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Correspondence to Issei Komuro: komuro-tky{at}umin.ac.jp
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12-fold) CSPs were migrated and homed in injured heart than in normal heart. CSPs in injured heart differentiated into cardiomyocytes, endothelial cells, or smooth muscle cells (4.4%, 6.7%, and 29% of total CSP-derived cells, respectively). These results suggest that CSPs are intrinsic cardiac stem cells and involved in the regeneration of diseased hearts.
Abbreviations used in this paper: ANF, atrial natriuretic factor; BMP, bone morphogenetic protein; Brcp, breast cancer resistance protein; CMP, cardiac MP cell; CSP, cardiac SP cell; cTnT, cardiac troponin T; HDAC, histone deacetylases; MDR, multidrug resistance; MEF, myocyte-enhancer factor; MLC, myosin light chain; MP, main population; OT, oxytocin; OTA, OT antagonist; PE, phycoerythrin; PY, Pyronin Y; SA, sarcomeric
-actinin; Sca-1, stem cell antigen 1; SMA, smooth muscle cell actin; SP, side population; TSA, trichostatin A; vWF, von Willebrand factor.
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Side population (SP) cells are first identified as mouse hematopoietic stem cells with long-term multilineage reconstitution abilities based on their unique ability to efflux the DNA-binding dye Hoechst 33342 (Goodell et al., 1996, 1997). SP cells exist in a variety of organs, such as bone marrow, skeletal muscle, liver, brain, lung, skin, and heart (Asakura and Rudnicki, 2002; Montanaro et al., 2003). Zhou et al. (2001) reported that the ATP-binding cassette transporter, ABCG2 (also known as breast cancer resistance protein 1 [Bcrp1]), is a molecular determinant of this SP phenotype in hematopoietic stem cells. In mouse lung and rat liver, the SP phenotype has been reported to be largely determined by the expression of ABCG2 (Shimano et al., 2003; Summer et al., 2003). Among the tissue-derived SP cells, bone marrow and skeletal muscle SP cells have been well investigated. Bone marrow SP cells were first identified as a primitive population of hematopoietic stem cells (Goodell et al., 1996). The bone marrow–derived SP cells show long-term multilineage reconstitution in lethally irradiated recipients and form hematopoietic colonies in vitro (Goodell et al., 1996, 1997; Asakura and Rudnicki, 2002). Jackson et al. (2001) have reported that bone marrow SP cells also differentiate into endothelial cells and cardiomyocytes in ischemic hearts. Gussoni et al. (1999) reported that transplantation of skeletal muscle SP cells into the irradiated mdx mouse results in the reconstitution of the hematopoietic compartment of the transplanted recipients and regeneration of donor-derived, dystrophin-positive muscle in the affected muscle. Skeletal muscle SP cells have the in vitro hematopoietic activity, and differentiate into skeletal myocytes when cocultured with satellite cell–derived myoblasts (Asakura et al., 2002). These results suggest that SP cells have features of somatic stem cells, and that cardiac SP cells (CSPs) may be a promising candidate for cardiac stem/progenitor cells.
CSPs from postnatal hearts have been reported to differentiate into cardiomyocytes when cocultured with cardiomyocytes (Hierlihy et al., 2002; Martin et al., 2004; Pfister et al., 2005). However, factors that induce differentiation of CSPs into cardiomyocytes have not been identified. Several growth or humoral factors have been reported to possess the ability to induce the differentiation of primitive cells into cardiomyocytes. During the development, bone morphogenetic proteins (BMPs) and fibroblast growth factors promote cardiogenesis in chick (Sugi and Lough, 1995; Schultheiss et al., 1997). Both canonical and noncanonical Wnts play an important role in the cardiac differentiation (Eisenberg et al., 1997; Pandur et al., 2002; Naito et al., 2003). Oxytocin (OT) and dynorphin B induce differentiation of embryonic stem cells and P19 embryonal carcinoma cells into cardiomyocytes (Ventura and Maioli, 2000; Paquin et al., 2002; Ventura et al., 2003). Besides growth or humoral factors, chemical compounds such as DMSO and 5'-azacytidine have been reported to promote the cardiomyocyte differentiation of embryonic or somatic stem cells (Makino et al., 1999; Xu et al., 2002). These findings suggest that both extracellular signals and epigenetic modification are capable of turning the fate of stem cells to cardiomyocytes. Recently, Linke et al. (2005) have reported that c-kit–, MDR-1–, or Sca-1–positive cardiac stem cells migrate and proliferate in response to hepatocyte growth factor and insulin-like growth factor-1, respectively. However, it is still elusive whether CSPs, by responding to the ischemia-induced factors, move to the injured area of the heart and differentiate into cardiomyocytes.
