.5 and Mesp1 overexpression on the yield of spontaneously beating ES cell-derived cardiomyocytes We next performed qRT–PCRs for known target genes of Nkx2.5 at day 10 of differentiation. These revealed a ninefold increase of mRNA encoding Mef2c and a 6.7-fold increase of ANF mRNA in three independent pooled clones (Figure 3) confirming the functionality of the overexpressed transcription factor in our ES cell clones. Again, our

Downloaded from cardiovascres.oxfordjournals.org at Islamic Azad University on August 28, 2011

evident from fluorescent activated cell sorting (FACS) analyses for Flk-1 (VEGFR-2, KDR), the earliest surface marker for the lateral mesoderm,16 which was not increased until day 4–6 of differentiation, when lateral and paraxial mesoderm have formed.15,17 Electrophysiological analysis of isolated beating cardiomyocytes showed all subtypes of cardiac ES cell differentiation albeit with a high proportion of yet uncommitted early/intermediate cell types.18,19 Whereas these earliest cardiovascular precursors may become important for innovative approaches such as reseeding whole decellularized hearts20 in order to reconstitute the whole myocardium including the vasculature, direct cell transplantation may rather be dependent on specific ventricular cells, e.g. to repair a typical infarction. Therefore, in long term, the specific generation of cardiovascular cellular subtypes has to be taken into account for various clinical applications or to transfer specific diseases to in vitro models. As a candidate for directed ventricular differentiation of stem cells, we now have chosen Nkx2.5/Csx because of its known importance for the underlying processes in the embryo, where this factor plays an important role in specification and maturation of ventricular cardiomyocytes.21 As a member of the NK homeobox gene family Nkx2.5/Csx is conserved in evolution and acts as a DNA-binding transcriptional activator.22,23 The high evolutionary conservation is underlined by the fact that Nkx2.5/Csx homologues have been identified in various vertebrate species from Xenopus to humans24–28 as well as in non-vertebrates: in Drosophila, tinman, the homologue of Nkx2.5/Csx in the fly is required for cell fate specification of the dorsal vessel, the equivalent of the vertebrate heart.29,30 In vertebrates, Nkx2.5/Csx plays an important role in lineage specification and maturation of ventricular cardiomyocytes as shown via ventricular restricted Nkx2.5/Csx knockout mice. Thereby cell type specific loss of Nkx2.5/Csx was achieved by MLC2v-Cre-mediated recombination21 of the floxed Nkx2.5/Csx allele. The importance of Nkx2.5/Csx in ventricular formation is further underlined by the fact that expression of the ventricular-specific homeobox gene Irx4 requires correct Nkx2.5/Csx and dHAND expression.31 Correspondingly, complete loss of Nkx2.5/Csx during embryogenesis leads to formation of only a single atrial chamber with complete ventricular dysgenesis.31 On the basis of this background, we compared the effect of forced expression of Nkx2.5/Csx with that of the cardiovascular fate inducer MesP1 on ES cell development. We hypothesized that overexpression of Nkx2.5/Csx specifically may enhance the yield of ventricular cells, whereas MesP1 may rather promote the earliest yet multipotent cardiovascular precursors. Our data show proof of principle for such cardiovascular subtype-specific programming of stem cells and confirm the molecular hierarchy for cardiovascular specification initiated via MesP1 with the key factor Nkx2.5 further downstream.

R. David et al.


Specific cardiovascular cell types from ES cells

265

Figure 2 Fluorescent activated cell sorting (FACS) analyses for Flk-1 (VEGFR-2), the earliest surface marker for the lateral mesoderm, at day 6 of ES cell differentiation. Results from EBs derived from Control (A), MesP1 (B), and Nkx2.5 (C ) overexpressing ES cells. An increased appearance of Flk-1 is not observed in Nkx2.5 as opposed to MesP1 cells.

established hMesP1 and EGFP transfected clones15 served as positive and negative controls. As a marker for the second vs. the primary heart fields, we analysed the mRNA levels for Isl1 and Tbx2032,33 and Tbx5,34 respectively. These were significantly increased in Nkx2.5 as well as MesP1 clones, whereas Tbx2 mRNA, a marker for blockage of chamber differentiation,35,36 was reduced in Nkx2.5 as opposed to MesP1 clones (Figure 3). As epicardial markers, we analysed Tbx18 and Wt137,38 mRNAs both of which were increased in Nkx2.5 and MesP1 clones (Figure 3). During differentiation Nkx2.5 overexpressing ES cell clones started to contract earlier and showed four- to six-fold more contracting areas (Supplementary material online, Figure S2 and Movies 1 and 2). This enhanced cardiac differentiation resembles the effect of MesP1

overexpression recently described by us,15 however the MesP1 programmed cells appeared to maintain their maximal beating activity slightly more robust (Supplementary material online, Figure S2). In correspondence with their beating activity, Nkx2.5 overexpressing cardiomyocytes showed normal patterns of the sarcomeric markers a-Actinin and TroponinI (Figure 4A and B) and Connexin43 expression revealed intercellular contacts (Figure 4C).

