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EMBRYONIC STEM CELLS

Differentiation In Vivo of Cardiac Committed Human Embryonic Stem Cells in Postmyocardial Infarcted Rats

^aInstitut National de la Santé et de la Recherche Médicale (INSERM), Unité Mixte de Recherche 861, I-Stem, Association Française contre les Myopathies, Evry, France;
^bINSERM, U 633; Assistance Publique-Hôpitaux de Paris, Ecole de Chirurgie, Paris, France;
^cINSERM U 633, Paris, France;
^dAssistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Department of Cardiology; University Paris-Descartes, Faculty of Medicine; INSERM, U 633, Paris, France;
^eAssistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Department of Pathology; University Paris-Descartes, Faculty of Medicine; INSERM, U 430, Paris, France;
^fAssistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Department of Cardiovascular Surgery; INSERM, U 633, Laboratoire de Recherches Biochirurgicales; University Paris-Descartes, Faculty of Medicine, Paris, France;
^gTechnion Institute, Haifa, Israel

Key Words. Human embryonic stem cells • Cell transplantation • In vivo • Cardiology

Correspondence: Michel Pucéat, Ph.D., Institut National de la Santé et de la Recherche Médicale/Evry University Unité Mixte de Recherche 861, I-Stem, Association Française contre les Myopathies, 5, rue Desbrières, Evry 91030, France. Telephone: (33) 169908527; Fax: (33) 169908521; e-mail: e-mail: mpuceat{at}istem.genethon.fr

Received on February 22, 2007; accepted for publication on May 23, 2007.

First published online in STEM CELLS EXPRESS  May 31, 2007.

	ABSTRACT

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Human embryonic stem (HES) cells can give rise to cardiomyocytes in vitro. However, whether undifferentiated HES cells also feature a myocardial regenerative capacity after in vivo engraftment has not been established yet. We compared two HES cell lines (HUES-1 and I6) that were specified toward a cardiac lineage by exposure to bone morphogenetic protein-2 (BMP2) and SU5402, a fibroblast growth factor receptor inhibitor. Real-time polymerase chain reaction (PCR) revealed that the cardiogenic inductive factor turned on expression of mesodermal and cardiac genes (Tbx6, Isl1, FoxH1, Nkx2.5, Mef2c, and -actin). Thirty immunosuppressed rats underwent coronary artery ligation and, 2 weeks later, were randomized and received in-scar injections of either culture medium (controls) or BMP2 (±SU5402)-treated HES cells. After 2 months, human cells were detected by anti-human lamin immunostaining, and their cardiomyocytic differentiation was evidenced by their expression of cardiac markers by reverse transcription-PCR and immunofluorescence using an anti-ß myosin antibody. No teratoma was observed in hearts or any other organ of the body. The ability of cardiac-specified HES cells to differentiate along the cardiomyogenic pathway following transplantation into infarcted myocardium raises the hope that these cells might become effective candidates for myocardial regeneration.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