We first report that CSPs from postnatal rat hearts differentiate into cardiomyocytes both in vitro and in vivo. Both OT and trichostatin A (TSA) induced postnatal CSPs to differentiate into beating cardiomyocytes. After intravenous transplantation of CSPs into normal adult rats, CSPs migrated and homed in the interstitial space of myocardium. When CSPs were intravenously transplanted into the cryoinjured heart, the number of CSPs was significantly larger in the border area than in the remote or infarct area after transplantation. Furthermore, CSPs differentiated into cardiomyocytes, endothelial cells, or smooth muscle cells in the border area. These findings suggest that CSPs are resident cardiac stem cells, which can migrate and regenerate myocardium in response to the ischemia-induced factors.
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4.0%, 2.0%, and 1.2% in fetal, neonatal, and adult hearts, respectively. In neonatal CSPs, 14% of the cells expressed CD45, 59% expressed CD29, and 8% expressed CD31 (Fig. 1 B, a–c). The percentage of CD31-positive cells was 13.1 ± 4.0% under the fluorescent microscope (Fig. 1 B, d and e). To examine whether CSPs were in a noncycling quiescent state, cardiac cells were stained with both Hoechst 33342 and Pyronin Y (PY). The percentage of cells in PY-negative G0 stage was significantly higher in CSPs (74.3 ± 1.4%) than in cardiac MP cells (CMP; 34.0 ± 2.6%; Fig. 2 A, a).
A comparable result was obtained from the bone marrow SP and MP cells (PY-negative G0 stage of bone marrow SP, 79.8 ± 3.1%; bone marrow MP, 41.7 ± 5.4%; Fig. 2 A, b). This suggests that CSPs represent a quiescent stem cell population in the heart.
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Localization of CSP in the heart
In neonatal rat hearts, most Bcrp1-positive cells (95.4%) were CD31 (Fig. 3 A, a [coronary artery] and b [capillary]), but there were some CD31-negative/Bcrp1-positive cells (Fig. 3 A, c, arrowheads).
Most of the CD31-negative/Bcrp1-positive cells (94.3 ± 9.8%) existed in the perivascular area (Fig. 3 A, c–e, arrowheads). There were also a few CD31-negative/Bcrp1-positive cells in the interstitial space (5.6% ± 9.8%; Fig. 3 A, f–i, arrowheads) between cardiomyocytes, which were stained with sarcomeric -actinin (SA; Fig. 3 A, h, arrows) and distant from CD31-positive vessels (Fig. 3 A, f, arrows). There were no significant differences in the percentage of CD31-negative/Bcrp1-positive cells per total Bcrp1-positive cells among apex, mid, and base of left ventricles (Fig. 3 B, a), and also among chambers (i.e., atrium, left, and right ventricles; Fig. 3 B, b). It has been reported that N-cadherin, CD29, and ß1 integrin mediate the adhesion of stem cells to specialized mesenchymal cells and extracellular matrix in the niche environment (Zhang et al., 2003; Wilson et al., 2004). Bcrp1-positive cells in the interstitial space coexpressed CD29 and N-cadherin around the surface of the cells (Fig. 4, a and b, arrowheads).
