Abstract:
Dedifferentiation is a mechanism whereby specialized cells regain properties of their ancestors, including, in the extreme, stemness. We found that highly-purified cardiomyocytes isolated from adult mammalian hearts dedifferentiated rapidly when cultured in mitogen-rich medium. Such myocytes reentered the cell cycle and proliferated, expressing stem cell surface markers such as c-kit and early cardiac transcription factors including GATA and NKx2.5. These myocyte-derived cells (MDC) were capable of re-differentiating into myocytes and endothelial cells. Contrary to prevailing dogma, cardiomyocyte dedifferentiation yields proliferative cells expressing stem cell markers and capable of multilineage differentiation. Cardiomyocyte dedifferentiation is a potential source of endogenous stem cells in the adult heart.

Description:
[0001]    This invention was made using funds from the United States government, which therefore retains certain rights in the invention. A grant from the National Institutes of Health, HL083109, was used. 
     
    
     TECHNICAL FIELD OF THE INVENTION 
       [0002]    This invention is related to the area of stem cells and stem-like cells. In particular, it relates to cardiac cells having regenerative uses. 
       BACKGROUND OF THE INVENTION 
       [0003]    The mammalian heart has long been considered to be a highly specialized organ unable to repair itself after injury. The recent recognition that the heart contains its own pool of stem cells has ushered in a new era of cardiac biology and therapeutics. Cardiac stem cells (CSCs) express a variety of stem cell antigens (e.g. c-kit, sca-1, is1-1, SSEA-1, ABCG2) and cardiac-specific markers (e.g. NKx2.5, GATA4, α-MHC) (Lyngbaek et al., 2007; Barile et al., 2007); when transplanted, they contribute to regeneration of injured myocardium and improve cardiac function. Nevertheless, little is known regarding the sources of cardiac stem cells. The focus to date has been on seeding from circulating blood pools (Yeh et al., 2003; Shyu et al., 2006) versus endogenous cardiac origin, e.g. as embryonic remnants (Torella et al., 2006). 
         [0004]    Here, we consider dedifferentiation as yet another potential source of CSCs. Dedifferentiation can change the phenotype and functions of specialized cells, rendering them closer to their ancestors with augmented plasticity. For instance, pigment cells derived from neural crest can dedifferentiate and reprogram to become multipotent self-renewing progenitors expressing early neural marker genes Sox10, FoxD3, Pax3 and Slug, and give rise to glial cells and myofibroblasts (Real et al., 2006). Dedifferentiation is a common occurrence in plants; plant protoplasts from tobacco leaves have been reported to undergo a transitory phase conferring pluripotentiality, that precedes signal-dependent re-entry into the cell cycle (Zhao et al., 2001). 
         [0005]    In adult cardiomyocytes, dedifferentiation has been investigated extensively at the phenotypic level. Compared to normal myocytes, dedifferentiated cells become physiologically more “neonatal”, while morphologically they flatten and spread in culture, with increased diameter and surface area (Ausma et al., 2001; Fredj et al., 2005). Sarcomeric structures are lost, with disorganized myofibrils (Bird et al., 2003; Horackova and Byczko, 1997) and dramatically altered expression of cardiac α-actinin,α-MHC, α-MLC, etc (Benardeau et al., 1997b; Bird et al., 2003). A phenomenon akin to in vitro dedifferentiation has also been described in vivo, in fibrillating atria (Rucker-Martin et al., 2002), chronically-ischemic myocardium, and in the border zone of myocardial infarcts (Dispersyn et al., 2002; Driesen et al., 2007). Such dedifferentiated myocytes are not apoptotic and presumably reflect adaptations to abnormal myocardial stress (Dispersyn et al., 2002). 
         [0006]    There is a continuing need in the art for new sources of regenerative cells for therapy of heart diseases. 
       SUMMARY OF THE INVENTION 
       [0007]    According to one embodiment of the invention a method for obtaining stem-cell-like myocyte-derived cells (MDCs) from atrial or ventricular heart tissue is provided. Cells are isolated from atrial or ventricular heart tissue to form a cell suspension. The cell suspension may be optionally purified to increase the proportion of myocytes in the cell suspension. The cells are cultured in a medium comprising a mitogen. A composition comprising MDCs is thereby formed. 