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Heart failure is becoming a predominant disease and a leading cause of death in most developed countries. Regardless of the origin of myocardial failure (i.e., ischemic or genetic), the clinical symptoms result mainly from the death of cardiomyocytes replaced by a fibrotic and noncontractile tissue. Pharmacological approaches to cure or relieve heart failure have been facing limitations. Because of a limited regeneration capability of the heart [1] and a shortage in donors for heart transplantation, an external source of cells has been envisioned as a therapeutic solution to bring a gain in function to diseased myocardium. For the last few years, hematopoietic stem cells had raised many hopes as a potential autologous cell source to repair diseased myocardium. However, the enthusiasm generated by the early noncontrolled phase I studies has been dampened by the more recent recognition that out of four randomized controlled trials entailing intracoronary infusions of bone marrow-derived cells shortly after myocardial infarction, three failed to meet their primary end point, that is, an improvement in left ventricular ejection fraction [2–5]. Combined with basic studies disproving the cardiogenic potential of these cells [6, 7], the outcome of these trials demonstrates that these cells do not really regenerate the diseased myocardium, and it is unlikely that their paracrine effects may be sufficient for restoring function of extensively scarred myocardium [8]. The same limitations apply to skeletal myoblasts [9]. Thus, these findings call for another stem cell source to achieve myocardial regeneration. Among various other cell types, embryonic stem (ES) cells [10–12] or ES cell-derived cardiomyocytes [13, 14] have turned out to be the most promising for replacing scar fibrosis by new contractile elements. However, the number of cells required to regenerate a postinfarcted human myocardium (i.e., several hundreds of million) is too high to be reasonably achieved by in vitro engineering of ES cell-derived cardiomyocytes. For the last few years, we and others have shown that proliferative mouse ES cells engrafted in a diseased myocardium further differentiate into functional cardiomyocytes following in vitro commitment using the cardiogenic morphogen bone morphogenetic protein-2 (BMP2) [10–12]. Cardiac-specified cells then complete their differentiation in response to the local cues present in the scar and do not generate any kind of tumors. Thus, although human ES cells do not share with mouse ES cells the same molecular mechanisms of self-renewal or capabilities of spontaneous differentiation [15], we designed this study to assess whether human ES cells could be specified toward a cardiac lineage in vitro and subsequently differentiate into cardiomyocytes in situ following their transplantation in infarcted myocardium.

Herein, we bring the proof of concept that human ES cells can also be directed toward a cardiogenic fate using the morphogen BMP2. Furthermore, the cells do differentiate into cardiomyocytes following engraftment into the myocardial scar without any sign of hyperproliferation. These data open the path for the use of early cardiac progenitors, which retain the capability to proliferate and repopulate the postinfarction scar.

	MATERIALS AND METHODS

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Real-Time Quantitative Polymerase Chain Reaction by SYBR Green Detection
RNA was extracted from human embryonic stem (HES) cells or slices of rat myocardium using a Qiagen (Hilden, Germany, http://www1.qiagen.com) kit. One µg of RNA was reverse-transcribed using the Mu-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and oligo(dT)16.

Real-time quantitative polymerase chain reaction (PCR) was performed using a Light Cycler (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) or a Chromo4 thermal cycler (Bio-Rad, Hercules, CA, http://www.bio-rad.com). Amplification was carried out as recommended by the manufacturers. Twelve or 22 µl reaction mixture contained 10 or 20 µl of Roche or ABgene (Surrey, U.K., http://www.abgene.com) SYBR Green I mix, respectively (including Taq DNA polymerase, reaction buffer, deoxynucleoside trisphosphate mix, and SYBR Green I dye, 3 mM MgCl₂), 0.25 µM concentration of appropriate primer, and 2 µl of cDNA. The amplification program included the initial denaturation step at 95°C for 15 or 8 minutes and 40 cycles of denaturation at 95°C for 10 seconds, annealing at 65°C for 8 seconds (Light Cycler) or 20 seconds (Chromo4), and extension at 72°C for 8 or 30 seconds. The temperature transition rate was 20°C/second (Light Cycler) or 4°C/second (Bio-Rad). Fluorescence was measured at the end of each extension step. After amplification, a melting curve was acquired by heating the product at 20°C/second or 4°C/second to 95°C, cooling it at 20°C/second or 4°C/second to 70°C, keeping it at 70°C for 20 seconds, and then slowly heating it at 20°C/second or 4°C/second to 95°C. Fluorescence was measured through the slow heating phase. Melting curves were used to determine the specificity of PCR products, which were confirmed using conventional gel electrophoresis. Data were analyzed according to Pfaffl [16]. Primers specific for human genes are described in Table 1.