At the junction of Bcrp1-positive cells and the neighboring cell, abundant coexpression of CD29 and N-cadherin was observed (Fig. 4, c and d, arrowheads). The perivascular Bcrp1-positive cells, which were localized adjacent to the smooth muscle cell actin (SMA)–positive cells, coexpressed CD29 (Fig. 4, e–g, arrowheads). These findings suggest that Bcrp1-positive cardiac stem or progenitor cells were localized in the specialized area of the myocardium, which may be similar to the stem cell niche in other organs, such as hematopoietic and gonad systems (Gonzalez-Reyes, 2003; Zhang et al., 2003; Wilson et al., 2004).
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10 d (Fig. 5 A, d and e). 3 wk after the treatment, some clusters of beating cells were recognized among flattened cells (Fig. 5 A, f, and Video 2). There were no differences between OT- and TSA-induced cardiomyocytes in regard to the percentage of beating cells (OT, 0.27 ± 0.2%; TSA, 0.50 ± 0.21%; Fig. 5 B, c, shaded bar) and the percentage of SA-positive cells (OT, 3.8 ± 3.8%; TSA, 5.5 ± 3.9%; Fig. 5 B, c, open bar). Low magnification immunofluorescent images of SA and nuclear DNA of OT- and TSA-induced cardiomyocytes are shown in Fig. 5 B (a [OT] and b [TSA]). Fine striation was observed in OT- and TSA-induced cardiomyocytes (Fig. S2 A, a–d). Nontreated SP cells never exhibited spindle-shaped morphology or beating, and MP cells treated with OT or TSA detached from culture dishes within 1 wk.
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To examine the expression of cardiac proteins, the CSPs treated with OT or TSA were stained with specific antibodies against cardiac proteins. The cells treated with OT or TSA expressed GATA4 (Fig. 5 D, a and d), atrial natriuretic factor (ANF; Fig. 5 D, b, c, e, and f), cardiac troponin T (cTnT; Fig. 5 D, c and f), MLC2v (Fig. 5 D, a), and SA (Fig. 5 D, b, d, and e). Notably, staining of each contractile protein showed a fine striated pattern, suggesting that treatments with OT or TSA induced differentiation of CSPs into mature cardiomyocytes.
Cardiac SP cells can differentiate into osteocytes and adipocytes
It has been reported that SP cells from skeletal muscle and bone marrow differentiate into various types of cells, such as adipocytes, endothelial cells, and skeletal muscle and cardiac myocytes (Asakura et al., 2002; Iijima et al., 2003; Tamaki et al., 2003). To determine whether CSPs from the heart have multipotency of differentiation, we examined whether these cells could differentiate into cells other than cardiomyocytes. When CSPs were cultured with osteogenic inducers, including ß-glycerophosphate, dexamethasone, and ascorbic acid-2 phosphate, some SP cells stained positive with alkaline phosphatase, which is one of the early markers of osteocytes (Fig. S1 A, a, available at http://www.jcb.org/cgi/content/full/jcb.200603014/DC1). RT-PCR analysis revealed that expression of alkaline phosphatase gene was induced in cardiac SP cells after treatment with osteogenic inducers (Fig. S1 A, b, lane O). On the other hand, cardiac SP cells treated with OT or TSA did not express alkaline phosphatase (Fig. S1 A, b, lanes OT and T). When cardiac SP cells were cultured in adipogenic induction with MDI-I mixture for 20 d, some SP cells showed cytoplasmic accumulation of oil droplets stained with Oil Red O, indicating that CSPs differentiated into adipocytes (Fig. S1 A, c).
CSPs migrate and home into the injured heart
When GFP+ CSPs were transplanted into the normal rat via the tail vein, GFP+ CSPs were distributed over the various organs, such as lung (Fig. 6 A, a), spleen (Fig. 6 A, b), liver (Fig. 6 A, c and d), skeletal muscle (Fig. 6 A, e and f), bone marrow (Fig. 6 A, g and h), and heart (Fig. 6 A, i–l).