         [0008]    According to some embodiments, cells are harvested at a plurality of time points from the medium comprising MDCs to form a plurality of samples of MDCs. The proliferative capacity of one or more of the samples of MDCs is assessed. One or more of the samples of MDCs is then clonally proliferated. One or more of the samples of MDCs is tested to confirm expression of one or more marker of stem cells selected from the group consisting of c-kit, sca-1, MCR1, CD34, CD33, alpha-MHC, NKx2.5, GATA4, and CD105. 
         [0009]    Also provided by the present invention is an isolated preparation of cardiac stem-like cells. The cells proliferate in culture and express a marker selected from the group consisting of c-kit, NKx2.5, and GATA4. The cells can be derived from adult cardiac atrial or ventricular myocytes. 
         [0010]    These and other embodiments which will be apparent to those of skill in the art upon reading the specification provide the art with methods and tools for regenerating cardiac tissue after disease has damaged cardiac tissue. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0011]      FIG. 1A-1C . Dedifferentiation and Proliferation of Cardiomyocytes. ( FIG. 1A ) Purified atrial myocytes were cultured as described in Experimental Procedures. Daughter cell budded from the mother atrial myocyte after 3.5 days; Arrow indicates the daughter cell. ( FIG. 1B ) Purified ventricular myocytes (insert) dedifferentiate remarkably after about 3 days of culture, and start to divide at day 6, showing significant cytoplasmic division. Scale bar, 100 μm. ( FIG. 1C ) Examples of proliferation of atrial myocytes culture for 6d Immunofluorescence shows the expression of Aurora B (green) at the cleavage gap (white arrow) between the myocyte that expresses weak cTnT (red; red arrow) and the newly divided cell without detectable cTnT (large white arrow); Both cells are positive to anti-BrdU immunostaining (white). Nuclei are stained with DAPI (blue). Scale bar, 20 μm. 
           [0012]      FIG. 2A-2C . Cell cycle Progression of Dedifferentiated Myocytes and the mechanisms. ( FIG. 2A ,  FIG. 2B ) Expression of cell cycle markers with antibodies against Ki67 ( FIG. 2A , green) and histone S3 pospho S10 (H3P) ( FIG. 2A , His, red), and BrdU ( FIG. 2B , red). Shown are the representative images of culture of ventricular myocytes at 8d. Plotted data (right panels) show the time course of expression of Ki67, H3P, and BrdU incorporation as percentages of cells. * p&lt;0.05; +p&lt;0.001 for atrial vs ventricular myocytes; n=151˜380 cells for different time points. ( FIG. 2C ) Mean data of fluorescence intensity for the expressions of 14-3-3 (left), and p21 and p53 (right) in freshly isolated (Ctl) atrial myocytes (Atr), which were significantly lower than in ventricular myocytes (Vent); Both decreased significantly after 5d culture. * p&lt;0.01 vs Ctl; +p&lt;0.01 vs Ctl Atr. 
           [0013]      FIG. 3A-3C . Myocyte-Derived Cells (MDC) express cardiac stem cell marker. ( FIG. 3A ) Example images show the clusters of small phase bright cells (MDC) arise from myocytes isolated from guinea pig atria (a, 10d culture; b, 4d after MDC 1 st  harvest), rat atria (c, 9d culture) and ventricle (d, 14d culture) in continuous culture. ( FIG. 3B ) Expression of c-kit in freshly harvested MDC (a) or plated for 18 hr (b); (c) Image shows the heterogenous MDC, expressing c-kit (green), CD34 (white) and cTnT (red); (d) After harvest of MDC, culture layer cells were incubated with c-kit-PE (red), indicating strong c-kit staining in cells located proximal around the MDC clusters being harvested. ( FIG. 3C ) RT-PCR amplification of stem cell and cardiac markers. H, heart tissue; BM, bone marrow cells; A.P., purified atrial myocytes; MDC, myocyte-derived cells; Sp, spheres formed from MDC. 
           [0014]      FIG. 4A-D . Re-differentiation of MDC. ( FIG. 4A ) Sphere formed from MDC loosely adhere on the culture layer (a) or detached and became suspension, and eventually (2-5d) beat spontaneously. Both MDC and spheres can be harvested and cultured for further tests. (b) Freshly harvested MDC sphere; (c) MDC sphere flattened on the culture vessel and cells crawled off the sphere 3 hr after plating. (d) MDC 18 hr after harvest from myocyte culture. Shown in here are rat myocyte culture. ( FIG. 4B ) Example image of immunohistochemical test showing the expression of c-kit and cardiac α-MHC in sphere (left) and cells off the sphere (right). ( FIG. 4C ) Expression of Cx43 (left) and CD31 (right) in spheres. ( FIG. 4D ) Green fluorescence in a sphere transduced with replication-defective lentivirus encoding eGFP driven by cardiac α-MHC promoter at 3d. 