3.3 Effect of Nkx2.5 and Mesp1 overexpression on the number of cardiovascular marker expressing cells We next investigated whether our observations are reflected by protein expression patterns and found two-fold increased

Downloaded from cardiovascres.oxfordjournals.org at Islamic Azad University on August 28, 2011

Figure 1 Functionality of the Nkx2.5 overexpression construct in ES cells (A and B). Stably transfected murine ES cell colonies showing EGFP fluorescence. (C ) qRT–PCR using cDNA from undifferentiated ES cell clones: relative expression levels of pluripotency markers in the presence of leukemia inhibitory factor (LIF) show no significant difference compared with hMesP1 or EGFP control transfected cells.


266

R. David et al.

Downloaded from cardiovascres.oxfordjournals.org at Islamic Azad University on August 28, 2011

Figure 3 Increased appearance of cardiomyocytes in Nkx2.5 and MesP1 overexpressing ES cells qRT–PCRs for cardiovascular markers at day 10 (panels 1–2) and day 6 (panels 3–8) of differentiation. hMesP1 and EGFP transfected clones served as controls (data are mean + SEM, n ¼ 3).

numbers of cells expressing a-Actinin, which is expressed in cardiomyocytes as well as skeletal myocytes (Figure 5A). However, the numbers of cells expressing the cardiac specific structural markers TnI and cardiac MLC-1 (Figure 5B and C) were four-fold increased, indicating that the specific induction of cardiogenesis in Nkx2.5 and MesP1

overexpressing ES cells. As opposed to MesP1,15 we did not find a Nkx2.5 driven increase of CD31 expressing cells representing endothelial progenitor cells (Figure 5D). This corresponds to the fact that MesP1 demarcates all progenitors of the cardiovascular lineage,39–43 whereas Nkx2.5 is primarily expressed in cells contributing to the myocardium.


Specific cardiovascular cell types from ES cells

267

Figure 4 Subcellular structures of Nkx2.5 programmed cardiomyocytes. (A) Confocal analysis of a-Actinin expression in Nkx2.5 overexpressing (red). Counterstaining against Actin (green). (B) Confocal analysis of TroponinI expression (red). Counterstaining against Actin (green). (C ) Confocal analysis of Cx43 expression (red). Counterstaining against Actin (green). Scale bar: 10 um.

3.4 Divergent effects of Nkx2.5 and Mesp1 overexpression on the action potential parameters of ES cell-derived cardiomyocytes We next performed single-cell patch clamp analyses to investigate action potential parameters of spontaneously beating cardiomyocytes in Nkx2.5 and MesP1 overexpressing ES cell clones. As established in our previous work,15 we chose day 6 þ 12 of EB development for these experiments. In general, all cell types described for isolated beating cardiomyocytes obtained from EB development, namely ventricle-like, atrial-like, and SA/AV (pacemaker-like) cells, as well as intermediate cells are present in preparations from Nkx2.5 and MesP1-ES cell clones as well as controls (Supplementary material online, Figure S3A; Table 1). The action potentials generated by the various cell types did not differ significantly between Nkx2.5, MesP1, and control cells with respect to their distinct parameters such as maximal diastolic potential (MDP), diastolic depolarisation rate (DDR), upstroke velocity or AP/plateau duration, or in their reaction to b-adrenergic (Isoprotenerol) and muscarinic (Carbachol) stimulation, supporting the notion of correct cardiomyocyte development (Figure 6A

and B; Supplementary material online, Figure S3B and C; Table 1). However, we found highly different distributions of the various cell types in Nkx2.5 vs. MesP1 expressing cells. Only one cell of 24 (4%) of the analysed Nkx2.5 cells could be classified as an early/intermediate cardiomyocyte, whereas in the MesP1-cell population, 57% (16 of 28 cells) was of the intermediate type. The controls were ranking in the middle with 24% of these early/intermediate cells (Figure 6A, B, E, F; Table 1). Even though there are various types of ‘early/intermediate type cells’,18 they could all be identified according to their fast, stable DDRs of 30–60 mV/s and short but distinct plateau phases. In addition, as recently described by us,15 a small If was present in all intermediate type cells, setting them clearly apart from pacemaker-like cells which have an at least three times higher If density, and from atrial- or ventricularlike cells from which basically no If could be recorded (Figure 6C and D; Table 1). As opposed to the distribution of these yet undifferentiated intermediate type cells, the near-mature differentiated ventricular cell type was found in 79% (19 of 24) of the analysed Nkx2.5 cells (Figure 6F; Table 1), whereas in

Downloaded from cardiovascres.oxfordjournals.org at Islamic Azad University on August 28, 2011

Figure 5 Divergent effects on the appearance of cardiovascular marker expressing cells in Nkx2.5 and MesP1 programmed EBs. (A) a-Actinin expressing cells are increased 2-fold in Nkx2.5 and MesP1 clones at day 6. (B) TroponinI expressing cells are increased 3.5-fold in Nkx2.5 and MesP1 clones at day 18. (C) MLC-1 expressing cells are increased 3.2-fold in Nkx2.5 and MesP1 clones at day 6. (D) CD-31 (PECAM) expressing cells are not increased in Nkx2.5 clones but are increased 4.3-fold in MesP1 clones.