In the lung and spleen, there were less GFP+ cells at 12 wk than at 1 wk after transplantation (12 wk/1 wk ratio; 0.19 for lung and 0.67 for spleen; Fig. 6 B). On the contrary, in the liver, skeletal muscle, and heart, more GFP+ cells existed at 12 wk than at 1 wk after transplantation (12 wk/1 wk ratio; 1.63 for liver, 2.0 for skeletal muscle, and 3.0 for heart; Fig. 6 B). 4 wk after transplantation, some GFP+ CSPs in the liver expressed albumin (Fig. 6 A, c and d). In skeletal muscle, GFP+ CSPs had multiple nuclei and expressed desmin (Fig. 6 A, e and f). However, there were no GFP+ CSPs positive for CD45 in the bone marrow (Fig. 6 A, g and h). GFP+ CSPs in the heart expressed CD29 (Fig. 6 A, i and j) and were localized in the interstitial space of myocardium, which was delineated by collagen type IV (Fig. 6 A, k and l). In the normal heart, transplanted GFP+ CSPs did not express cTnT (not depicted).
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Discussion |
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Two groups have reported expression of cardiac proteins in CSPs when cocultured with primary cardiomyocytes (Hierlihy et al., 2002) or with CMP (Martin et al., 2004). Because both groups did not examine the contractile ability of SP-derived cells, it has remained unclear whether CSPs differentiate into mature cardiomyocytes. In addition, by the coculture method, it is difficult to distinguish if cardiomyocyte differentiation is accomplished by transdifferentiation or fusion. Recently, Pfister et al. (2005) that CD31-negative CSPs also differentiate into functionally beating cardiomyocytes by coculture with adult rat cardiomyocytes. In this study, we first demonstrated that CSPs could differentiate into mature cardiomyocytes, which showed not only cardiac gene expression but also sarcomere formation and spontaneous beating, by single reagents such as OT and TSA.
There were more CSPs in the rat heart of the early developmental stage. Fetal rat CSPs account for 4% of total isolated cells, 2% of neonatal rat CSPs, and 1.2% of adult rat CSPs. Our result of the developmental change of the CSP fraction is similar to the previously reported one in mouse hearts (Tomita et al., 2005). The percentage of CSPs from adult mouse was 0.24% in our experiments (unpublished data). The percentage varied from 0.02% to 2% in previous reports (Hierlihy et al., 2002; Oh et al., 2003; Martin et al., 2004; Tomita et al., 2005). Although there may be a difference in the percentage of CSPs among the species, the cell surface markers of isolated CSPs were variable among the reports. Pfister et al. (2005) and Tomita et al. (2005) reported that a large portion of isolated CSPs from adult mouse are CD31 positive. In this study, CD31-positive cells were only 7.6% in isolated CSPs from neonatal rats. Our immunohistochemical analysis indicated that most Bcrp1-positive cells in the heart are CD31-positive endothelial cells. The reason for these variations may be attributed to distinct isolation techniques and to the fact that most endothelial cells were lost during the step of cell isolation discussed in this study. Considering the conclusion of Pfister et al. (2005) that CD31-negative CSPs represent a distinct cardiac progenitor cell population, our CSPs isolated from neonatal rats are a condensed population of cardiac progenitors.
The ability to induce CSPs into the mature cardiomyocytes is comparable between OT and TSA (Fig. 5 B). There were only a few studies showing quantitative analysis of the frequency of monocultured CSP-derived cardiomyocytes. Pfister et al. (2005) reported that 10% of CD31–/Sca-1+/CSP expressed disorganized -actinin and troponin I, but they did not show the characteristic sarcomeric organization and spontaneous beating, suggesting immature cardiomyocytes. Tomita et al. (2005) have reported that when CSP-derived cardiosphere was dissociated and cultured, 0.28% of the total cells differentiated into cardiomyocytes, which were positive for -actinin and sarcomeric myosin (Tomita et al., 2005). In this study, 5% of CSPs differentiated into cardiomyocytes with fine sarcomere structures and spontaneous beating (Fig. S2 A, a–f). Therefore, both OT and TSA possess more powerful cardiogenic activity against CSPs than the previously reported methods.
