Author + information
- Received August 20, 2007
- Revision received October 26, 2007
- Accepted November 27, 2007
- Published online March 4, 2008.
- Joo-Yong Hahn, MD, PhD⁎,†,
- Hyun-Ju Cho, MS⁎,
- Hyun-Jae Kang, MD, PhD⁎,†,⁎ (, )
- Tack-Seung Kim, MS‡,
- Mi-Hyung Kim, PhD‡,
- Jung-Hwa Chung, MD⁎,
- Jang-Whan Bae, MD, PhD⁎,
- Byung-Hee Oh, MD, PhD⁎,†,
- Young-Bae Park, MD, PhD⁎,† and
- Hyo-Soo Kim, MD, PhD⁎,†,⁎ ()
- ↵⁎Reprint requests and correspondence:
Dr. Hyo-Soo Kim Dr. Hyun-Jae Kang, Department of Internal Medicine, Seoul National University College of Medicine, 28 Yeongun-dong, Chongro-gu, Seoul, 110-744, Korea.
Objectives The goal of this study was to investigate the effect of pre-treatment of mesenchymal stem cells (MSCs) with growth factors (GFs) on cardiomyogenic differentiation, cytoprotective action on cardiomyocytes (CMCs), and their therapeutic efficacy in myocardial infarction.
Background Mechanisms of myocardial repair with MSC transplantation have not been fully elucidated, and therapeutic efficacy needs to be enhanced.
Methods The MSCs obtained from the bone marrow of Fisher344 rats were treated with fibroblast growth factor-2, insulin-like growth factor-1, and bone morphogenetic protein-2. The expression of cardiac specific markers and the cytoprotective effect of MSCs with its mechanism were evaluated. Efficacy of MSCs transplantation was studied in rat myocardial infarction model.
Results Treatment of MSCs with cocktails of GFs enhanced expression of cardiac transcription factors and survival. Induction of cardiac specific markers by coculture with CMCs and gap junctional communication with CMCs was more active in GF-treated MSCs than untreated MSCs. The GF-treated MSCs reduced apoptosis of neighboring CMCs in a hypoxic condition and enhanced the phosphorylated Akt and phosphorylated c-AMP response element binding protein expression of CMCs, which was markedly reduced by gap junction blockade. In a rat myocardial infarction model, transplantation of GF-treated MSCs resulted in smaller infarct size and better cardiac function than transplantation of untreated MSCs. Additionally, GF treatment enhanced gap junction formation of transplanted MSCs, which did not aggravate arrhythmia.
Conclusions Pre-treatment of MSCs with GFs enhanced cytoprotective effects on neighboring CMCs through gap junction and improved the therapeutic efficacy of MSC transplantation for myocardial repair. “Priming of MSCs with GFs” before transplantation might improve the therapeutic efficacy of cell therapy.
Mesenchymal stem cells (MSCs) have been reported to repair damaged myocardium and improve cardiac function after myocardial infarction (MI) in pre-clinical studies (1,2). Bone marrow cells containing MSCs were most widely studied in clinical trials; however, their therapeutic benefit needs to be improved (3,4). Although the underlying mechanisms have not been fully elucidated, the differentiation of MSCs into cardiomyocytes (CMCs) might explain, at least partly, their therapeutic effect. Additionally, MSCs can exert cytoprotective effects on CMCs in a paracrine way. A substantial portion of the salutary effects of MSC implantation might be attributable to salvage of endangered ischemic myocardium (5). Although MSCs secrete large amounts of angiogenic and anti-apoptotic factors (6), the mechanisms of these cytoprotective effects are not fully understood. Gap junctions allow exchange of small molecules, including secondary messengers between adjacent cells (7), and might be a potential route for MSCs to exert cytoprotective effects.
To improve efficacy of MSC transplantation, interventions that facilitate differentiation and enhance the cytoprotective effects of MSC can be a rational approach. Recently, the role of growth factors (GFs) in the differentiation of stem cells into CMCs has been studied, including fibroblast growth factor (FGF)-2, insulin-like growth factor (IGF)-1, and bone morphogenetic protein (BMP)-2 (8–11). Combination of these GFs might facilitate differentiation of MSCs into CMCs. Furthermore, many GFs stimulate an anti-apoptotic signal, and FGF-2 and IGF-1 have been reported to increase the expression of connexin-43 (12,13). Pre-treatment of MSCs with GFs might enhance transfer of anti-apoptotic signal to CMCs.