12.3 33.96 1.38 + 1.05 3.53 + 2.75

28.2 189 12.6 8.3 102 1.3 26.6 + 6.0 133.7 + 32.6 0.9 + 0.4 32.8 + 5.5 272.1 + 95.2 18.7 + 7.6

9.7 + 1.18 30.5 + 1.39 2.6 + 2.03 1.5 + 1.33 (pA/pF) 2110 mV

If density

23.5 + 9.0 203.3 + 58.9 7.25 + 7.5 9.8 + 2.8 129.8 + 19.3 0.56 + 0.25 22.8 + 4.0 130.7 + 44.0 0.76 + 0.23 28.8 + 5.5 241.1 + 35.8 19.6 + 3.9 Overshoot (mV) AP duration (ms) Repolarising velocity (V/s)

Single cell patch clamp and If current density analyses at –110 mV activation reveal all cell types described for cardiac ES cell development in Nkx2.5-, MesP1-ES cell clones, and controls. On the basis of these analyses, over 79% (19 of 24) of the analysed Nkx2.5 cells could be classified as near-mature differentiated ventricular-like (ventr.) cells whereas in the MesP1-cell population, only 25% (seven of 28 cells) and in the control-cell population, 45% (13 of 29) ventricular-like cells could be found. On the other hand, in the MesP1 cell population, the main cell type is the intermediate/early type (Inter)19 (57%, compared with 24% among the controls and 4% among Nkx2.5 cells). These are typical for developing mouse embryonic cardiomyocytes, found about day 9–12 p.c. Values given are mean of n cells + SD. AP, action potential; MDP, maximal diastolic potential; DDR, diastolic depolarisation rate; p.c. post conception.

13.1 + 5.5 36.5 + 9.6 1.41 + 0.24 1.53 + 0.77

24.4 + 8.8 182.5 + 36.0 6.77 + 6.6 10.8 + 1.8 132 + 22.6 0.4 + 0.28 20.0 + 3.1 133.7 + 22.9 0.47 + 0.28 33.4 + 8.7 216.9 + 44.3 24.9 + 16.7

258.5 + 2.4 103 + 31.1 2.2 + 0.7 271.9 + 5.6 2.7 + 0.6 45.9 + 9.6 272.7 + 7.8 2.34 + 2.0 83.3 + 27.1

MDP (mV) DDR (mV/s) Upstroke velocity (V/s) Overshoot (mV) AP duration (ms) Repolarising velocity (V/s) If density 2110 mV (pA/pF) 261.9 92.7 4.5 273.7 + 4.2 3.75 + 2.4 73.2 + 13.4 MDP (mV) DDR (mV/s) Upstroke velocity (V/s)

273.4 + 7.6 4.1 + 1.4 53.4 + 29.3

257.2 + 4.6 99.5 + 16.5 2.9 + 0.56

261.1 + 7.4 48.2 + 5.7 29.3 + 17.6

MDP (mV) DDR (mV/s) Upstroke velocity (V/s) Overshoot (mV) AP duration (ms) Repolarising velocity (V/s) If density 2110 mV (pA/pF)

274.4 + 9.8 4.24 + 3.2 71.5 + 19.9

266.7 + 5.3 8.1 + 7.2 31.5 + 18.1

265.9 44.1 14.5

SA/AV (n 5 2) atr. (n 5 3) ventr. (n57) MesP1 Inter (n 5 1) SA/AV (n 5 1) atr. (n 5 3) ventr. (n 5 19) Nkx2.5 Inter (n 5 7) SA/AV (n 5 6) atr. (n 5 3) ventr. (n 5 13) Controls