OT, a hypothalamic neuropeptide, induces uterine contraction and milk ejection. In recent years, however, functional OT receptors have been found in various organs, such as kidney, ovary, testis, thymus, heart, vascular endothelium, osteoclasts, myoblasts, pancreatic islet cells, adipocytes, and several types of cancer cells (Gimpl and Fahrenholz, 2001). OT receptors and OT biosynthesis are detected in atria and ventricles of the rat heart, and OT is thought to be involved in ANF release from cardiomyocytes (Gutkowska et al., 1997; Jankowski et al., 1998). CSPs are a heterogenous population of the cells, including cardiac stem/progenitor cells, endothelial progenitor cells, and other unknown cells. When CSPs are treated with OT or TSA, mesenchymal-like cells were observed near cardiomyocytes. Presently, we do not have the evidence to indicate that OT receptors are expressed in cardiac stem/progenitor cells, but not in other cells. It has recently been reported that elevated OT and OT receptor protein levels in growing fetal hearts and OT receptor immunostaining were predominantly detected in cardiomyocytes and endothelial cells (Jankowski et al., 2004). These observations suggest that OT acts on cardiomyogenesis, but it remains to be determined whether OT has direct effects on cardiac stem cells.
We have recently reported that OT induces differentiation of adult cardiac Sca-1 cells into mature cardiomyocytes (Matsuura et al., 2004). Because of the lack of Sca-1 in rats and the unavailability of decent antibodies against rat c-kit, we could not determine the relationship between CSPs and other cardiac stem cells populations, such as Sca-1+ or c-kit+ cells. The expression of cardiac transcription factors was absent in freshly isolated CSPs. We performed semiquantitative RT-PCR, showing the expression levels of Nkx-2.5 in CSPs were negligible (Fig. S1C, a and b). Therefore, CSPs may be more primitive stem or progenitor cells in comparison with cardiac Sca-1+ cells, in which faint but substantial expressions of cardiac transcription factors were observed. Our findings suggest that the OT-mediated signaling may play a pivotal role in the differentiation of various cardiac stem cells into cardiomyocytes.
Histone deacetylases (HDAC) catalyze the deacetylation from conserved lysine residues in the N-terminal tails of histones (Hassig and Schreiber, 1997). Silencing of genes has been shown to be accomplished by histone deacetylation, and inhibition of HDAC reverses the silencing effect. HDAC are critically involved in cell cycle regulation, cell proliferation, cancer development, and cell differentiation (Marks et al., 2003; Legube and Trouche, 2003). Recently, HDAC inhibitors such as TSA, valproic acid, and butyric acid have been reported to modulate cell type–specific gene expression. The lymphoid lineage-determining factor Ikaros is repressed under the circumstances with hypoacetylation of core histones at promoter sites, and this repression is relieved by TSA (Koipally et al., 1999). Hsieh et al. (2004) reported that valproic acid induces neural differentiation of adult hippocampal neural progenitors through the induction of neuroD. In this study, TSA induced de novo expressions of Nkx2.5, GATA4, and MEF2C, suggesting that acetylation of chromatin activates specific master genes, products of which promote the expression of a series of cardiac transcription factors. It remains to be determined what genes are activated and involved in cardiomyocyte differentiation by the treatment of TSA.