           [0015]      FIG. 5A-5B  (S1). Purity of myocyte preparation and myocyte dedifferentiation. ( FIG. 5A ) Immunocytochemical tests for cardiac α-MHC, CD90, CD34, CD31 or CD90 (all color-coded) in purified atrial (Atr) or ventricular (Vent) myocytes, showing the preparation is highly pure for cardiomyocytes; ( FIG. 5B ) Time-lapse tracking of guinea pig myocyte dedifferentiation, showing significant weaker expression of cTnT. 
           [0016]      FIG. 6A-6D  (S2). Electrophysiology of Dedifferentiated myocytes and myocyte-derived cells (MDC). ( FIG. 6A ) Example recording of inward rectifier potassium current (I K1 ) in fresh (Ctl) and 4d or 7d cultured myocytes, and MDC; ( FIG. 6B ) I-V relationship of I K1  in fresh or cultured myocytes or in MDC. Digits in bracket denote cell numbers. * p&lt;0.05. ( FIG. 6C ) Resting membrane potential (RMP); p&lt;0.001 for all vs Ctl. ( FIG. 6D ) Capacitance as a means to measure cell size 
           [0017]    FIG.  7 A(a-d)- 7 B(a-c). (S3) Mitosis and Cytokinesis of cardiomyocytes. 
           [0018]    ( FIG. 8A-8C ) (S4). Time for 1 st  confluent of myocyte culture ( FIG. 8A ), MDC diameter ( FIG. 8B ), and time for SP beating ( FIG. 8C ). 
           [0019]      FIG. 9  (S5). RT-PCR detection of other transcripts. RT-PCR amplification of other markers of rat cells. M, DNA ladder; H, heart; BM, bone marrow; VS, aorta vessel; AP, purified atrial myocytes; VP, purified ventricular myocytes; MDC, myocyte- derived cells; Sphere, sphere formed from MDC. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0020]    We investigated dedifferentiation of adult atrial and ventricular myocytes. The salient results are that in vitro cell culture conditions can promote dedifferentiation that is associated with down-regulation of cell cycle inhibitors 14-3-3η and p21, and that the dedifferentiated cells can divide and generate cardiac precursor cells that are positive for c-kit, Nkx2.5 and GATA4. The dedifferentiated adult mammalian cardiomycytes are an abundant source of cells for use in cardiac cell regenerative therapies. 
         [0021]    Surprisingly, applicants have found that adult myocytes, derived from the atrium or ventricles, can dedifferentiate and become stem-cell like (MDCs). The stem-cell likeness is reflected in the expression of c-Kit (detectable by RT-PCR), which adult myocytes do not express. When these MDCs differentiate, they lose expression of c-Kit. We have not detected expression of Sca-1 under current conditions in the MDCs, although conditions may be found in which it would be expressed. One distinguishing feature of the MDCs is their cell size. The MDCs (10-30 um) are bigger than regular cardiac stem cells (approx 6-10 um diameter) or bone marrow stem cells (6-8 um). 
         [0022]    Myocytes can be isolated from either atrial or ventricles of the heart. These can be obtained from any source, for example from biopsies (endomyocardial or surgical specimens), cadavers, animal donors, etc. As is known in the art, the tissue can be mechanically macerated to produce and liberate myocytes. Enzymes, such as proteases, can also be used to liberate myoctyes from the tissue. Purification of adult myocytes can be by any means known in the art. These include differential centrifugation, culturing under selective conditions, differential harvesting of cultured cells, and gradient centrifugation. The purification, however, is optional. 
         [0023]    In order to dedifferentiate isolated adult cardiac myoctyes, one can culture them in the presence of mitogens. Proliferating cells results which have altered properties. Any mitogen can be used. Mitogens present in serum can be used, including bovine, fetal bovine, human, porcine, and ovine sera. Any amount between 0.1 to 20% serum can be used, for example, from 0.1 to 1%, from 1% to 5%, from 5% to 10%, from 10% to 15%, and from 15% to 20%. The amount can be increased, in steps increases or in a gradient, as growth progresses. Purified growth factors can be used as mitogens, including but not limited to VEGF, HGV, IGF, FGF, EGF, GCSF, GMCSF, MCSF, CSF-1, and PDGF. Changes in proliferation markers, proliferative index, and marker expression can be seen in as little as 3, 5, 7, 9, 11 days. Culturing can be carried out from 1 to 60 days. Cultures can be reseeded to maintain a high proliferative index. Cell cycle inhibitor expression decreases and proliferative index increases from the initial. 