In this study, we investigated whether treatment of MSCs with a combination of GFs (FGF-2, IGF-1, and BMP-2) enhances cardiomyogenic differentiation and the cytoprotective effect of MSCs, elucidating the potential role of gap junction in cytoprotective action of MSCs. Furthermore, to confirm the therapeutic applicability of GF-primed MSCs, we examined therapeutic efficacy of GF-treated MSCs in improving myocardial repair after transplantation in a rat MI model.
All animal experiments were performed under approval from the Institutional Animal Care and Use Committee of Seoul National University Hospital and complied with the National Research Council’s “Guidelines for the Care and Use of Laboratory Animals” (revised 1996).
Isolation and culture of MSCs from rat bone marrow
The MSCs were isolated from the femur of 8-week-old male Fischer 344 rats (200 to approximately 250 g, Daehan Biolink Co., Chungbuk, Korea). Bone marrow plugs were extracted and suspended in MSC culture medium (Dulbecco’s Modified Eagle Medium [DMEM, Invitrogen, Carlsbad, California], 10% fetal bovine serum [FBS, Hyclone, Logan, Utah], ascorbic acid [Sigma, St. Louis, Missouri], dexamethasone [Sigma], and mouse leukemia inhibitory factor [Sigma]) and then incubated at 37°C for 3 days before the first medium change. The mesenchymal population was isolated on the basis of its ability to adhere to the culture plate (14). At 90% confluence, the cells were trypsinized and subcultured. Second passage MSCs were used in all experiments. The MSCs were labeled with vibrant 1.1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, Molecular Probes, Eugene, Oregon) or with 4′,6-diamino-2-phenylindole (DAPI, Sigma) for detection and labeled with vibrant DiO (Molecular Probes) for separation after coculture with CMCs.
Isolation of neonatal CMCs
The CMCs were isolated from the hearts of neonatal Fisher 344 rats as previously described with minor modifications (15). Briefly, 2- to 4-day-old rats were killed, and then hearts were excised. Ventricles were minced and trypsinized, and supernatant was obtained. And then CMCs were separated, placed in serum-free DMEM for 24 h, and then cultured in 10% FBS-DMEM.
Viability assay of MSCs
Viability of MSCs was measured after exposure to 0.5% hypoxia for 30 h with trypan blue exclusion assay.
Differentiation of MSCs into CMCs: treatment with GFs and coculture with CMCs
To define optimal treatment condition, we tested differentiation efficacy of BMP-2, FGF-2, IGF-1, and their combination. First, differentiation efficacy was measured by degree of myocyte enhancer factor (MEF)-2 expression (MEF2-positive cells/total MSCs) on immunofluorescent staining with anti-MEF2 (1:200, Santa Cruz Biotechnology, Santa Cruz, California). Treatment of MSCs with a combination of BMP-2, FGF-2, and IGF-1 resulted in the higher percentage of MEF2-positive cells compared with BMP-2 or FGF-2 or IGF-1 alone or any combination of 2 GFs. Expression of MEF2 reached a peak at day 7 and then showed a plateau (Online Figs. I and II). Second, in western blot analysis, expression of CMC-specific markers such as Nkx2.5, GATA4, and cTnI was stronger with combination of BMP-2, FGF-2, and IGF-1 compared with BMP-2 or FGF-2 or IGF-1 alone or any combination of 2 GFs (Online Fig. III). Again, expression of CMC-specific markers increased until day 7 and then showed a plateau. On the basis of the results of these experiments, differentiation was induced by 7 days’ incubation with differentiation media (DMEM supplemented with 2% FBS, ascorbic acid, and dexamethasone) and after 2 days’ coculture with CMCs. For GF treatment, FGF-2, IGF-1, and BMP-2 (all from R&D Systems, Minneapolis, Minnesota) were added to the differentiation medium. The concentration was 50 ng/ml for FGF-2, 2 ng/ml for IGF-1, and 10 ng/ml for BMP-2. To evaluate the expression of cardiac transcription factors and cardiac specific markers, immunoblot analysis was performed at 1, 3, 5, and 7 days after differentiation induction. After coculture with CMCs, immunofluorescence staining was performed to define the phenotype of MSCs with anticardiac troponin I (Santa Cruz Biotechnology) and anticonnexin-43 (Santa Cruz Biotechnology), which were detected with goat antirabbit IgG antibodies (Molecular Probes) conjugated with fluorescein isothiocyanate. Meanwhile, in another experiment, DiO-labeled MSCs were separated with fluorescence-activated cell sorter (FACS) from CMCs by detecting DiO in MSCs. Immunoblot was performed as previously described (16). The MSCs were harvested in lysis buffer. Protein was separated on SDS-polyacrylamide electrophoresis gel and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, Massachusetts). The membrane was blocked and incubated with primary antibodies against GATA-4, NKx-2.5, cardiac troponin-I, connexin-43, and antialpha tubulin (all from Oncogene, Cambridge, Massachusetts) were used at a dilution of 1:500. As secondary antibody, antimouse IgG HRP (Promega, Madison, Wisconsin) or antirabbit IgG HRP (Promega) was used at a dilution of 1:2,500. A chemiluminescent detection reagent ECL (Amersham, Piscataway, New Jersey) was used for detection. To characterize the cell type of MSCs after GF treatment and coculture with CMCs, reverse transcriptase polymerase chain reaction (RT-PCR) was done as previously described (17). Total ribonucleic acid (RNA) was isolated by Trizol (Invitrogen) method and reverse transcribed with reverse transcription system (Clontech, Mountain View, California), and complementary deoxyribonucleic acid (cDNA) was amplified with CMC or myocyte-specific markers (connexin-43, cardiac troponin-I, alpha- and beta-MHC, alpha-sarcomeric actin, and atrial natriuretic peptide) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) by PCR. Primers used were as follows:
Forward primer 5′-TCCTTGGTGTCTCTCGCTTT-3′ and reverse primer 5′-GTTCACCCAATGCGACTCTT-3′ for connexin-43
Forward primer 5′-CACCCTTCTAAGACCCTCCA-3′ and reverse primer 5′-CCTCCTTCTTCACCTGCTTG-3′ for cardiac troponin-I
Forward primer 5′-CATAGGGGACCGTAGCAAGA-3′ and reverse primer 5′-CTGCCCCTTGGTGACATACT-3′ for alpha-MHC
Forward primer 5′-GCACTGGCCAAGTCAGTGTA-3′ and reverse primer 5′-GGACACGGTCTGAAAGGATG-3′ for beta-MHC
Forward primer 5′-GACCACAGCTGAACGTGAGA-3′ and reverse primer 5′-CATAGCACGATGGTCGATTG-3′ for alpha-sarcomeric actin
Forward primer 5′-AGGCCATATTGGAGCAAATC-3′ and reverse primer 5′-CCTTAATATGCAGAGTGGGAGA-3′ for atrial natriuretic peptide
Forward primer 5′-CGTGGAAGGACTCATGAC-3′ and reverse primer 5′-CAAATTCGTTGTCATACCAG-3′ for GAPDH
Intercellular communication between MSCs and CMCs through gap junction: dye transfer experiment
The MSCs were labeled with DiI, which cannot pass through the gap junction, and calcein-AM (2.5 μmol/l; Calbiochem, San Diego, California), which can spread only through the gap junction. And then labeled MSCs were cocultured with unlabeled CMCs. At 48 h after coculture, dye transfer was evaluated under fluorescence microscope. To selectively block gap junctional communication, heptanol (0.5 mmol/ml) was used.
Hypoxia exposure to CMCs with or without MSCs coculture
Hypoxic conditions were created by incubating CMCs at 37°C in airtight chambers with 0.5% oxygen concentration under coculture with DiI-labeled MSCs. After 24-h hypoxic exposure, cocultured MSCs were eliminated by FACS. Then, apoptosis of CMCs was evaluated by flow cytometry (FACStar plus, Becton Dickinson, Franklin Lakes, New Jersey) as described previously (18). The CMCs exposed to hypoxia without coculture were used as a control. To evaluate the role of gap junction in the cytoprotective effect of MSCs, heptanol (0.5 mmol/ml) was added during hypoxia. To investigate underlying mechanism of cytoprotective effect, the expression of phosphorylated Akt and phosphorylated c-AMP response element binding protein (CREB) in CMCs were evaluated with immunoblot analysis.
Assay of IGF-1 and Hepatocyte GF for evaluating paracrine function of MSCs
After 7 days’ incubation of MSCs in differentiation medium followed by 30 h of incubation in conditioned medium (low glucose DMED with 10% FBS), IGF-1 and hepatocyte growth factor (HGF) levels in conditioned medium were measured by enzyme-linked immunosorbent assay (rat IGF-1 enzyme immunoassay, Diagnostic Systems Laboratory, Webster, Texas; and rat HGF enzyme immunoassay, Institute of Immunology, Tokyo, Japan). To exclude the possibility of cross reaction, antirat IGF-1 antibody that has no reactivity to human IGF-1 was used for this assay.