SP cells are thought to be a population of quiescent stem cells, which reside in the niche of the organs and contribute to life-long maintenance or repair of the tissue (Asakura and Rudnicki, 2002; Montanaro et al., 2003). Quiescence of CSPs was confirmed by PY staining. Stem cell niches play a pivotal role in controlling the self-renewal and differentiation of stem cells (for review see Moore and Lemischka, 2006). Niches consist of stem cells, niche stromal cells, and extracellular matrix, and the interaction between stem cells and the cellular microenvironments through adhesion molecules is important, as are paracrine factors. Bcrp1-positive cells in the heart coexpressed CD29 and N-cadherin on their cell surface and were located in the interstitial space and perivascular area. Although the niche stroma cells for cardiac Bcrp1-positive cells were not specified in this study, the fact that most Bcrp1-positive cells existed in the perivascular area suggests that pericytes or adventitial mesenchymal cells may be a component of the stem cell niches. During the preparation of this manuscript, Urbanek et al. (2006) reported that c-kit–positive cardiac stem cells and lineage-committed cells are clustered together, forming their niches in adult mouse heart. In their paper, 4ß1 integrin–mediated adhesion to laminin and fibronectin, as well as E- and N-cadherin–mediated cell–cell communications are supposed to be the fundamental structure of the cardiac stem cell niches. Some groups have reported that the frequency of cardiac stem cell clusters, including MDR1-positive cells, is inversely related to the hemodynamic load sustained by the anatomical regions of the heart; they accumulate in the atria and apex and are less numerous at the base and mid portion of the left ventricle (Leri et al., 2005). However, the frequency of CD31-negative/Bcrp1-positive cells in neonatal hearts did not show significant difference in the anatomical regions in this study. The reason for this discordant result may be that the left ventricle of neonatal hearts is under less hemodynamic load than that of adult hearts.
Intravenously transplanted CSPs were trapped in the lung and spleen, but redistributed in heart, liver, and skeletal muscle. CSPs in the heart were localized in the basal membrane between the myocardium and expressed CD29 on their cell surface, suggesting that CSPs penetrate the fenestrated endothelium, migrate into the basal lamina, and reside along with cardiomyocytes. Although some CSPs in liver and skeletal muscle expressed tissue-specific proteins such as albumin and desmin, respectively, transplanted CSPs in the normal heart did not express cardiac contractile proteins. It has been reported that transplanted bone marrow cells fuse with hepatocytes and skeletal muscle and regenerate the tissues (Camargo et al., 2003; Corbel et al., 2003; Vassilopoulos et al., 2003; Wang et al., 2003). Therefore, highly fusogenic hepatocytes and myotubes may fuse with transplanted CSPs and express differentiated marker proteins, whereas CSPs homing to the heart may not fuse with cardiomyocytes, and thus maintain stem or progenitor status.
Tissue damage, such as total body irradiation or chemotherapy, leads to secretion of chemokines and cytokines and facilitates hematopoietic stem cell migration and repopulation (Lapidot et al., 2005). Torrente et al. (2003) reported that skeletal muscle–derived stem cells home and migrate to the perivascular space of a damaged muscle of mdx mice after intravenous transplantation, and that the molecules involved in this process are L-selectin and mucosal addressin cell adhesion molecule-1. CSPs distributed in lung, spleen, liver, and skeletal muscle, but did not home specifically to the normal heart tissue. However, CSPs infused into rats with cryoinjured hearts homed in the heart, suggesting that the factors inducing migration and homing of stem cells may be released from injured heart. Further studies are necessary to understand the molecular mechanisms of differentiation, expansion, and migration of cardiac stem cells.
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Materials and methods |
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Cell culture
CSPs were cultured on gelatin-coated dishes with Iscove's Modified Dulbecco's Medium supplemented with 10% FBS. 24 h after seeding, the cells were treated with 10 pg/ml TSA or 100 nM of OT (both Sigma-Aldrich) for 72 h.
CSP transplantation of cryoinjured heart model
Male Wistar rats were anesthetized with 50 mg/kg ketamine i.p. and xylidine (10 mg/kg, i.p.) and a 6-mm aluminum rod, which was cooled to –190°C by immersion in liquid nitrogen, applied to the left ventricular free wall to produce cryoinjury, after the tail vein injection of 3 x 105 CSPs or CMPs derived from neonatal GFP transgenic into syngenic wild-type adult rats (CSP transplantation, n = 3; CMP transplantation, n = 3). As control groups, normal rats, which were subjected to the injection of 3 x 105 of CSPs (n = 3) or CMP (n = 3) were prepared. 4 wk after injection, rats were killed and lung, spleen, liver, skeletal muscle, and heart were fixed according to the periodate-lysine-paraformaldehyde fixative methods and snap-frozen in liquid nitrogen.