         [0024]    The electrophysiology of the cells also changes as they are cultured. Inward rectifier potassium current and membrane resting potential decreased as cells dedifferentiated. In addition, electrical capacitance of the cells decreased. 
         [0025]    Cardiomyocytes can be isolated from any mammals. These include rodents and primates. Exemplary animal sources include rat, mouse, guinea pig, goat, rabbit, pig, and human. Cardiomyocytes can be obtained from laboratory animals, cadavers, or patients. If human cardiomyocytes are used, they can be delivered back to the same patient or to different patients. They can be stored at any stage in the process, before dedifferentiation, after dedifferentiation, and after redifferentiation. 
         [0026]    The MDCs demonstrate the ability to differentiate. For example, they form spheres. The spheres express less CD34 and c-Kit than the MDCs. 
         [0027]    Because the MDCs have the ability to redifferentiate, they are useful for treating patients and animals with heart disease or heart disease models. Such diseases include chronic heart failure, post-myocardial infarction, right ventricular failure, pulmonary hypertension, ventricular dysfunction induced by a cytotoxic agent, and ventricular dysfunction induced by an anti-neoplastic agent. The MDCs can be introduced by any means known in the art, including but not limited to intracoronary infusion via a catheter, intramyocardial injection via a catheter, and intramyocardial injection during surgery. 
         [0028]    The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention. 
       EXAMPLES 
     Example 1 
     Dedifferentiated Cardiomyocytes Re-Enter Cell Cycle and Proliferate 
       [0029]    We purified enzymatically-separated cardiomyocytes from hearts of adult rats, guinea pigs or mice using multiple differential centrifugation and Percoll gradient separation steps. Tests of morphology ( FIG. 1 ), immunoreactivity (FIG. S 1 A), and RT-PCR ( FIG. 3 , FIG. S 5 ) confirmed the purity of the isolated cardiomyocytes. Visually, the primary cells look homogeneously large and striated despite that atrial myocytes have variable shapes when plated to culture; more importantly, there is no detectable expression of proteins or transcripts characteristic of fibroblasts, endothelial cells or stem cells. To track individual cells in culture, atrial and ventricular myocytes were cultured at low density in grid-culture dishes or on coverslips. Shortly after plating, myocytes dedifferentiated, losing striations, rounding up and, often, beating spontaneously. Immunocytochemical studies demonstrated that after 3 days of culture, myocytes dedifferentiated, with significantly reduced expression of α-MHC or cTnT (Fig S1B). Inward rectifier potassium current (I K1 ) and membrane resting potential, characters of cardiomyocytes, were dramatically reduced in dedifferentiated myocytes. Electrical capacitance as a means of assessing cell size (Zhang et al., 2003) was also significant smaller with culture prolonged and dedifferentiation and proliferation progressed (Fig S2). 
         [0030]    In addition to these long-recognized morphological and physiological changes, we found that plated myocytes begin to divide and give rise to daughter cells within 3-7 days in culture. Expression of aurora B in the cleavage gap between cells indicates that new divided, BrdU-positive cells with barely detected cTnT are from cardiomyocytes which typically express cTnT ( FIG. 1 ). In addition, atrial myocytes showed greater plasticity and produced daughter cells earlier than ventricular myocytes, but the phenomena are generally similar in myocytes from either chamber. A subgroup of dedifferentiated round myocytes that budded off new daughter cells continued to demonstrate spontaneous contractions. In other cases, cells rounded up before flattening and spreading, did not show spontaneous beating, but gave rise to phase-bright daughter cells. 