Rat MI model and MSCs transplantation
Rats were anesthetized with ketamine hydrochloride (100 mg/kg, Yuhan Corp., Seoul, Korea) and xylazine (10 mg/kg, Bayer, Shawnee Mission, Kansas) by intraperitoneal injection. Experimental MI was induced by temporary ligation of the left anterior descending artery for 45 min, as previously described (19). Development of MI was confirmed by echocardiography 7 days after the procedure. Then rats were randomized into 3 groups: rats receiving control media only (control group), untreated MSCs (untreated MSC group), and GF-treated MSCs (treated MSC group). Before transplantation, MSCs were labeled with red DiI for detection at later time points. Under anesthesia, each rat received 3 injections (total of 106 cells/heart) into the border zone of MI, approximately 1 to 2 mm apart.
Functional assessment of the infarcted rat heart by echocardiography
Transthoracic echocardiography was performed at just before coronary ligation and 1, 4, and 8 weeks after coronary ligation, as described previously (19) with an echocardiographic system (Acuson 128-XP system, Acuson, Florida) equipped with a 7-MHz linear–array transducer. Left ventricular end-diastolic and end-systolic dimensions (LVEDD and LVESD) were measured according to the leading-edge method of the American Society of Echocardiography. The LV percent fractional shortening (%FS) was calculated as 100 × (LVEDD − LVESD)/LVEDD.
Induction of arrhythmia
Arrhythmia was induced by aconitine (Sigma) infusion. At 8 weeks after coronary ligation, rats were anesthetized and 24-gauge intravenous catheter was inserted into the femoral vein for drug administration. After a 30-min period of stabilization, aconitine was intravenously infused at a rate of 0.1 ml/min by infusion pump for 510 s. After aconitine infusion, rats were observed for 20 min. Electrocardiography (ECG, Surgivet, Waukesha, Wisconsin) was continuously recorded throughout the experiment. The incidence of arrhythmias was analyzed in accordance with the Lambeth Conventions (20). An arrhythmia score was used to indicate the incidence and duration of arrhythmias, where 0 indicates no arrhythmia; 1: an arrhythmia duration of <10 s; 2: an arrhythmia duration of 11 to 30 s; 3: an arrhythmia duration of 91 to 180 s or reversible ventricular fibrillation (VF) for <10 s; 5: an arrhythmia duration longer than 180 s or reversible VF for more than 10 s; and 6: an irreversible VF or death of the animal.
Histological analysis of the infarcted rat heart with or without MSCs transplantation
Rats were killed at 8 weeks after coronary ligation for the assessment. Heart sections embedded in paraffin were cut into 4-μm slices, and the size of area of fibrosis was determined by image analysis system (Image Pro version 4.5; MediaCybernetics, Bethesda, Maryland) on Masson’s trichrome-stained slides. The measurements of area of fibrosis were performed on 2 separate sections of each heart, and the averages were used for statistical analysis. DiI-positive area was measured with automated color detection software (Image Pro version 4.5, MediaCybernetics). For immuno-histochemical analysis, the rat hearts were cryopreserved in OCT compound (Tissue-Teck, Sakura, Torrance, California). To evaluate apoptosis, the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay (Chemicon S7100 kit, Chemicon, Temecula, California) and immunohistochemistry for caspase-3 (Abcam 1:50, Abcam, Cambridge, Massachusetts) were performed. The TUNEL or caspase-3 positive cells were counted in 10 different microscopic fields of at least 3 different sections from each animal. Immunofluorescence staining was performed with anticardiac troponin I (Santa Cruz Biotechnology) or anticonnexin-43 (Santa Cruz Biotechnology), which was detected with goat antirabbit IgG antibodies (Molecular Probes) conjugated with green fluorescein isothiocyanate. Blue DAPI staining was done to localize the nucleus. Colocalization of the red DiI-labeled MSCs and green connexin-43 or cardiac troponin I expression were examined with a confocal microscope (LSM 510 META, Carl Zeiss, Peabody, Massachusetts).
All data are presented as mean ± SD. Continuous variables were compared by the Student t test, and multiple comparisons were performed by analysis of variance with a Bonferroni correction. A value of p < 0.05 was considered significant, and all analyses were performed with SPSS version 11.0 (SPSS, Chicago, Illinois).