Electrophysiological parameters of (near-)terminally differentiated cardiomyocytes at developmental day 6þ12

the MesP-cell population only 25% (seven of 28 cells) (Figure 6F; Table 1), and in the control-cell population, 45% (13 of 29) mature ventricular-like cells were found (Figure 6E; Table 1). These cells could be clearly distinguished from other more differentiated cell types by a combination of the following action potential parameters (Figure 6A, B; Table 1): (i) they revealed a distinct plateau phase, at least one-third of total AP duration, which was shorter in atrial as well as intermediate cells and missing in SA/AV-cells. (ii) Ventricular-like cells had a MDP less than 270 mV, whereas SA/AV cells showed a considerable more positive MDP of more than 260 mV. (iii) Even though ventricular-like cells showed spontaneous contraction activity, these cells had the slowest DDR (,5 mV/s) of all cell types, followed by atrial cells with 5–10 mV/s and intermediate cells with 30–60 mV/s as stated above. In SA/AV cells, this value typically exceeded 60 mV/s. (iv) On the contrary, the upstroke/fast depolarization velocity was fastest in ventricular-like cells (70–90 V/s) but slowest in SA/AV cells (,5 V/s). (v) Ventricular-like cells showed a largepositive overshoot of about þ30 mV, whereas SA/AV cells showed the smallest overshoot with a maximum of þ10 mV. Importantly, the ventricular-like cells responded to isoproterenol as expected leading to a prolongation of their plateau phase (Supplementary material online, Figure S3B and C; Table 1) and a slowdown of the AP rate, whereas in SA/AV cells an acceleration was observed.

4. Discussion Stem cell based cardiac repair will require sufficient yields of cardiac cell types for transplantation and tissue engineering as well as for innovative approaches such as reseeding whole decellularized hearts.20 Whereas the latter likely requires the earliest cardiovascular precursors to reconstitute the whole myocardium including the vasculature, direct cell transplantation may rather be dependent on specific ventricular cells, e.g. to repair a typical infarction. We here demonstrate that cell type specific ‘cardiovascular forward programming’ via overexpression of different early cardiovascular transcription factors with the goal to highly enrich the desired cell type is feasible in pluripotent stem cells. In particular, forced expression of Nkx2.5 enhanced cardiogenesis in murine ES (mES) cells reflected by a fivefold increased appearance of beating foci as well as upregulated cardiac mRNA and protein expression. In contrast to the bHLH transcription factor MesP1 no increase of the endothelial lineage within the cardiovasculogenic mesoderm was observed. In detailed patch clamping analyses, the electrophysiological characteristics corresponded to all subtypes of cardiac ES cell differentiation in EBs programmed with Nkx2.5 as well as MesP1. However, the distribution of cardiomyocytic subtypes had distinct characteristics: MesP1 enhanced the appearance of early/intermediate type cardiomyocytes, whereas Nkx2.5 led to preferentially differentiated ventricular cells. In our experimental setting using Nkx2.5 overexpressing ES cells under LIF administration, we did not find a significant influence on Oct4, Nanog and Rex-1 expression in the undifferentiated colonies (Figure 1C). This corresponds to our previous findings for MesP1 overexpression15 and shows that neither of the two early cardiac factors investigated is alone sufficient to induce cardiac stem cell

Downloaded from cardiovascres.oxfordjournals.org at Islamic Azad University on August 28, 2011

Table 1

264.1 + 7.0 45.1 + 11.0 23.4 + 5.6

R. David et al.

Inter (n 5 16)

268


Specific cardiovascular cell types from ES cells

269

differentiation. Therefore, similar to MesP1-based cardiogenesis likewise Nkx2.5 function requires the initial presence of general mesoderm inducing factors.15,44 However, a difference between the hierarchic position of the two factors became evident from FACS analyses for Flk-1 (VEGFR-2, KDR), the earliest surface marker for the lateral mesoderm.16 Thereby, the Flk-1 positive population in MesP1 overexpressing ES cells first did not significantly increase until days 4–6 of differentiation, when lateral and paraxial mesoderm have formed.15,17 However, after that time point the enhanced cardiovascular differentiation in MesP1 overexpressing clones was reflected in three-fold larger Flk-1 expressing populations.15 We concluded that MesP1-based cardiogenesis depends on initial general mesoderm formation.44 We now included Nkx2.5 in these analyses to further address its function during cardiovascular differentiation in relation to that of MesP1. Thereby, as opposed to MesP1 we did not observe an increased appearance of

Flk-1 expressing cells induced via Nkx2.5 overexpression (Figure 2). This indicates that Nkx2.5 alone is not sufficient to shift nascent mesoderm towards a cardiovascular fate but rather requires initial cardiovascular induction via MesP1. In fact Nkx2.5 subsequently promotes terminal differentiation of preferentially ventricular cardiomyocytes as evident from our extensive electrophysiological analyses (Figure 6). Therefore, our findings confirm that cardiovascular specification is initiated via MesP1 with key differentiation factors such as Nkx2.5 further downstream.15,45,46 Because direct downstream targets for Nkx2.5/Csx have been identified, such as MEF2-C47 and ANP,48 we verified the functionality of our overexpression construct in qRT– PCRs after LIF withdrawal indeed revealing increases of mRNA levels for these genes (Figure 3). In addition, ANP mRNA levels are more abundant in ventricles than in atria during embryonic development and the appearance of increased ANP expression in adult ventricles has become

