Human umbilical cord blood derived mesenchymal stem cells improve cardiac function in cTnTR141W transgenic mouse of dilated cardiomyopathy

European Journal of Cell Biology 95 (2016) 57–67

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Research paper

Human umbilical cord blood derived mesenchymal stem cells improve cardiac function in cTnTR141W transgenic mouse of dilated cardiomyopathy Xuhe Gong a , Pengbo Wang a , Qingqing Wu b , Sijia Wang a , Litian Yu a , Guogan Wang a,b,∗ a Emergency and Critical Center, Department of Cardiology, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China b Departments of Obstetrics and Gynaecology, Fuxing Hospital, Capital Medical University, Beijing, China

a r t i c l e

i n f o

Article history: Received 29 August 2015 Received in revised form 15 November 2015 Accepted 16 November 2015 Keywords: Umbilical cord blood mesenchymal stem cells Dilated cardiomyopathy Akt Paracrine effects

a b s t r a c t Cell transplantation is a promising strategy in regenerative medicine. Beneficial effects of bone marrow mesenchymal stem cells (BM-MSCs) on heart disease have been widely reported. However, the MSCs in these studies have been mainly derived from autologous animals, and data on MSCs from human umbilical cord blood (UCB-MSCs) are still scarce. We investigated whether intramyocardial xenogeneic administration of UCB-MSCs is beneficial for preserving heart function in a cTnTR141W transgenic mouse of dilated cardiomyopathy (DCM). Cultured UCB-MSCs, which were identified by there morphology, differentiation and cell surface markers, were transplanted into cTnTR141W transgenic mice to examine apoptosis, fibrosis, vasculogenesis and the associated Akt pathway. Moreover, we measured the expression levels of VEGF and IGF-1, which are growth factors required for differentiation into cardiomyocytes, and are also involved in cardiac regeneration and improving heart function. One month after transplantation, MSCs significantly decreased chamber dilation and contractile dysfunction in the cTnTR141W mice. MSCs transplanted hearts showed a significant decrease in cardiac apoptosis and its regulation by the Akt pathway. Cardiac fibrosis and cytoplasmic vacuolisation were significantly attenuated in the MSCs group. Importantly, the levels of VEGF and IGF-1 were increased in the MSCs transplanted hearts. In vitro, the MSC-conditioned medium displayed anti-apoptotic activity in h9c2 cardiomyocytes subjected to hypoxia. These results further confirm the paracrine effects of MSCs. In conclusion, UCB-MSCs preserve cardiac function after intramyocardial transplantation in a DCM mouse, and this effect may be associated with reductions in cellular apoptosis, inflammation, hypertrophy and myocardial fibrosis; in addition to; up-regulation of Akt, VEGF and IGF-1; and enhanced angiogenesis. © 2015 Elsevier GmbH. All rights reserved.

1. Introduction Dilated cardiomyopathy (DCM) causes a major health burden worldwide. Despite advances in medicine and interventional therapy, the prognosis of patients with DCM remains poor, and they have high rates of hospitalisation, morbidity and mortality (Richardson et al., 1996). Hence, there is a need for methodologically sound studies to improve therapies for clinical practice.

∗ Corresponding author at: Emergency and Critical Center, Department of Cardiology, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, People’s Republic of China. Tel.: +86 10 88398471; fax: +86 10 88398471. E-mail address: [email protected] (G. Wang). http://dx.doi.org/10.1016/j.ejcb.2015.11.003 0171-9335/© 2015 Elsevier GmbH. All rights reserved.