Pre-treatment of MSCs with GFs potentiated their viability in hypoxic condition
The effects of pre-treatment with GFs combination of FGF-2, IGF-1, and BMP-2 on the viability of MSCs were evaluated. Viability of MSCs was measured after exposure to 0.5% hypoxia for 30 h. The GF-treated MSCs showed better survival than untreated MSCs under hypoxia (viable cell proportion in trypan blue exclusion assay: 67 ± 11% vs. 47 ± 14%, p < 0.05).
Pre-treatment of MSCs with GFs augmented the expression of various cardiac specific markers and connexin-43, which was induced by coculture with CMCs
We evaluated the effects of GF treatment on differentiation of MSCs into CMCs. After 7-day treatment with GFs, RT-PCR showed that expression of alpha-sarcomeric actin was higher in GF-treated MSCs than untreated MSCs. However, in the single MSCs culture condition, most of the markers did not express irrespective of treatment with GFs. Although connexin-43 expressed weakly in both GF-treated MSCs and untreated ones, there was no significant difference between the 2 groups. But, after coculture with CMCs, the messenger RNA (mRNA) expression of CMC- or myocyte-specific markers such as atrial natriuretic peptide, troponin-I, alpha and beta myosin heavy chain as well as connexin-43 was augmented in GF-treated MSCs compared with untreated ones (Fig. 1A).
Immunoblot analysis showed that treatment with GFs increased the expression of cardiac transcription factors. The protein expression of GATA-4 and NKx-2.5 were induced distinctively during 7 days of culture with GFs although not without GFs (Fig. 1B). After an additional 2 days of coculture with CMCs, connexin-43 protein was expressed, which was enhanced in GF-treated MSCs compared with untreated ones (Figs. 1C and 1D). In immunofluorescent staining, connexin-43 was expressed linearly between MSCs and CMCs, which suggests the formation of gap junction. It was more frequently observed in the GF-treated MSCs (Fig. 1E). Although western blot showed that the expression of cardiac troponin I was increased in GF-treated MSCs, the frequency of troponin I positive-cells in immunofluorescent staining was not significantly different between GF-treated and untreated MSCs.
Pre-treatment of MSCs with GFs enhanced gap junctional communication between MSCs and CMCs
To evaluate gap junctional communication, we examined direct dye transfer from MSCs to adjacent CMCs. We labeled MSCs both with red DiI and green calcein-AM and cocultured them with the unlabeled CMCs (Fig. 2). Only calcein-AM, not DiI, is transmittable through gap junction. We observed cells stained with green calcein-AM without red DiI, which indicated CMCs that have taken up calcein-AM via functioning gap junctions from MSCs. Quantitative analysis showed that transfer of calcein-AM from MSCs to the surrounding CMCs through the gap junction was more frequent in coculture of CMCs with GF-treated MSCs than with untreated MSCs (Fig. 2D). After the addition of a gap junction blocker, calcein-AM transfer was blocked (Fig. 2C). This suggested that MSCs were linked to CMCs via functioning gap junctions. Therefore, pre-treatment with GFs enhanced not only the expression of connexin-43 in MSCs but also the gap junctional communication with CMCs.
GF-treated MSCs protect CMCs through a gap junction-mediated mechanism
We investigated whether GFs potentiated the cytoprotective effect of MSCs and whether this effect is mediated through the gap junction (Fig. 3). After 24 h of hypoxia, the apoptotic fraction of CMCs was significantly reduced in coculture condition with MSCs compared with CMCs single culture (Figs. 3A and 3B). Such antiapoptotic action of MSCs on CMCs was significantly potentiated in the coculture with GF-treated MSCs (Fig. 3C). Treatment of heptanol partially inhibited antiapoptotic effects of treated MSCs on CMCs (Fig. 3D). These data suggest that MSCs exert a protective effect on CMCs via gap junctions, which is augmented by GF treatment of MSCs. Additionally, expression of phospho-Akt and phospho-CREB increased in CMCs cocultured with GF-treated MSCs compared with untreated MSCs, which was also reversed by heptanol (Fig. 3E). However, there was no significant difference in the capacity to secrete IGF-1 or HGF between untreated and treated MSCs (Fig. 3F). Collectively, these results suggest that, at least partially, cytoprotective effect of MSCs was mediated through gap junction and that GF pre-treatment of MSCs potentiated the gap junction-mediated cytoprotective effect of MSCs. The increase of intercellular communication through gap junction activated Akt and CREB pathway in CMCs.