Downloaded from cardiovascres.oxfordjournals.org at Islamic Azad University on August 28, 2011

Figure 6 Nkx2.5 vs. MesP1 overexpression in ES cells promotes divergent subtypes of electrophysiologically functional cardiomyocytes. (A and B) Mean + SD for two of the action potential parameters used to discriminate between the cell types displayed in Supplementary material online, Figure S3A. For controls, Nkx2.5and MesP1-clones, the diastolic depolarisation rates ‘DDR’ (A) and AP durations (B) did not differ significantly within the same group, i.e. Vent., Atr., SA/AV or Inter. demonstrating correct differentiation. Refer to Table 1 for further values and n-numbers for each column. The individual parameters could be used to determine the AP type. (C and D) If recordings to discriminate between vent./atr. cells and SA/AV cells or inter. cells. (C) Example If, recorded from a ventricularlike (middle panel) and an intermediate cell (lower panel). (D) Mean + SD If current densities at –110 mV activation. A large If was recorded from SA/AV cells, an intermediate-sized If from intermediate type cells. (E and F ) Distribution of cardiomyocyte subtypes after Nkx2.5 and MesP1 overexpression (F) compared with a controls (E) at day 18, according to the analyses described above.


270

MesP1 overexpression does not interfere with the potency to generate all types of functional cells described for isolated beating cardiomyocytes from EBs.15,18,19 The same holds true for preparations from Nkx2.5 cell clones, where likewise ventricle-like, atrial-like, and SA/AV (pacemakerlike) cells as well as intermediate cells are found in our single cell patch clamp analyses (Suppl. Figure S3A; Table 1). Importantly, correct cardiomyocytic development was not impaired as the action potentials and pharmacological responses of the various cell types did not differ significantly between ‘programmed’ and control cells (Figure 6A, B; Supplementary material online, Figure S3B and C; Table 1). These findings are confirmed via normal patterns of sarcomeric markers (Figure 4A and B). However, one major finding of our work described here is the quantitative distribution of the cellular subtypes in Nkx2.5 vs. MesP1 programmed cells, which was highly different: MesP1 promoted the appearance of early/intermediate type cardiomyocytes, whereas Nkx2.5 led to over 75% near terminally differentiated ventricular cells (Figure 6; Table 1). These findings account for the more robust maintenance of spontaneously beating foci in MesP1 programmed cells (Supplementary material online, Figure S2). Future stem cell based cardiovascular repair faces a number of hurdles to be overcome. Whereas the cardiac differentiation of classical adult stem cells such as bone marrow derived cells is at present highly questionable, recently a number of promising novel pluripotent cell types have been described. These include spermatogonial and parthenogeneic stem cells as well as reprogrammed cells (iPS)1–11 While these cells are comparable to ES cells regarding their pluripotency, they are devoid of important ethical issues inescapably connected with human ES cells. The novel cell types have recently been shown to differentiate into true cardiomyocytes,13 but their use in future therapy or disease models in vitro will require an in-depth understanding of molecular mechanisms underlying cardiovascular stem cell differentiation. In this regard, our present work shows proof of principle for cardiovascular subtype-specific programming of pluripotent stem cells towards a cardiovascular fate without impairing their functionality. Therefore, it will be of great interest to transfer this approach to the various types of native or induced pluripotent stem cells from these novel sources1–11 using various early cardiovascular transcription factors. This may facilitate the achievement of high yields of the desired cardiac cellular subtype as initially shown here for Nkx2.5 and MesP1. It will be of great interest to use these preprogrammed cells for transplantations into infracted myocardium regarding their beneficial effects. Ultimately, this approach may also help to overcome the hurdles yet existing for cardiovascular differentiation of adult multipotent stem cells in their native state.

Supplementary material Supplementary material is available at Cardiovascular Research online. Ref. 55 is cited in the supplementary material.

Acknowledgements We are very grateful for expert technical assistance to Christiane Gross.

Downloaded from cardiovascres.oxfordjournals.org at Islamic Azad University on August 28, 2011