         [0031]    Although the dedifferentiation mechanism has been studied intensively and better elucidated in myocytes from amphibians and zebrafishes (Straube and Tanaka, 2006b; Lien et al., 2006; Ahuja et al., 2007), it is poorly understood in mammalian cardiomyocytes (Engel et al., 2005; Driesen et al., 2006; Montessuit et al., 2004). We analyzed cell cycle progression in this cell culture model by studying the active cell cycle markers Ki67, histone H3 and BrdU incorporation by immunocytochemistry. Ki-67 is a vital molecule for cell proliferation that is expressed in proliferating cells at all phases of the active cell cycle, but is absent in resting (G0 phase) cells. After 2d in culture, 11±8% and 6±2% of atrial and ventricular myocytes, respectively, re-entered active cell cycle and expressed Ki-67, with gradually increased levels, reaching to 80±11.9% and 46±11% at 11 d for atrial and ventricular myocytes, respectively (p&lt;0.001) ( FIG. 2A ). We assessed the proportion of dedifferentiated myocytes entering the S phase by incubating the cells with BrdU for various periods. Cells in M phase were detected using an antibody against phospho histone H3 at S10 (H3P). We found a progressive increase in the numbers of BrdU- and H3P-positive cells, reaching a maximum at about 1 week. Interestingly, the proportion of BrdU- and H3P-positive cells was always higher in cultures of atrial myocytes than in that of ventricular myocytes ( FIG. 2A ,  2 B). Besides the cytokinesis, we also found cells in anaphase and telophase (Fig S3), demonstrating the progression of proliferation of the dedifferentiated myocytes. 
         [0032]    To further decipher the mechanisms underlying the cell cycle progression and their differences between atrial and ventricular myocytes, we investigated the expressions of interrelated factors like 14-3-3 (YWHAH), p21 and p53 that are critical checkpoint regulators in cell cycle progression (Ahuja et al., 2007) by immunocytochemical detection of cells cultured for 5 days. Expression of the negative cell cycle regulator 14-3-3 has been shown to prevent the cell cycle progression and serum-induced proliferation (Du et al., 2005; Yang et al., 2006). As predicted, the expression of 14-3-3η, an abundant isoforms in the heart (He et al., 2006), was significantly lower in freshly isolated atrial myocytes than in fresh ventricular myocytes. Furthermore, on day 5, when was the faster response period of cell cycle progression for both types of cells, expression of 14-3-3η was dramatically reduced ( FIG. 2C ). p21 (WAF1/CIP1), a downstream target of 14-3-3 and key inhibitory factor involving in all phases of cell cycle (Li and Brooks, 1999), was also reduced significantly in cultured dedifferentiating/proliferating myocytes. Its endogenous level was 61% higher in freshly isolated ventricular myocytes than in atrial myocytes. Furthermore, p53 expressed much less in fresh atrial myocytes than in ventricular myocytes, and decreased significantly in atrial myocytes but not much in ventricular myocytes. Taken together, the data suggest the weaker inhibitory signals in atrial myocytes facilitate their faster and easier cell cycle progression and the diminution of the inhibitory factors render the cell into cell cycle progression and proliferation. 
       Example 2 
     Myocyte-Derived Cells Exhibit Cardiac Stem Cell Markers 
       [0033]    Myocytes cultured in normal density become confluent after 1-2 weeks (Fig S4A) and thereafter clusters of loosely-adherent phase-bright round cells emerged above the monolayer of dedifferentiated/proliferating cells ( FIG. 3 ). These cells, seemed to be heterogenous in size (Fig S4B), can be harvested by gently pipetting without trypsinization and are referred to as myocyte-derived cells (MDC). 
         [0034]    Dedifferentiation, e.g., in pigment cells, has been demonstrated to contribute to stem cells and tissue regeneration (Real et al., 2006). We asked if MDC that is distinct from cardiomyocytes in morphology and electrophysiology, have any characteristics of cardiac stem cells (Smith et al., 2007; Boyle et al., 2006). By direct and indirect fluorescent immunostaining, we found that rat MDC do indeed express stem cell markers c-kit and CD34, but little or weak, if any, sca-1 or CD90 (data not shown); 61±19.7% freshly harvested MDC were positive to c-kit. Furthermore, in the area of MDC clusters, there were cells in the layer strongly positive for c-kit immunostaining ( FIG. 3B ), implicating the source of MDC. 
         [0035]    To further confirm the expression of stem cell markers in MDC, we performed RT-PCR to test the expression of different transcripts. c-kit was expressed in heart tissue, bone marrow cells, and MDCs. In addition, the other cardiac stem cell transcript sca-1 was undetectable in MDC; endothelial precursor marker gene CD34 was present in MDC. Cardiac transcripts α-MHC, Nkx2.5, and GATA4 were all detected in MDC, heart tissue and purified myocytes as well ( FIG. 3C ; Fig S5). 