Transplantation of GF–pre-treated MSCs showed a superior myocardial salvage of rat infarcted heart to that of naïve ones
The area of fibrotic scar in the infarcted rat heart was significantly smaller when GF-treated MSCs were transplanted than when untreated MSCs were at 8 weeks after MI (Figs. 4A and 4B). Left ventricular %FS, LVESD, and LVEDD were similar among all 3 groups at baseline. At 8 weeks, the motion of the LV anterior wall was obviously better when transplanted with the GF-treated MSCs than with the untreated MSCs or with control media (Fig. 4C). Although LVEDD was not significantly different among 3 groups (Fig. 4D), %FS were significantly higher when transplanted with the GF-treated MSCs than with the untreated MSCs or with control media at 4 and 8 weeks after MI (Fig. 4E).
Pre-treatment of MSCs with GFs enhanced engraftment of MSCs and gap junction formation with CMCs and reduced apoptosis in infarcted myocardium
Transplanted MSCs were detected until 8 weeks after MI in the infarct border zone. At 2 and 8 weeks, the proportion of MSCs expressing connexin-43 was higher in the heart transplanted with GF–pre-treated MSC than with untreated ones. The gap junction formation was more frequently observed between MSCs themselves or MSCs and surrounding CMCs in the GF-treated MSC group compared with the untreated MSC group (Figs. 5A to 5C). In addition to quantitative increase in the expression of connexin-43, expression pattern was more organized in the heart transplanted with GF–pre-treated MSC than with untreated ones (Online Fig. IV). However, the expression of troponin-I in MSC was similar in the treated MSC group and untreated ones (Online Fig. V). Area of surviving MSCs was significantly greater in the GF-treated MSC group compared with the untreated group at 2 weeks (Figs. 5D and 5E). Apoptosis was significantly reduced in the infarcted heart when transplanted with the GF-treated MSCs than with the untreated ones at 2 weeks after MI (1 week after MSC implantation) (Figs. 5F to 5H). Taken together, pre-treatment of MSCs with GFs enhances gap junction formation and cell survival in the infarcted heart.
Transplantation of GF–pre-treated MSCs did not aggravate susceptibility to drug-induced arrhythmia after MI
The arrhythmia induction experiment was performed in 20 rats (10 rats transplanted with the GF-treated MSCs and with untreated MSCs, respectively) at 8 weeks after MI. During and after aconitine infusion, we were able to observe premature ventricular contraction (PVC), ventricular tachycardia, and VF (Fig. 6A). There were 5 rats in the GF-treated MSC group and 8 in the untreated MSC group showing any kind of arrhythmia. Ventricular tachycardia was induced in 2 in the GF-treated MSC group and 3 in the untreated group. One rat in each group died, owing to irreversible VF. Rats transplanted with the treated MSCs had a tendency toward a lower arrhythmia score and less frequent PVCs, which did not reach statistical significance (Figs. 6B and 6C). These data suggest that enhanced expression of connexin-43 in MSCs and gap junctional communication with CMCs by GF pre-treatment of MSCs at least did not aggravate arrhythmia after transplantation of MSCs to the infarcted myocardium.
In this study, we showed that pre-treatment of MSCs with a combination of GFs (FGF-2 + IGF-1 + BMP-2) enhanced the expression of cardiac transcription factors and that these primed MSCs, compared with naïve MSCs, showed the augmented induction of cardiac specific genes by coculture with CMCs. With the greater expression of connexin-43 and gap junction formation between CMCs, the primed MSCs showed better cytoprotective effects on CMCs than naïve ones. Furthermore, the cytoprotective effect on CMCs was mediated through gap junctions, which is a novel mechanism to explain the therapeutic effect of MSCs. In rat MI model, transplantation of GF–pre-treated MSCs after MI enhanced gap junction formation and cell survival in the infarcted myocardium and reduced infarct size, leading to the improved LV function.
GF treatment and cardiomyogenic differentiation of MSCs
Bone morphogenetic protein, a member of the transforming growth factor (TGF) family, and FGF have been reported to play important roles in induction of cardiac transcription factors in developing embryos (21) and to induce cardiomyogenic differentiation from noncardiac mesodermal cells (11,22). The BMP-2 in vivo induced expression of cardiac-specific transcription factors (8,11) and is known to play an important role in the process of heart development (23,24). The FGF-2 also plays a pivotal role in the differentiation process of resident cardiac progenitor cells into functional CMCs (10). The IGF-1 was shown to enhance cellular engraftment and host organ-specific differentiation of embryonic stem cells after injection in the area of acute myocardial injury (9) and to enhance survival and proliferation of cardiac stem cells (25).