established as a marker for the induction of the embryonic gene program in ventricular hypertrophy.49 Therefore our findings confirm an increased ventricular differentiation driven via Nkx2.5. To analyse potential divergent effects of Nkx2.5 vs. MesP1 on second vs. primary heart field induction, we analysed expression of Isl1 and Tbx20 vs. Tbx5,32–34 all of which were significantly increased via either of the two factors (Figure 3). In addition, analysing epicardial induction we found Tbx18 and Wt1 mRNAs increased37,38 (Figure 3). For MesP1, these findings are in agreement with its key role to induce the entire cardiovasculogenic population.15,39–43,45,46 Likewise, Nkx2.5 is highly expressed in the early cardiogenic cells in both primary and secondary heart fields.24,50,51 Thereby, Nkx2.5 expressing progenitors do also contribute to the proepicardium expressing Wt1 and Tbx18, whereas Nkx2.5 knockout results in abnormal proepicardial development and decreased expression of Wt1 supporting our findings.38 Interestingly, Tbx2 mRNA levels were reduced in Nkx2.5 cells when compared with controls and increased in MesP1 cells (Figure 3). Tbx2 suppresses terminal differentiation of chamber myocardial cells as shown for mice and zebrafish.35,36 Therefore, the enhanced Nkx2.5-based ventricular differentiation (Figure 6) is reflected in reduced Tbx2 expression. Nkx2.5 overexpressing clones behaved similar to those described by us for MesP1 overexpression with respect to the rather crude criterion of spontaneous beating: they started to contract earlier and showed more beating areas (Supplementary material online, Figure S2 and Movies). Our findings correspond to subsequent quantitative FACS analyses, which showed two-fold increased a-Actinin positive cells. This marker is expressed in cardiomyocytes as well as skeletal myocytes (Figure 5A). Importantly, cells expressing the cardiac specific sarcomeric proteins TnI and cardiac MLC-1 (Figure 5B and C) were three- to four-fold increased, which shows that cardiogenesis was specifically induced via Nkx2.5 as well as MesP1 overexpression. Therefore, the effects of Nkx2.5 as well as MesP1 clearly exceed previous reports describing an increased yield of ES cell-derived cardiomyocytes by treatment with retinoic acid,52 Nitric oxide (NO) and by an inducible NO synthase53 as well as via loss of RBP-J, a downstream element in the Notch pathway.54 However, as opposed to our previous findings using MesP1 for cardiovascular forward programming,15 no increase of CD31 expressing cells representing endothelial progenitor cells was induced via Nkx2.5 (Figure 5D). This difference between the two transcription factors is in agreement with the knowledge that MesP1 is sufficient to induce the entire cardiovasculogenic cell population.15,39–43 On the other hand, Nkx2.5 is primarily expressed in cells contributing to the myocardium. Likewise, these findings confirm the molecular hierarchy for the early cardiovascular specification highly conserved in chordates initiated via MesP genes with subordinate factors such as Nkx. The specificity of the cardiogenic effects is further reflected in the numbers of cells expressing a-Actinin, expressed in cardiomyocytes as well as skeletal myocytes as a-Actinin positive cells were only two-fold increased (Figure 5A). To ultimately analyse the suitability of our ‘cardiac forward programming’ approach, we compared the Nkx2.5 and MesP1 derived cardiomyocytes with control cells at an electrophysiological level. We have recently described that

R. David et al.


Specific cardiovascular cell types from ES cells

Conflict of interest: none declared.

Funding R.D. is funded by the DFG (FR 705/11-3). C.B. and F.S. are funded by the Fo ¨FoLe program of the LMU Munich. Additional funding was granted by the Fritz-Bender-Stiftung and by the Helmut-Legerlotz-Stiftung.