       Example 3 
     Myocyte-Derived Cells Re-Differentiate 
       [0036]    MDC self-organized into spheres 3-5 days after the cluster cells became more confluent. There were 0˜4 spheres in each well of a 6-well culture plate, depending on the condition of cells. MDC spheres either loosely adhered to the culture layer or became suspended in medium, and show slow spontaneous activity within 2-5 days of sphere stage (Fig S4C. The semi-adherent spheres could be harvested by gentle pipetting. Semi-adherent or suspending spheres flattened onto the bottom when seeded into fibronectin-coated plates, and gave rise to cells off the spheres, which eventually stopped beating while turning into monolayer cells ( FIG. 4A ). Moreover, myocyte cultures could provide 3˜4 harvests of MDC or spheres. New daughter cells emerged again always around the area where previous MDC were produced. 
         [0037]    In the spheres, most cells were positive for α-MHC, connexin 43 (Cx43), and CD31 immunostaining, and some positive for c-kit. Some cells off the sphere also express cTnT and others express c-kit ( FIG. 4B ). When transduced with replication-defective lentivirus encoding enhanced green fluorescent protein (eGFP) driven by the cardiac α-MHC promoter, MDC spheres exhibited focal green florescence within 3-5 days along with spontaneous contraction ( FIG. 4D ). RT-PCR revealed that in the spheres, there was weaker stem cell transcript signal of c-kit, but stronger signal of cardiac transcripts α-MHC, Nkx2.5, and GATA4, suggesting the cardiogenesis and re-differentiation of MDC when entering in sphere phase. In addition, endothelial precursor marker gene CD34, present in MDC, tended to decrease in the spheres; endothelial marker CD31 (PECAM-1) expresses in both MDCs and the spheres (Figure S5). 
       Example 4 
     Experimental Procedures 
       [0038]    Isolation, Purification, and Primary Culture of Cardiomyocytes 
         [0039]    Cardiomyocytes were isolated from adult male Wistar-Kyoto rats (4-8 weeks, 70-120 g), Hartley guinea pigs (3-5 weeks, 300-380 g) or C57BL/6 mice (4-6 weeks, 17-21 g) by enzymatic digestion of the whole heart on a Langendorff apparatus with similar protocol as previously described. (Zhang et al., 2006; Kizana et al., 2007) Heparinized animals were anaesthetized by sodium pentobarbital (Ovation Pharmaceuticals Inc, Deerfield, Ill.). Hearts were rapidly excised and cleaned to remove blood in ice-cold Tyrode&#39;s solution before mounted to a Langendorff apparatus conjugating to a pressure monitoring device, and perfused retrogradely with the following four oxygenated solutions in sequential order: modified Tyrode&#39;s solution containing 1.0 mM Ca 2   +  (2 min), Ca 2   + -free Tyrode&#39;s solution (2-3 min), Ca 2   + -free Tyrode&#39;s solution containing 0.2 Wunsch unit/ml of collagenase made from Liberase Blendzyme 4 (Roche Molecular Biochemicals, Indianapolis, Ind.) for 10-20 min depending on species and digest conditions. Digested atrium and ventricles were cut off and minced in Kruftbrühe (KB) solution, then filtered through a 200 um nylon mesh to remove big piece of undigested tissues. Isolated cells were rinsed in KB solution and let settled by gravity for 3 times to remove debris and non-cardiomyocytes. Resuspended cells in KB solution were loaded above the top layer of Percoll gradient which was formed by 20%, 40%, and 70% of Percoll to separate myocytes from debris and other types of cells. After three washes in KB solution, myocytes were resuspended in KB solution or in culture media for further experiments. Modified Tyrode&#39;s solution contained (mM): NaCl 105, KCl 5.4, KH2PO4 0.6, NaH2PO4 0.6, NaHCO3 6, KHCO3 5, CaCl2 1, MgCl2 1, HEPES 10, glucose 5, taurine 20 (pH 7.35 with NaOH), and KB solution had (mM): KCl20, KH2PO4 10, K-glutamate 70, MgCl2 1, glucose 25, β-hydroxybutyric acid 10, taurine 20, EGTA 0.5, HEPES 10, and 0.1% albumin (pH 7.25 with KOH). 