Recently, cell therapy for DCM has been gaining attention because of inspiring achievements in research of cell therapy for ischaemic cardiomyopathy. Accumulating experimental and clinical evidence support cell therapy as a promising therapeutic strategy to improve cardiac function (Carmeliet, 2000; Seth et al., 2006; Zlatkovic-Lindor et al., 2010). For example, Vrtovec et al. (2013) have investigated the long-term (5-year follow-up) effects of intracoronary CD34+ cell transplantation in DCM, and they have found that stem cell transplantation may be associated with improved ventricular function, exercise tolerance, and longterm survival in patients. The proposed underlying mechanisms of this improvement include differentiation into cardiomyocytes, paracrine effects and angiogenesis. However, the stem cells used in previous experiments were mainly derived from autologous sources, such as autologous bone marrow derived MSCs (BM-MSCs) (Martin-Rendon et al., 2008),

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which have a low proliferative ability, age quickly, have an invasive collection method and other shortcomings. Thus, we propose a xenogeneic stem cell therapy approach, using human umbilical cord blood derived MSCs (UCB-MSCs), These stem cells may be optimal for treating DCM because of their lower immunogenicity (Perea-Gil et al., 2015; Lee et al., 2014) and greater degree of cardiomyocyte reprogramming than other xenogeneic stem cells (Yannarelli et al., 2013). Moreover, UCB-MSCs have the distinct advantages of being easy to obtain, minimally invasive and without ethical issue (Oran and Shpall, 2012), these stem cells have been applied in the research of spinal cord injury (Cui et al., 2014) and renal ischemia-reperfusion injury (Jang et al., 2008), demonstrating their promising clinical application. Currently, the animal model used to study DCM is often druginduced (e.g., Doxorubicin and Adriamycin). However, the causes of DCM are multifactorial. Genetic factors are a major contributor to DCM, affecting 30–48% of patients (Jefferies and Towbin, 2010). However, little is known about the impact of stem cell therapy on the outcomes of this type of DCM. The cTnTR141W transgenic mouse is a relatively ideal model of DCM caused by genetic inheritance, and it may be a highly representative model to study this condition. Furthermore, the effects of xenogeneic stem cells may be explored using this model. Accumulating evidence has shown that marked cardiac endothelial derangements are associated with DCM progression and clinical outcomes; and these derangements are apparent in the DCM model, hence, angiogenesis is the one of the key research focuses; Moreover, many studies have shown that MSCs therapy has a powerful and comprehensive paracrine effect. Thus, in this study, we also examined the paracrine effect of MSCs in vivo and vitro. In the present study, a specific population of human MSCs derived from UCB was isolated and identified. We investigated the effects and mechanism of xenogeneic UCB-MSCs in the cTnTR141W transgenic mouse of DCM. The results showed that the intramyocardial transfer of UCB-MSCs improved heart function through anti-apoptotic, anti-inflammatory and proangiogenic mechanisms. Moreover, in vitro, MSC-conditioned medium protected h9c2 cells from hypoxia-induced apoptosis through paracrine mechanisms. 2. Materials and methods 2.1. Animals The cTnTR141W transgenic male mice were established at the Laboratory of Animal Science of Peking Union Medical College and maintained on a C57BL/6J genetic background. The transgenic mice expressed high levels of the mutant human cTnTR141W protein and showed ventricular chamber enlargement, systolic dysfunction, myocardial hypertrophy, and interstitial fibrosis at 4 months of age (Juan et al., 2008). All experimental protocols were approved by the Ethics Committee for Animal Research of Fuwai Hospital and conformed to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996). 2.2. Isolation, culture and characterisation of UCB-MSCs Human UCB was obtained from Fuxing Hospital, Beijing. The isolation procedure was performed as previously described (Phuc et al., 2011). Briefly, UCB was collected with the consent of the mother and the ethical committee of Fuxing Hospital. The study was performed according to the guidelines of the Declaration of Helsinki. To isolate mononuclear cells (MNCs), each UCB sample was diluted to a 1:1 ratio with phosphate buffered solution (PBS)