References

21. Hirota H, Chen J, Betz UA, Rajewsky K, Gu Y, Ross J et al. Loss of a gp130 cardiac muscle cell survival pathway is a critical event in the onset of heart failure during biomechanical stress. Cell 1999;97:189–198. 22. Kim Y, Nirenberg M. Drosophila NK-homeobox genes. Proc Natl Acad Sci USA 1989;86:7716–7720. 23. Harvey RP. NK-2 homeobox genes and heart development. Dev Biol 1996; 178:203–216. 24. Lints TJ, Parsons LM, Hartley L, Lyons I, Harvey RP. Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants. Development 1993;119:969. 25. Tonissen KF, Drysdale TA, Lints TJ, Harvey RP, Krieg PA. XNkx-2.5, a Xenopus gene related to Nkx-2.5 and tinman: evidence for a conserved role in cardiac development. Dev Biol 1994;162:325–328. 26. Schultheiss TM, Xydas S, Lassar AB. Induction of avian cardiac myogenesis by anterior endoderm. Development 1995;121:4203–4214. 27. Chen JN, Fishman MC. Zebrafish tinman homolog demarcates the heart field and initiates myocardial differentiation. Development 1996;122: 3809–3816. 28. Shiojima I, Komuro I, Mizuno T, Aikawa R, Akazawa H, Oka T et al. Molecular cloning and characterization of human cardiac homeobox gene CSX1. Circ Res 1996;79:920–929. 29. Azpiazu N, Frasch M. tinman and bagpipe: two homeo box genes that determine cell fates in the dorsal mesoderm of Drosophila. Genes Dev 1993;7:1325–1340. 30. Bodmer R. The gene tinman is required for specification of the heart and visceral muscles in Drosophila. Development 1993;118:719–729. 31. Yamagishi H, Yamagishi C, Nakagawa O, Harvey RP, Olson EN, Srivastava D. The combinatorial activities of Nkx2.5 and dHAND are essential for cardiac ventricle formation. Dev Biol 2001;239:190–203. 32. Brade T, Gessert S, Kuhl M, Pandur P. The amphibian second heart field: Xenopus islet-1 is required for cardiovascular development. Dev Biol 2007;311:297–310. 33. Cai CL, Liang X, Shi Y, Chu PH, Pfaff SL, Chen J et al. Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Dev Cell 2003;5: 877–889. 34. Sakabe M, Matsui H, Sakata H, Ando K, Yamagishi T, Nakajima Y. Understanding heart development and congenital heart defects through developmental biology: a segmental approach. Congenit Anom (Kyoto) 2005; 45:107–118. 35. Aanhaanen WT, Brons JF, Dominguez JN, Rana MS, Norden J, Airik R et al. The Tbx2þ primary myocardium of the atrioventricular canal forms the atrioventricular node and the base of the left ventricle. Circ Res 2009; 7:7. 36. Ribeiro I, Kawakami Y, Buscher D, Raya A, Rodriguez-Leon J, Morita M et al. Tbx2 and Tbx3 regulate the dynamics of cell proliferation during heart remodeling. PLoS ONE 2007;2:e398. 37. Kraus F, Haenig B, Kispert A. Cloning and expression analysis of the mouse T-box gene Tbx18. Mech Dev 2001;100:83–86. 38. Zhou B, von Gise A, Ma Q, Rivera-Feliciano J, Pu WT. Nkx2.5- and Isl1-expressing cardiac progenitors contribute to proepicardium. Biochem Biophys Res Commun 2008;375:450–453. 39. Kitajima S, Takagi A, Inoue T, Saga Y. MesP1 and MesP2 are essential for the development of cardiac mesoderm. Development 2000;127: 3215–3226. 40. Kitajima S, Miyagawa-Tomita S, Inoue T, Kanno J, Saga Y. Mesp1nonexpressing cells contribute to the ventricular cardiac conduction system. Dev Dyn 2006;235:395–402. 41. Saga Y, Hata N, Kobayashi S, Magnuson T, Seldin MF, Taketo MM. MesP1: a novel basic helix-loop-helix protein expressed in the nascent mesodermal cells during mouse gastrulation. Development 1996;122:2769–2778. 42. Saga Y, Miyagawa-Tomita S, Takagi A, Kitajima S, Miyazaki J, Inoue T. MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube. Development 1999;126:3437–3447. 43. Saga Y, Kitajima S, Miyagawa-Tomita S. Mesp1 expression is the earliest sign of cardiovascular development. Trends Cardiovasc Med 2000;10: 345–352. 44. Liu Y, Asakura M, Inoue H, Nakamura T, Sano M, Niu Z et al. Sox17 is essential for the specification of cardiac mesoderm in embryonic stem cells. Proc Natl Acad Sci USA 2007;104:3859–3864. 45. Lindsley RC, Gill JG, Murphy TL, Langer EM, Cai M, Mashayekhi M et al. Mesp1 coordinately regulates cardiovascular fate restriction and epithelial-mesenchymal transition in differentiating ESCs. Cell Stem Cell 2008;3:55–68.

Downloaded from cardiovascres.oxfordjournals.org at Islamic Azad University on August 28, 2011