In the present study, we demonstrated that a combined treatment with FGF-1, IGF-1, and BMP-2 increased the expression of cardiac transcription factors in MSCs. The expression of CMC-specific genes, however, was not induced by treatment with GFs in the MSCs single culture condition. But in coculture condition with CMCs, GF-treated MSCs showed a greater expression of cardiac specific genes including connexin-43 and a better gap junction formation with CMCs than naïve MSCs. We think that MSCs after treatment with GFs are “primed” MSCs expressing cardiac transcription factors or “committed” MSCs into myocyte lineage but not mature or immature CMCs. But these “primed” MSCs have a capability to differentiate to CMC-like cells in coculture with CMCs in vitro, to form gap junction, and to survive in the infarcted myocardium in vivo more than naïve MSCs. Our data suggested that GFs have the potential to induce MSCs to express CMC-specific genes and connexin-43 and to form functioning gap junction, in response to appropriate stimuli such as coculture with CMCs. Although the underlying mechanism responsible for increased expression of connexin-43 by GFs in MSCs remains unclear, FGF-2 and IGF-1 have been shown to increase connexin-43 expression and intercellular communication (12,13).
Our data suggest that contact with CMCs would be needed to differentiate MSCs into CMC lineage. It was reported that differentiation of bone marrow stromal cells into cells with cardiac phenotype requires intercellular communication with myocytes (26). However, whether intercellular communication is essential for cardiomyogenic differentiation is unknown. Li et al. (27) reported that TGF-beta induced the myogenic differentiation of stem cells without coculture.
Gap junction and cytoprotective effect
In this study, GF–pre-treated MSCs exerted better protective effects on CMCs than untreated MSCs, and these protective effects were mediated through gap junctions. Currently, the cytoprotective effect of MSCs is regarded as one of the mechanisms to explain the therapeutic effects of MSCs. Previous studies have focused on paracrine effects of MSCs. The MSCs secrete large amounts of various angiogenic and anti-apoptotic factors (6), and the conditioned medium from cultured MSCs were shown to reduce apoptosis of CMCs exposed to hypoxia (5). We also observed that MSCs secreted IGF-1 and HGF in this study. However, the capacity to secrete cytokines is not different between GF-treated and untreated MSCs in this study. Therefore, paracrine effects can not explain the difference in cytoprotective effects between GF-treated and untreated MSCs.
In this study, formation of functional gap junctions was more efficient in GF-treated MSCs compared with untreated MSCs, and the blockade of gap junctions reversed the protective effects of MSCs on CMCs and abolished the difference in protective effects on CMCs between GF-treated and untreated MSCs. These data suggest that the cytoprotective effects of MSCs are mediated not only by paracrine actions but also by direct cell-to-cell communication through gap junctions and that treatment with GFs might potentiate gap junction-mediated cytoprotective effect. Recent data support the hypothesis that gap junction might propagate cell survival and death signals (28). In our study, connexin-43 expression that indicated the formation of gap junction was more prominent in GF-treated MSC group in vitro and vivo. And dye transfer experiments demonstrated that metabolic coupling with CMCs was more efficient in GF-treated MSCs than in untreated MSCs. Although we could not confirm which material was transferred from MSCs to CMCs, second messengers, which have been known to pass through the gap junction, are potential candidates. Cyclic adenosine monophosphate is a well-known second messenger that can pass through gap junction (7) and phosphorylate CREB, which in turn exerts an antiapoptotic effect (29). In the present study, we showed increased phosphorylation of CREB in CMCs cocultured with treated MSCs. Moreover, the Akt pathway was activated in CMCs cocultured with treated MSCs. Enhanced expression of CREB and Akt in CMCs cocultured with GF-treated MSCs were inhibited by blocking gap junction. In short, MSCs have a cytoprotective effect on CMCs through the gap junction, which is potentiated by treatment of MSCs with GFs that facilitate the expression of connexin-43 and formation of functional gap junctions.