Immunocytochemistry and histochemistry
Cells were fixed with 4% paraformaldehyde and preblocked with PBS containing 2% donkey serum, 2% BSA, and 0.2% NP-40 for 30 min. Primary antibodies in PBS containing 2% donkey serum, 2% BSA, and 0.1% NP-40 were applied overnight at 4°C. FITC-, Cy3-, or Cy5-conjugated secondary antibodies were applied to visualize expression of specific proteins. Nuclear staining was performed with TOPRO-3 (Invitrogen). To detect expression of Bcrp1, fresh isolated cells were fixed in methanol/ethanol (1:1) for 1 min, and the cells were incubated with rabbit anti–mouse Bcrp1 antibodies for 2 h at room temperature. After washing three times with PBS containing 2% donkey serum, the secondary antibody was added for 1 h.
6-µm cryostat sections of fresh-frozen or fixed rat heart were prepared. Fresh-frozen sections were fixed with 1% formaldehyde for 15 min at room temperature. Blocking and staining procedures were performed according to the protocol described in the previous paragraph. Confocal images were acquired at room temperature using a microscope (Radiance 2000; Bio-Rad Laboratories) with Plan Apo 60x/1.40 NA oil immersion objective (Nikon) and Laser Sharp 2000 confocal software (Bio-Rad Laboratories). For Fig. 1 B (d and e), Fig. 5 B (a and b), Fig. S1 A (a and c), and Fig. S3 A (a–f), Axioscop 2 Plus (Carl Zeiss MicroImaging, Inc.) with Plan-NEOFLUAR 100x/1.30 NA oil immersion and 40x/0.75 NA objectives (Carl Zeiss MicroImaging, Inc.).
RNA extraction and RT-PCR analysis
SP cells were isolated from cardiac cells using EPICS ALTRA flow cytometric sorting. Total RNA was obtained from SP cells, TSA-treated SP cells, and the neonatal rat heart by RNA-Bee reagent (TEL-TEST). RT-PCR was performed using 0.1 mg of total RNA. For semiquantitative analysis, reverse transcribed products were pooled and fivefold serial dilutions were used for PCR. PCR was performed in a reaction volume of 20 µl with 200 nM deoxynucleoside triphosphates, 500 nM each of sense and antisense primers, and 2.5 U/100 µl Taq polymerase (Roche). Every PCR condition was confirmed to be within the linear range and within semiquantitative range for these specific genes and primer pairs. The primers used in this study and the PCR conditions are described in Table II.
To confirm that the obtained bands were not derived from contaminated genomic DNA, a negative experiment was done for each sample without reverse transcriptase before PCR. Amplified samples were electrophoresed on 2% agarose gels and stained with ethidium bromide. For semiquantitative RT-PCR analysis, PCR was performed on undiluted cDNA and on fivefold serial dilutions of cDNA, and the intensity of the ethidium bromide–stained bands was quantified using the Image program (Wayne Rasband, National Institutes of Health). Diluted pools showing the same intensity for ß-actin were used for further PCR and quantification of Nkx-2.5 gene expression.
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Statistical analysis
The significance of differences among mean values was determined by t test. P values were corrected for multiple comparisons by the Bonferoni correction. The accepted level of significance was P < 0.05.
Online supplemental material
Fig. S1 shows the osteogenic and adipogenic differentiation of CSPs. Fig. S2 shows the fine sarcomeric patterns of OT- and TSA-induced CSP-derived cardiomyocytes. Live images of beating cells were taken with an inverted microscope (Carl Zeiss MicroImaging, Inc.) equipped with chilled charge-coupled device camera (Hamamatsu) using I-O DATA Videorecorder software. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200603014/DC1.
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Acknowledgments |
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Submitted: 3 March 2006
Accepted: 20 December 2006
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