HES cells were treated for 48 hours with 10 ng/ml BMP2 in the presence or absence of 1 µM SU5402, a FGF receptor inhibitor, in low KOSR (5%) containing KO-DMEM. Embryoid bodies were generated after trypsinization (HUES-1) or collagenase (I6) dissociation of HES cell colonies and cell aggregation in low attachment dishes (Nunc, Rochester, NY, http://www.nuncbrand.com) in Dulbecco's modified Eagle's medium, 10% fetal calf serum.

Myocardial Infarction Model
Myocardial infarction was induced in female Wistar (mean weight of 250 g) by ligation of the left coronary artery. Rats were operated on under general anesthesia with isoflurane (Baxter, Deerfield, IL, http://www.baxter.com) 3% at induction and 2% for maintenance. After tracheal intubation, mechanical ventilation (Minerve; Alphalab) was set at a rate of 70/minute and with a 0.2-ml average insufflate volume. Analgesia was performed with a 10 mg/kg subcutaneous injection of ketoprofen (Merial, Duluth, Georgia, http://www.merial.com). The heart was exposed through a left thoracotomy, and the left coronary artery was permanently snared between the pulmonary artery trunk and the left atrial appendage.

Rat Randomization and Myocardial Cell Injection
On the 15th day following infarction, the rats were reoperated on by median sternotomy and randomized to receive injections of BMP2-treated HUES-1 cells (3 x 10⁶ HUES-1 cells, n = 11 rats) in suspension of single cells, BMP2-treated I6 ES cells (3 x 10⁶ I6 cells, n = 11 rats) in suspension of small-cell clusters, or control medium (n = 9 rats). Additional animals (n = 5 rats) received in-scar injections of 3 x 10⁶ HUES-1 cells that had been exposed to both BMP2 and SU5402. We selected the HUES-1 cell line for the latter experimental situation since this is the one that is not already committed to the mesoderm. One rat of each group (HUES-1 cell- and I6 cell-transplanted) died within 48 hours after cell injection. Immunosuppressive therapy, consisting of one daily 10 mg/kg subcutaneous shot of cyclosporine A, was started on the same day and continued until sacrifice.

Histopathology
Myocardial sections were stained with eosin and hematoxylin using a standard protocol. Two months after myocardial injection, rats were euthanized after general anesthesia. Transverse-cut rat hearts were immediately fixed in OTC (Tissue-Tek; Sakura, Torrance, CA, http://www.sakuraus.com) and frozen at –180°C nitrogen. Eight-µm sections were cut on an ultramicrotome (LM 1850; Leica, Heerbrugg, Switzerland, http://www.leica.com). Potential tumor growth was assessed with 8-µm standardized sections stained with hematoxylin and eosin.

Immunofluorescence of myocardial cryosections was performed after paraformaldehyde fixation and permeabilization using Triton X-100 with an anti-human ventricular ß myosin heavy chain (MHC) (Chemicon, Temecula, CA, http://www.chemicon.com), anti-human lamin A/C (Novocastra Ltd., Newcastle upon Tyne, U.K., http://www.novocastra.co.uk), anti-atrial natriuretic peptide (ANP; Abgent, San Diego, http://www.abgent.com), and anti-Connexin 43 (Cx43) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) antibodies. The proteins were revealed using Alexa-conjugated antibodies. Sections were observed in confocal microscopy (LSM-510 META; Carl Zeiss, Jena, Germany, http://www.zeiss.com). In addition, a whole-body autopsy of each transplanted rat, including brain, lungs, liver, spleen, pancreas, kidneys, periaortic lymph nodes, thymus, spine, and ovaries, was systematically performed for the detection of a tumor.

	RESULTS

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Phenotype of Undifferentiated I6 and HUES-1 Cell Lines
We used both HUES-1 and I6 HES cell lines to test their cardiogenic potential in vitro and in vivo. Indeed, a real-time PCR amplification of a few mesodermal and cardiac genes in both cell lines showed that the I6 cell line featured a higher basal expression of both mesodermal (Tbx6, SRF, Mesp1, brachyury) and early cardiac (Isl1, Mef2c, Tbx20) genes. GATA4 was weakly expressed in I6 but not in HUES-1 cells. Nkx2.5 was barely detected in either I6 or HUES-1 cell lines. Oct-4 level was not significantly different between both cell lines (Fig. 1).