and 15 ml of the diluted blood was gently loaded onto a 20 ml Ficoll Paque PLUS gradient (1.077 g/ml, GE). After centrifugation at 600 × g for 20 min, MNCs were harvested from the interface and suspended in DMEM/F12 medium supplemented with 10% foetal bovine serum (Gibco) and 1% penicillin/streptomycin. The medium was replaced after five days and every three days thereafter until putative MSC colonies were observed. 2.3. In vitro identification and labelling of MSCs For the differentiation experiments, human MSC osteogenic and adipogenic medium (Cyagen, China) was used. MSCs at the third passage were differentiated for 14–21 days, then osteogenic differentiation was evaluated via alizarin red S staining, and adipogenic potential was assessed via Oil Red O staining. Antibodies against the human antigens CD34-FITC, CD44-PE, CD45-PerCP and CD105-APC (Miltenyi Biotec) were analysed by FACS Calibur, and data were analysed with CellQuest software (BD). To track the transplanted cells in the recipient myocardium, MSCs were labelled with eGFP (enhanced green fluorescence protein) using lentiviral vectors before transplantation. The survival of transplanted eGFP-positive cells was detected at one month after transplantation using immunofluorescence staining. 2.4. Conditioned medium collection and processing UCB-MSCs were grown to 80% confluence, and they were then washed with PBS. Next, DMEM/F12 (without serum) was added, and the cells were exposed to normoxia (37 ◦ C, 5% CO2 ) or hypoxia (in a sealed, hypoxic GENbox jar fitted with a catalyst). After 12 h, the media was collected, centrifuged at 300 × g for 10 min to remove cell debris, filtered with a 0.22 ␮m cellulose syringe filter, and labelled as UCB-MSC conditioned medium (UCM), normoxia (UCM-N) and hypoxia (UCM-H). The UCM was used to treat h9c2 cells that had been subjected to 24 h of hypoxia to determine its effects on hypoxia-induced apoptosis. 2.5. Detection of H9c2 cell apoptosis in vitro Before subjecting h9c2 cells to hypoxia for 24 h, the medium was changed to (A) DMEM/F12, (B) UCM-N, (C) UCM-H, or (D) UCMN + LY294002 (an Akt inhibitor, 40 ␮M). After exposure, the cells were harvested by trypsinisation and washed twice with cold PBS. Cell apoptosis was assessed using an Annexin V-FITC/PI Kit (Keygen Biotechnology, China) according to the manufacturer’s instructions. Briefly, 5 ␮l of Annexin V was added to the cells and incubated for 30 min at room temperature. Then, 500 ␮l of binding buffer and 5 ␮l of PI were added. Stained cells were immediately analysed using a FACS Calibur flow cytometer and Cell Quest software (BD). 2.6. MSCs preparation and transplantation The mice (aged 4 months) were randomly divided into one of the following groups: DCM, DCM + PBS, or DCM + MSC. Transgenenegative mice were used as controls. Each group contained 6 animals. The eGFP-labelled MSCs were washed with PBS and separated using 0.05% trypsin. After the addition of culture medium, the cell suspension was centrifuged at 1000 rbp for 5 min. The cell pellet was then re-suspended in PBS at a concentration of 108 cells/ml. A total of 1.5 × 106 MSCs (15 ␮l of the cell suspension) were injected with a microsyringe into the left ventricular anterior free wall at five locations. For the DCM + PBS group, the same volume of PBS was injected. The animals were sacrificed at one month after the injection.