1. Guan K, Nayernia K, Maier LS, Wagner S, Dressel R, Lee JH et al. Pluripotency of spermatogonial stem cells from adult mouse testis. Nature 2006; 440:1199–1203. 2. Mai Q, Yu Y, Li T, Wang L, Chen MJ, Huang SZ et al. Derivation of human embryonic stem cell lines from parthenogenetic blastocysts. Cell Res 2007;17:1008–1019. 3. Xing F, Fang Z, Qin B, Li Y, Hou J, Chen X. Parthenogenetic embryonic stem cells derived from cryopreserved newborn mouse ovaries: a new approach to autologous stem cell therapy. Fertil Steril 2008;17:17. 4. De Sousa PA, Wilmut I. Human parthenogenetic embryo stem cells: appreciating what you have when you have it. Cell Stem Cell 2007;1:243–244. 5. Shao H, Wei Z, Wang L, Wen L, Duan B, Mang L et al. Generation and characterization of mouse parthenogenetic embryonic stem cells containing genomes from non-growing and fully grown oocytes. Cell Biol Int 2007;31:1336–1344. 6. Aoi T, Yae K, Nakagawa M, Ichisaka T, Okita K, Takahashi K et al. Generation of pluripotent stem cells from adult mouse liver and stomach cells. Science 2008;321:699–702. 7. Meissner A, Wernig M, Jaenisch R. Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat Biotechnol 2007; 25:1177–1181. 8. Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 2008;26:101–106. 9. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131:861–872. 10. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126:663–676. 11. Kossack N, Meneses J, Shefi S, Nguyen HN, Chavez S, Nicholas C et al. Isolation and characterization of pluripotent human spermatogonial stem cell-derived cells. Stem Cells 2008;16:16. 12. Nir SG, David R, Zaruba M, Franz WM, Itskovitz-Eldor J. Human embryonic stem cells for cardiovascular repair. Cardiovasc Res 2003;58:313–323. 13. Mauritz C, Schwanke K, Reppel M, Neef S, Katsirntaki K, Maier LS et al. Generation of functional murine cardiac myocytes from induced pluripotent stem cells. Circulation 2008;118:507–517. 14. David R, Groebner M, Franz WM. Magnetic cell sorting purification of differentiated embryonic stem cells stably expressing truncated human CD4 as surface marker. Stem Cells 2005;23:477–482. 15. David R, Brenner C, Stieber J, Schwarz F, Brunner S, Vollmer M et al. MesP1 drives vertebrate cardiovascular differentiation through Dkk-1-mediated blockade of Wnt-signalling. Nat Cell Biol 2008;10: 338–345. 16. Izumi N, Era T, Akimaru H, Yasunaga M, Nishikawa S. Dissecting the molecular hierarchy for mesendoderm differentiation through a combination of embryonic stem cell culture and RNA interference. Stem Cells 2007; 25:1664–1674. 17. Sakurai H, Era T, Jakt LM, Okada M, Nakai S, Nishikawa S. In vitro modeling of paraxial and lateral mesoderm differentiation reveals early reversibility. Stem Cells 2006;24:575–586. 18. Maltsev VA, Rohwedel J, Hescheler J, Wobus AM. Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusnodal, atrial and ventricular cell types. Mech Dev 1993;44:41–50. 19. Maltsev VA, Wobus AM, Rohwedel J, Bader M, Hescheler J. Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents. Circ Res 1994;75: 233–244. 20. Ott HC, Matthiesen TS, Goh SK, Black LD, Kren SM, Netoff TI et al. Perfusion-decellularized matrix: using nature’s platform to engineer a bioartificial heart. Nat Med 2008;14:213–221.

271


272 46. Bondue A, Lapouge G, Paulissen C, Semeraro C, Iacovino M, Kyba M et al. Mesp1 acts as a master regulator of multipotent cardiovascular progenitor specification. Cell Stem Cell 2008;3:69–84. 47. von Both I, Silvestri C, Erdemir T, Lickert H, Walls JR, Henkelman RM et al. Foxh1 is essential for development of the anterior heart field. Dev Cell 2004;7:331–345. 48. Biben C, Harvey RP. Homeodomain factor Nkx2.5 controls left/right asymmetric expression of bHLH gene eHand during murine heart development. Genes Dev 1997;11:1357–1369. 49. Cameron VA, Ellmers LJ. Minireview: natriuretic peptides during development of the fetal heart and circulation. Endocrinology 2003;144: 2191–2194. 50. Kasahara H, Bartunkova S, Schinke M, Tanaka M, Izumo S. Cardiac and extracardiac expression of Csx/Nkx2.5 homeodomain protein. Circ Res 1998;82:936–946. 51. Stanley EG, Biben C, Elefanty A, Barnett L, Koentgen F, Robb L et al. Efficient Cre-mediated deletion in cardiac progenitor cells conferred by a

R. David et al.

52.

53.

54.

55.

30 UTR-ires-Cre allele of the homeobox gene Nkx2.5. Int J Dev Biol 2002;46:431–439. Wobus AM, Kaomei G, Shan J, Wellner MC, Rohwedel J, Ji G et al. Retinoic acid accelerates embryonic stem cell-derived cardiac differentiation and enhances development of ventricular cardiomyocytes. J Mol Cell Cardiol 1997;29:1525–1539. Kanno S, Kim PK, Sallam K, Lei J, Billiar TR, Shears LL II. Nitric oxide facilitates cardiomyogenesis in mouse embryonic stem cells. Proc Natl Acad Sci USA 2004;101:12277–12281. Schroeder T, Fraser ST, Ogawa M, Nishikawa S, Oka C, Bornkamm GW et al. Recombination signal sequence-binding protein Jkappa alters mesodermal cell fate decisions by suppressing cardiomyogenesis. Proc Natl Acad Sci USA 2003;100:4018–4023. Muller M, Fleischmann BK, Selbert S, Ji GJ, Endl E, Middeler G et al. Selection of ventricular-like cardiomyocytes from ES cells in vitro. FASEB J 2000;14:2540–2548.

Downloaded from cardiovascres.oxfordjournals.org at Islamic Azad University on August 28, 2011


Stem Cell Biology, clinical application, ethical issues and more