View larger version (39K): [in this window] [in a new window]   Figure 1. Comparison of mesodermal and cardiac gene expression in HUES-1 and I6 cells. RNA was extracted and reverse transcribed from undifferentiated HUES-1 (passages 22–25) and I6 cell lines (passages 27–32) cultured for at least five passages after thaw-out on mouse embryonic fibroblasts prior to stimulation with bone morphogenetic protein-2. Differentiated colonies were cut out of the plate before RNA extraction. Gene expression was estimated by real-time polymerase chain reaction and expressed as a ratio between expression in I6 and expression in HUES-1. Data are normalized to ß-tubulin expression and expressed as means ± SEM (n = 3). *, statistically significant (p .01).

View larger version (45K): [in this window] [in a new window]   Figure 2. Cardiogenic effect of BMP2. (A): HUES-1 and I6 cells were treated for 48 hours with 10 ng/ml BMP2 in the presence or absence of fibroblast growth factor inhibitor SU5402 and RNA extracted and reverse transcribed. Gene expression was monitored by real-time quantitative polymerase chain reaction (PCR). Results are expressed as fold stimulation in gene expression when compared with untreated embryonic stem cells. (B): HUES-1 cells treated or not with BMP2 and SU5402 were allowed to aggregate to form EBs. EBs were kept in suspension for 5 days (D5) before RNA extraction and real-time PCR. Data are normalized to ß-tubulin expression and expressed as means ± SEM (n = 3–5). *, statistically significant (p .01). Abbreviations: BMP2, bone morphogenetic protein-2; EB, embryoid body; D5, day 5.

Engraftment of Cardiac Committed Cells in Postinfarcted Rat Heart
Two months after coronary artery ligation, human -actin mRNAs were identified in transplanted hearts but not in those injected with the control medium (Fig. 3). In contrast, we could not detect any mRNA encoding Oct-4, Pax6 (an early ectodermal marker), or -fetoprotein, an early endodermal marker (data not shown). Immunostaining with anti-ANP and anti-human lamin antibodies revealed the presence of lamin-positive human ES cell derived-cardiomyocytes (Fig. 4A).

View larger version (22K): [in this window] [in a new window]   Figure 3. Bone morphogenetic protein-2-treated human embryonic stem cells were engrafted into postmyocardial infarcted rats, and their fate was examined 2 months later by real-time polymerase chain reaction of -actin mRNA following reverse transcription of mRNA extracted from myocardial sections. The figure shows both the profile of the melting curves of amplicons and the amplicons on gel. Human DNA was used as a positive control. *, statistically significant (p .01). Abbreviations: Ctrl, control; HuDNA, human DNA.

View larger version (28K): [in this window] [in a new window]   Figure 4. Differentiation of human embryonic stem cells in vivo. Immunostaining of cryosections from human embryonic stem (HES) cell-engrafted myocardium using an anti-human lamin antibody (A) (note that the anti-lamin antibody stained HES-derived cells within the scar area but not the surrounding endogenous rat cells) and an anti-human ß myosin antibody (B). The antibody did not recognize the adult rat endogenous -myosin heavy chain (MHC) (left panel [B]), although it bound human ß-MHC. Images were acquired in confocal microscopy. (C): Quantification (in %) of the human ß-MHC positive regions in the scars of myocardium engrafted with BMP2-treated or BMP2 with SU5402-treated HUES-1 or BMP2-treated I6 cell lines; *, statistically significant (p .025). The area of ß-MHC positive area within the scar was calculated using the threshold function of MetaMorph software. (D): A transversal section stained by the anti-ß myosin antibody revealed some sarcomeric structures. The size was calculated using the scaling system of the Zeiss software driving the confocal microscope. Abbreviation: BMP2, bone morphogenetic protein-2.