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2.7. Cardiomyocyte apoptosis assay Apoptotic nuclei in heart sections were identified using an In Situ Cell Death Detection Kit (Roche) according to the manufacturer’s instructions. The samples were fixed in 10% formalin and embedded in paraffin. TUNEL-positive cells were examined in 3 randomly chosen high-powered fields. 2.8. Western blot analysis of Akt; Bcl-2 and Bax in myocardium Western blotting was performed using the cardiac tissue homogenates of all four groups. Protein concentrations were quantified using BCA protein assays. Protein samples were separated using 10% SDS–PAGE and transferred onto a polyvinylidene difluoride membrane. Nonspecific proteins were blocked in blocking buffer (5% nonfat dry milk in TTBS containing 0.05% Tween 20) for 2 h. Immunoblotting was performed with antibodies specific for total Akt, p-Akt, Bcl-2, and Bax (1:1000; CST, USA) overnight. Horseradish peroxidase-conjugated anti-rabbit IgG was applied as a secondary antibody for 2 h at room temperature. The signals were quantified using a chemiluminescence imaging system. Sample loading was normalised with GAPDH. 2.9. Determination of VEGF and IGF-1 in heart tissue and hs-CRP in serum Heart homogenates were prepared, and commercially available kits were used to measure the vascular endothelial growth factor (VEGF; Ray Biotech, Atlanta, USA) and insulin-like growth factor-1 (IGF-1; RD, Minneapolis, USA) levels in the different groups. Serum high-sensitivity C-reactive protein (hs-CRP) levels were measured using a mouse hs-CRP ELISA Kit (Bio Vendor, EUR) following the manufacturer’s instructions. 2.10. Heart weights and histological analysis Mice aged 5 months were euthanised, and their hearts were excised, washed in PBS, blotted dry on tissue paper, and weighed. The HW/BW (heart weight/body weight) ratio was then calculated. Cardiac tissue was fixed in 4% formaldehyde and mounted in paraffin blocks, and then 4 ␮m sections were prepared for HE and Masson staining. Additionally, anti-mouse CD31 and ␣-sma (1:400, Abcam, Cambridge, MA) were used to stain vessels and small arterioles separately. 2.11. Quantitative real time PCR Collagen I, Col1␣1 and Acta1 mRNAs were detected in myocardial tissues via qRT-PCR, using Actin for normalisation. Total RNA was isolated from the heart using TRIzol Reagent (Invitrogen, USA). First-strand cDNA was synthesised from 2 ␮g of total RNA using an All-in-OneTM First-Strand cDNA Synthesis Kit (GeneCopoeia, Inc) according to the manufacturer’s protocol. The forward and reverse primers are shown in a supplementary table. 2.12. Echocardiographic analysis Cardiac function was analysed using a small animal echocardiography analysis system (Vevo770, Canada) at one month after MSC transplantation. M-mode echocardiography of the left ventricle was recorded at the tip of the mitral valve apparatus with a 30 MHz transducer. The left ventricular end-diastolic diameter (LVEDD) and end-systolic diameter (LVESD), left ventricular posterior wall thickness at end-systole (LVPWs) and diastole (LVPWd), and left ventricular end-systolic volume (LVESV) and end-diastolic volume (LVEDV) were determined using the Teichholz formula

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(Seitz and Spiel, 1983). Finally, the ejection fraction (EF) and fractional shortening (FS) were calculated. 2.13. Statistical analysis Values were expressed as the mean ± SD. Data analyses were performed using ANOVA with the Bonferroni test for comparisons between multiple groups. Statistical analysis was performed with SPSS 20.0. A value of p < 0.05 was considered significant. 3. Results 3.1. Isolation and morphological analysis of UCB-MSCs UCB-derived MNCs formed adherent cell populations, which consisted of round and spindle shaped cells, after 5–7 days in culture. The number of adherent cells increased with time; ultimately, the cultures became homogeneous with fibroblast-like shaped cells. After 15–20 days, a number of classical MSC colonies appeared, displaying morphological characteristics observed in MSCs (Fig. 1A). 3.2. In vitro differentiation results After 3 weeks of osteogenic induction, MSCs displayed osteogenic phenotypes, as detected by alizarin red staining. After 3 weeks of culture in the adipogenic medium, neutral lipid vacuoles were apparent and stained with Oil Red O, confirming an increased fat content (Fig. 1B). 3.3. Characterisation by flow cytometry The levels of MSC-specific cell-surface antigens were analysed after three passages. The MSCs were strongly positive for specific surface markers such as CD105 (97.01%) and CD44 (95.84%), whereas they were negative for CD34 (0.02%) and CD45 (0%). These results further confirmed the presence of MSC characteristics (Fig. 1C), as defined by the International Society for Cellular Therapy (Dominici et al., 2006). 3.4. MSCs transplantation improves heart function in DCM Hearts in the DCM and DCM + PBS groups were grossly enlarged and heavier than those from the control group. After MSC treatment, the cardiac sizes decreased (Fig. 2A). The HW/BW ratio was increased by 30% in the DCM group (p < 0.01) and reduced by 10% in the DCM + MSC group (p < 0.05). Cardiomyocyte hypertrophy is indicated by increased expression of Acta1; the Col1␣1 levels were increased in the DCM and DCM + PBS groups compared with the control group, but these levels were decreased after MSC treatment (Fig. 2D). Systolic function deteriorated in the transgenic hearts, which exhibited enlarged ventricular chamber and decreased EF and FS. One month after MSC injection, there was an increase in EF (56.96 ± 3.54 vs 47.40 ± 6.64%) and FS (32.26 ± 2.93 vs 23.68 ± 4.00%). Moreover, the LVEDD was also significantly reduced in the MSC-transplanted mice compared with the DCM and DCM + PBS mice (p < 0.05 for all). There was no significant difference in heart rate among the four groups (Table 1). 3.5. Reduction of myocardial fibrosis and cytoplasmic vacuolisation by MSC transplantation In Fig. 2C, the blue areas show fibrotic tissue. Our quantitative data suggest that the DCM and DCM + PBS hearts demonstrated