         [0040]    Purified myocytes were resuspended in Medium 199 (Invitrogen, Carlsbad, Calif.) supplemented with 110 mg/L sodium pyruvate, 0.1 mM β-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin, and 5% FBS (Invitrogen) and cultured in laminin-coated 6-well culture plates or 100 mm dishes in normal density of 6000 and 9000 cells/cm 2  for ventricular and atrial myocytes respectively, at 37° C. for 1 hr before wash to remove dead and non-adherent cells, and repeated once after 1 hr of culture. Serum concentration in medium was gradually increased to 10% and 20%. On the second and third day of plating, medium was replaced to remove dead cells, and then maintained for prolonged culture while partially changed about every 5 days. 
         [0041]    Cell Imaging and Tracking 
         [0042]    In order to verify the proliferation of dedifferentiated myocytes, cells were plated in lower density as compared to normal dense culture for MDC production. Numeric grid-marked coverslips (Bellco Biotechnology, Vineland, N.J.) coated with laminin were used to identify the cellular changes during the culture, under time-lapse microscope (Nikon TE-2000E inverted microscope) for continuous analysis, or under regular inverted microscope (Nikon TE-2000U), with phase contrast objectives and images were captured with a monochrome CCD camera (Q-Imaging, Surrey, BC, Canada) with a program suite Image Pro Plus (Media Cybernetics, Bethesda, Md.). At the end of the tracking, cells were subjected to analysis of markers related to cell cycle progression and stem cell when needed. A 3CCD Color video camera (Sony) connected to a personal computer was used to capture real-time images and videos of beating cells and spheres. 
         [0043]    Culture of Myocyte-Derived Cells 
         [0044]    At about 10 days to 2 weeks after the culture, the loosely adherent myocytes-derived cells (MDC) were harvested by gentle pipetting for 3 times with a disposable transfer pipette. Cells were cultivated in same medium as of the serum-rich myocyte culture medium, for the experiments detecting the markers in fresh isolated cells. Alternatively, MDC culture medium which was DMEM/F 12 supplemented with 0.1 mM β-mercaptoethanol, bFGF 0.1 ng/ml, TGF- β 1 ng/ml, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FBS, was used to maintain the cells in 95% humidity, 5% CO2, at 37° C. 
         [0045]    Labelling of Myocytes with BrdU 
         [0046]    Cells were loaded with 3-bromo-2-deoxyuridine (BrdU; 5 μM) for various periods before immunocytochemical assay (Engel et al., 2005). 
         [0047]    Fluorescent Immunocytochemistry 
         [0048]    Cellular phenotypes in the cultures were analyzed similarly as previously described (Smith et al., 2007; Zhang et al., 2006) using immunofluorescence. To test the expression of stem cell markers, rabbit polyclonal antibody (pAb) against c-kit (CD117) (Santa Cruz Biotechnology, Santa Cruz, Calif.) or Oct-4 (Abcam, Cambridge, Mass.), mouse monoclonal antibody (mAb) against Sca-1 (Invitrogen), goat pAb against Thy-1 (CD90) were used as primary antibodies. Expression of cardiac markers were tested using antibodies included mouse mAb of cardiac specific α-MHC from Abcam, α-actin from Sigma, and rabbit pAb Cx43 and GATA4 from Invitrogen, goat pAb Nkx2.5 from Santa Cruz Biotechnologie, Inc. Primary antibodies against cell cycle-specific molecules included: Ki67, Histone H3 (phosphor S10) and anti-bromodeoxyuridine (BrdU) were from Abcam. The specificity of antibodies was confirmed by blocking peptides or control cells. Donkey anti-mouse, anti-rabbit, or anti-goat antibodies with fluorescent conjugation were used as secondary antibodies. 
         [0049]    Direct immunostaining were also performed to test the expression of stem cell markers in freshly harvested MDC using PE-conjugated mouse mAbs against c-kit (BD Biosciences), Sca-1 (Invitrogen), or FITC-conjugated CD90 (Abeam). 
         [0050]    In MDC spheres, stem cell and cardiac markers were detected using whole-mount immunofluorescent techniques and examined with standard and Z-stack confocal laser scan microscope (LSM 510; Zeiss). The acquisition settings were optimized to avoid false positive or false negative staining Images were processed by LSM 510 software suite. 
         [0051]    RT-PCR 
         [0052]    Reverse-transcription Polymerase Chain Reaction (RT-PCR) was performed to test the mRNA expression of both stem cell and cardiac markers. Extraction of total RNA from rat heart tissue, bone marrow cells flushed from femurs, purified myocytes, MDC, and MDC spheres, and one-step RT-PCR were carried out with commercially available kits (Qiagen, Valencia, Calif.). Primer pairs for c-kit, sca-1, Oct 4, α-MHC, GATA4, and NKx2.5, β-actin are listed in Table S1. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE S1 
               