Eosin-hematoxylin stained sections did not show any sign of inflammation or cell hyperproliferation 2 months post-transplantation (Fig. 5). Likewise, whole-body autopsies failed to disclose any tumor in peripheral organs.

View larger version (151K): [in this window] [in a new window]   Figure 5. Eosin-hematoxylin stained section from human embryonic stem cell-engrafted myocardium. The scar area does not show any sign of cell infiltration or cell proliferation.

	DISCUSSION

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BMP2 is a potent mesodermal and cardiogenic instructor when used at low concentration. Its cardiogenic potential is a well conserved property throughout evolution. Dpp, the Drosophila homolog of BMP2, favors formation of the mesoderm, including the heart [18]. Similar effects have been observed in zebrafish [19], Xenopus [20, 21], and chicken [22]. Our data obtained in two separate cell lines uncovered that BMP2 function is conserved in human species. Although I6 cells were more prone to give rise to a mesodermal lineage (Fig. 1), maximal BMP2 response was not significantly different from the one observed with HUES-1 cells, although maximal extent of gene expression was higher in I6 than in HUES-1 cells. Used alone, in a defined KOSR medium, BMP2 effect was weak, whereas its instructive action was dramatically enhanced by addition of the FGF receptor inhibitor, SU5402. Indeed, human ES cells are grown on feeder cells that secrete many factors, including FGF2, which is known to antagonize the BMP2 signaling pathway. SU5402 could act through at least two mechanisms to unmask the BMP2 transcriptional effect. First, FGF2 phosphorylates smad2/3, thereby preventing the BMP2 signaling cofactor from translocating into the nucleus and thus from exerting its transcriptional action [23]. Second, FGF2 is also known to act as a paracrine factor on both MEF and HES cells to regulate expression of Cerberus, a nodal and BMP antagonist enriched in HES cells [24]. Finally, it might be that SU5402 blocks self-renewal of cells and favors nonspecific differentiation, which is further directed to the mesoderm by BMP2. By blocking all or one of these pathways, the FGF inhibitor is required to unravel the BMP2 transcriptional response of HES cells.

In keeping with previous observations made in hearts transplanted with mouse [10, 11, 25] and human [26] ES cells, no hyperproliferation (teratoma) was observed in any of the rats injected with cardiac-committed HES cells. As intramyocardial injections in a beating heart are also known to cause leakage of a substantial proportion of cells [27], it is also noteworthy that we failed to document any extracardiac tumor. In fact, it was known for a long time that grafts of embryonic tissue also lose the capacity to form tumors very early after differentiation [28] when they acquire control of their proliferation by extracellular signal-regulated kinases. It is thus not surprising that a similar scenario takes place after cardiac commitment of HES cells. As such, our findings are not in contradiction with the previous observation [11] that injection of HES cells into a normal immunocompetent myocardium results in teratoma formation, since the latter results primarily suggest that such an environment is unlikely to provide enough cardiogenic factors required for differentiation of ES cells. Of note, the rather reassuring safety data yielded by our experiments were obtained despite the lack of pretransplantation sorting targeted at eliminating nonspecified cells. This suggests that the environment of the diseased myocardium (i.e., scar) enriched in growth factors is sufficient to drive primed ES cells toward a cardiac fate [25]. In a clinical perspective, however, such a selection step remains a major goal.

So far, two studies have assessed the effects of intramyocardial transplantation of HES cells. Both have entailed the use of embryoid body-derived cardiomyocytes into either normal myocardium [26] or acutely infarcted myocardium [29]. To make the protocol more clinically relevant, we selected a delayed timing of in-scar transplantation that tends to mimic the clinical scenario of heart failure and injected cardiac-specified but not yet fully differentiated monolayer-cultured cardiac progenitors. Altogether, the engraftment patterns seen after 2 months support the advantage of this cardiac commitment process before transplantation into the target scar where local signals are then expected to drive the fate of the graft further down the cardiomyocytic differentiation pathway.