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Fig. 1. Characterisation of UCB-MSCs. (A) Morphology of cultured MSCs at various time points. All MSCs exhibited spindle-shaped morphology. 100×, scale bars = 500 ␮m. (B) Differentiation capability of UCB-MSCs. Osteogenic differentiation demonstrated by the deposition of mineralised matrix detected by alizarin red staining (left). Adipocytic differentiation was shown with oil-red O staining (right). 100×, scale bars = 500 ␮m. (C) Surface marker expression of UCB-MSCs. Flow cytometry of MSCs at passage 3 demonstrated positive labelling for the mesenchymal markers CD105 and CD44, but these cells were negative for the haematopoietic markers CD34 and CD45. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

significant (p < 0.05) increases in the sizes of these blue areas. However, these expanded fibrotic areas were reduced following MSC transplantation (Fig. 2F). The expression levels of the procollagens Col1␣1 and collagen I in the MSC-treated mice were decreased, further substantiating the ability of MSCs to reduce fibrosis (Fig. 2D). Cytoplasmic vacuolisation was also examined. The hearts in the control group had full-length cardiac myocytes with intact cytoplasmic structures. However, in the DCM and DCM + PBS groups, cytoplasmic vacuolisation, a phenomenon of cytoplasmic disintegration, was slightly increased. This effect was dramatically attenuated in the DCM + MSC group (Fig. 2B and E).

3.6. Identification of implanted MSCs and CD31+ cells in LV myocardium Before transplantation, the green fluorescence of the MSCs was detected under fluorescence microscopy (Fig. 3A). One month following MSC injection, the mice were sacrificed to identify the implanted MSCs in the LV myocardium. Histological analyses revealed the engraftment of numerous eGFP-stained, undifferentiated MSCs in the DCM + MSC group (Fig. 3B and C). However, cellular staining of eGFP could not be identified by troponin I. By contrast, numerous areas of CD31+ staining were identified in the DCM + MSC group (Fig. 3D), and this staining was significantly

Table 1 M-Mode echocardiographic analysis. Variables

Control

HW/BW (mg/g) LVEDD (mm) LVESD (mm) LVPWd (mm) LVPWs (mm) LVEDV (ul) LVESV (ul) LVEF (%) LVFS (%) HR

4.99 4.17 3.45 0.75 0.81 61.5 22.1 64.70 34.14 358

± ± ± ± ± ± ± ± ± ±

DCM 0.38 0.18 0.37 0.09 0.15 8.13 5.12 4.62 2.72 29

6.45 4.93 4.01 0.70 0.76 124.6 65.7 47.66 25.84 385

DCM + PBS ± ± ± ± ± ± ± ± ± ±

0.48a 0.19a 0.52a 0.14 0.12 10.71a 7.21a 5.16a 4.64a 30

6.47 5.02 3.91 0.72 0.79 120.4 63.1 47.40 23.68 378

± ± ± ± ± ± ± ± ± ±

0.30a 0.28a 0.27a 0.10 0.23 8.73a 5.29a 6.64a 4.00a 26

DCM + MSC 5.80 4.59 3.61 0.71 0.77 94.5 40.8 56.96 32.26 368

± ± ± ± ± ± ± ± ± ±

0.34b 0.93b 0.31b 0.12 0.09 10.26b 6.23b 3.54b 2.93b 36

HW/BW: heart weight/body weight; LVEDD (LVESD): left ventricular end-diastolic dimension (end-systolic dimension), LVPWd (LVPWs): left ventricular posterior wall during diastole (systole); LVESV (LVEDV): left ventricular end-systolic volume (end-diastolic volume); LVEF: left ventricular ejection fraction; LVFS: left ventricular fractional shortening, HR: heart rate. Data are expressed as the mean ± SD; n = 6 for each group; (a) p < 0.01 VS Control, (b) p < 0.05 vs DCM and DCM + MSC.