Reduced infarct size and improved systolic function by transplantation of GF–pre-treated MSCs
There are several possible mechanisms that could explain the difference between GF-treated MSCs and untreated ones in reduction of infarct size and preservation of LV systolic function. First, GF treatment might make MSCs stronger against apoptosis. The GF–pre-treated MSCs showed better survival against hypoxia. The DiI-positive area was greater in the infarcted myocardium transplanted with GF–pre-treated MSCs than with naïve MSCs in the early post-transplantation period. Surviving MSC can contribute to cardiac repair through direct differentiation and paracrine and cytoprotective action. Thus the more MSCs survive, the bigger improvement of cardiac function can be expected (30). Anti-apoptotic effects of GF treatment on MSCs can be explained by at least 2 mechanisms: 1) FGF-2 prolonged telomerase length and life span of MSCs and is useful in obtaining a large number of cells with preserved differentiation potential (31); and 2) FGF and IGF-1 activate PI3-kinase/Akt pathway, which is known to mediate anti-apoptotic signaling (32,33). However, the survival benefit of MSCs was attenuated at 8 weeks, and absolute amounts of surviving MSCs were also markedly decreased. Thus, this mechanism can not solely explain the beneficial effects of GF treatment observed at 8 weeks. Second, as discussed previously, GF treatment enhanced gap junction-mediated cytoprotective effect of MSCs on CMCs. Finally, the better LV systolic function in the treated MSC group might be due to synchronized contraction. Enhanced intercellular communication through gap junction might lead to electrical synchronization and then synchronous contraction (34,35). Therefore, better-coordinated LV systolic contraction can be expected when GF–pre-treated MSCs are transplanted.
Effects of MSC transplantation on arrhythmogenecity of infarcted myocardium
Since proarrhythmic effects of skeletal myoblasts have been reported, there have been concerns about proarrhythmic potential of stem cell transplantation. Mixtures of MSCs and neonatal rat ventricular myocytes can produce an arrhythmogenic substrate (36). Whether increased gap junctional communication is favorable remains uncertain. However, increased expression of connexin-43 has been reported to reduce inducible arrhythmia in infarcted heart (37). Concordant with the previous study, transplantation of GF–pre-treated MSCs, which can make higher expression of connexin-43 and functional gap junctions, did not aggravate arrhythmic risk after MI in our study.
There are several limitations to this study. First, we used human GFs for pre-treatment of MSCs isolated from rat bone marrow. However, structural homology of these GFs is very high between humans and rats (38,39). Second, because we treated MSCs with a combination of 3 GFs, the effect of individual GF and the underlying mechanism of individual effect were not understood. Third, DiI is a membrane dye and might have limitation for detection of transplanted MSCs 8 weeks later. However, Dai et al. (40) reported that the DiI-positive cells were observed at 3 and 6 months after transplantation of DiI-labeled MSCs into the scar of a 1-week-old infarcted myocardium. And DiI diffuses within the plasma membrane, resulting in staining of the entire cell, which was observed in our study as well as a previous one (40).
We found that the priming of MSCs with a combination of FGF-2, IGF-1, and BMP-2 enhanced commitment of MSCs to CMC lineage in the coculture with CMCs. These GF-primed MSCs with better expression of connexin-43 showed a greater cytoprotective effect on CMCs, which was mediated through gap junction. Moreover, coupling through gap junction might explain the improvement of LV systolic function after transplantation of primed MSCs to the infarcted myocardium compared with naïve MSCs. Treatment of MSCs with GFs might be a physiological and feasible strategy to increase therapeutic efficacy of MSC transplantation to the infarcted myocardium, which has a significant clinical impact.
For supplementary figures, please see the online version of this article.
Pre-Treatment of Mesenchymal Stem Cells With a Combination of Growth Factors Enhances Gap Junction Formation, Cytoprotective Effect on Cardiomyocytes, and Therapeutic Efficacy for Myocardial Infarction
This study was supported by a grant from Stem Cell Research Center (SC3150) and from Innovative Research Institute for Cell Therapy, Republic of Korea. Drs. Hahn and Cho contributed equally to this work.
- Abbreviations and Acronyms
- bone morphogenetic protein
- Dulbecco’s Modified Eagle Medium
- fluorescence-activated cell sorter
- fetal bovine serum
- fibroblast growth factor
- fractional shortening
- growth factor
- insulin-like growth factor
- left ventricle/ventricular
- left ventricular end-diastolic dimension
- left ventricular end-systolic dimension
- myocyte enhancer factor
- myocardial infarction
- mesenchymal stem cell
- reverse transcriptase-polymerase chain reaction
- ventricular fibrillation
- Received August 20, 2007.
- Revision received October 26, 2007.
- Accepted November 27, 2007.
- American College of Cardiology Foundation
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