             
             
               
                   
               
               
                 Primers used for RT-PCR detection 
               
             
          
           
               
                   
                   
                 Primer 
                   
                 Prod 
                   
                   
                   
               
               
                 Molecule 
                 Access # 
                 sense oligo 5′ . . . 3′ 
                 antisense oligo 5′ . . . 3′ 
                 start 
                 stop 
                 length 
               
               
                   
               
             
          
           
               
                 c-Kit* 
                 NM022264 
                 AGCCGTCTCCACCATCCATCCAG 
                 GCGGACCAGTGCGTCGTTGTCTT 
                 142 
                 449 
                 308 
                   
               
               
                   
                   
                 (SEQ ID NO: 1) 
                 (SEQ ID NO: 10) 
               
               
                   
               
               
                 sca-1* 
                 XM_343263. 
                 CATCTTTCTCCTGGCCCTACT 
                 GAGGACTGAGCCCAGGATGAA 
                 46 
                 390 
                 345 
               
               
                   
                   
                 (SEQ ID NO: 2) 
                 (SEQ ID NO: 11) 
               
               
                   
               
               
                 CD90/Thy1 
                 NM_012673. 
                 CCTGCCTGGTGAACCAGAACCTT 
                 GCAGGCTTATGCCACCACACTTG 
                 125 
                 451 
                 327 
               
               
                   
                   
                 (SEQ ID NO: 3) 
                 (SEQ ID NO: 12) 
               
               
                   
               
               
                 CD31 
                 NM_031591 
                 AGAAGGAAGAGACGGTGTTG 
                 TTAGGAGGCGGTAAGTGATG 
                 1241 
                 1498 
                 258 
               
               
                   
                   
                 (SEQ ID NO: 4) 
                 (SEQ ID NO: 13) 
               
               
                   
               
               
                 CD34 
                 XM_001070343 
                 TCAGAGACCACGGTCAACTT 
                 ACTCCTCGGATTCCTGAACA 
                 417 
                 721 
                 305 
               
               
                   
                   
                 (SEQ ID NO: 5) 
                 (SEQ ID NO: 14) 
               
               
                   
               
               
                 GATA4 
                 NM_144730 
                 TCTAAGACACCAGCAGGTCCTC 
                 TTGGAGCTGGCCTGTGAT 
                 1540 
                 1823 
                 284 
               
               
                   
                   
                 (SEQ ID NO: 6) 
                 (SEQ ID NO: 15) 
               
               
                   
               
               
                 NKx2.5 
                 NM_053651 
                 TTATCCGCGAGCCTACGGTGA 
                 CTGCCGCTGTCGCTTACACTT 
                 366 
                 684 
                 319 
               
               
                   
                   
                 (SEQ ID NO: 7) 
                 (SEQ ID NO: 16) 
               
               
                   
               
               
                 aMHC 
                 NM_017239. 
                 AGTCAGAGAAGGAGCGCCTA 
                 TAGATCATCCAGGCCGCATA 
                 87 
                 378 
                 292 
               
               
                   
                   
                 (SEQ ID NO: 8) 
                 (SEQ ID NO: 17) 
               
               
                   
               
               
                 b-actin* 
                 NM_031144.2| 
                 ATATCGCTGCGCTCGTCGTC 
                 CGTCCCAGTTGGTGACAATG 
                 92 
                 322 
                 231 
               
               
                   
                   
                 (SEQ ID NO: 9) 
                 (SEQ ID NO: 18) 
               
               
                   
               
             
          
         
       
     
         [0053]    Statistics 
         [0054]    Data were expressed as mean±SEM, and paired or un-paired Student t-test were used to exam the significance of difference between groups, with a p&lt;0.01 considered as significant different. 
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