We should, however, point out that the phenotype of HES cell-derived cardiomyocytes in situ was rather close to a fetal one. Indeed, the cells still expressed ß-MHC and ANP, two known markers of the early stage of cardiac differentiation. The short length of the sarcomere is still characteristic of a fetal myocyte. Several reasons could account for this immature phenotype. HES cells may require a longer time (more than 2 months) to fully differentiate. Alternatively, the paracrine environment of the infarction scar may not allow the factors (some FGFs, neuregulin, retinoic acid, BMP10...) [30] or signals taking place in embryogenesis to ensure a full differentiation process.

Another interesting observation is that I6 cells gave rise to larger engraftment areas than HUES-1 cells. Although both cell lines respond with the same efficiency to BMP2, I6 cells feature a higher basal expression of mesodermal cardiac genes (Fig. 1). This indicates that the cell line is already committed to the mesoderm, which is likely to account for the better cardiogenic potential in vivo. Of note, HUES-1 cells pretreated with BMP2 together with SU5402 also featured a better engraftment than HUES-1 challenged by BMP2 alone. This further emphasizes that the stage of specification is crucial to ensure a proper differentiation of ES cells in situ. The finding that the rates of scar repopulation by the grafted cells were low overall probably reflects a combination of initially insufficient cell dosing, extracardiac cell leakage at the time of injections, and possible death of retained cells. Clearly, optimization of the functional benefits of ES cell transplantation will require that each of these issues be thoughtfully addressed.

Finally, and in contrast to what has been reported in our previous studies using mouse ES cells [10–31], we could not detect Cx43 mRNA or protein in HES cell-derived cardiomyocytes. HES-cell derived differentiated cardiomyocytes [13] did not either express Cx43 when transplanted in injured left ventricle, although they did express it when cocultured with neonatal rat cardiomyocytes [31]. The reason for this discrepancy with mouse ES cells or the ex vivo situation is still unclear and might involve line-specific differences in the cardiogenic potential, a still early-stage of cell development, a level of expression below the threshold of detection by immunostaining, a mistargeting of the protein, or inhibitory signals coming from the fibrotic scar of infarcted rat myocardium to which HES cells might be highly sensitive. Finally, human ESC were transplanted into rat hearts, and some of the cues required for the full differentiation of the cardiac-specified cells into Cx43-expressing cardiomyocytes may have been missing. This issue is under investigation in the laboratory.

Expression of Cx43 remains, however, critical to establish unequivocally. Indeed, true cardiac regeneration implies that the donor-derived cardiomyocytes can establish gap junction-supported electromechanical connections with those of the host. The formation of such a syncytium allowing graft-host synchronized beats, which is critical for enhancement of contractility, has not yet been achieved with adult cells, whether myogenic [32] or bone marrow-derived [33]. The demonstration that HES cells could fill this unmet need would likely be a major step for rationalizing their use in situations where patient outcomes are critically dependent on the replenishment of a new pool of contractile cells.

Altogether, our findings bring a proof of concept of the feasibility of cardiac commitment of human ES cells. Although many challenges remain, our data are promising as to the safe use of human ES cells in clinics.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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We thank the French association against myopathies, the Foundation LeDucq, and the National Research Agency (ANR) for funding, the French Agency of Biomedicine for its efficacious support in delivering the authorizations to use the HES cell lines, Drs. C. Cowan and D. Melton (Harvard University, Boston, U.S.) for providing the HUES-1 cell line and advice for its culture, Dr. R.A. Levine (MGH, Boston) for discussion, and the NIH (Grant NHLBI Supplement for Human Embryonic Stem Cell Research under Program Announcement PAR-04-037; Parent Grant R01 HL72265-01) for partial support. A.T. and J.L. contributed equally to this work.

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