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Fig. 2. Effect of MSC on cardiac size, hypertrophy, fibrosis and cytoplasmic vacuolisation in the cTnTR141W mice with DCM. (A) Heart size in the Control, DCM, DCM + PBS and DCM + MSC mice. (B) The arrow head indicates cytoplasmic vacuolisation, which was very obvious in the DCM and DCM + PBS groups. However, MSC treatment reduced this phenomenon. (C) Masson trichrome staining showing a markedly increased fibrotic area (green areas) in the DCM and DCM + PBS groups compared with the normal control and DCM + MSC groups. (D) RT-PCR analysis of the mRNA expression levels of the hypertrophic marker genes Acta1 and Col1␣1 and the fibrotic marker collagen I. (E and F) The histogram quantitatively shows cytoplasmic vacuolisation and cardiac fibrosis. 200×, scale bars = 50 ␮m. * p < 0.01 vs Control, # p < 0.05 vs DCM + MSC.

increased compared with the DCM and DCM + PBS groups in the LV myocardium.

DCM + MSC group, this number increased significantly (Fig. 4C and D). These results indicate that MSC treatment contributes to vascular regeneration.

3.7. Effects of transplanted MSCs on apoptosis in DCM Significant (p < 0.05) increases in TUNEL-positive nuclei were observed in the DCM and DCM + PBS groups compared with the normal controls. The significant increase in apoptotic nuclei was attenuated in the DCM + MSC group, suggesting a protective effect of the MSC treatment (p < 0.05, Fig. 4A and B). 3.8. Small arteriolar density The numbers of small arterioles in the LV myocardium were lower in the DCM and DCM + PBS groups; however, in the

3.9. UCM inhibits h9c2 cells apoptosis by a paracrine protective effect in vitro The MSC-mediated protective effect against apoptosis observed in the transplanted hearts led us to examine whether MSCconditioned medium produced similar effects in vitro. As shown in Fig. 5, after 24 h of incubation in a hypoxic environment, h9c2 cells increased their rate of apoptosis from 6.5 ± 1.2% (in normoxia) to 33.8 ± 5.7%. However, when UCM-N was added to h9c2 cells during hypoxia, the apoptosis rate decreased significantly from 33.8 ± 5.7% to 16.7 ± 3.1% (p < 0.01). More importantly, UCM-H

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Fig. 3. Identification of implanted MSCs and CD31+ cells in LV myocardium. (A) eGFP-labelled MSCs in vitro before transplantation. 10×, scale bars = 500 ␮m. (B) Fluorescence microscopy of eGFP-labelled undifferentiated MSCs (red arrows) showing their survival in the myocardium one month after transplantation. 100×, scale bars = 50 ␮m. (C) MSCs seen in the myocardium using confocal microscopy. 400×, scale bars = 12.5 ␮m. (D) Immunohistochemistry results of CD31 expression in different groups. (E) The area covered by CD31+ cells in the MSC implanted group at one month was larger than the other groups, demonstrating increased vascularity in the treated group. 400×, Scale bars = 50 ␮m. * p < 0.05 vs DCM, DCM + PBS and control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

further decreased cell apoptosis by 4.5 ± 1.4% compared with UCMN (p < 0.05). However, the Akt inhibitor LY294002 suppressed the UCM-N-mediated protection of h9c2 cells (p < 0.05). 3.10. MSCs regulate Akt pathways in DCM The protein levels of Akt and its activated form, p-Akt, were detected in mouse heart tissue homogenates. p-Akt expression was significantly decreased by 44% (p < 0.01) in the cTnTR141W hearts compared with the control hearts, whereas its expression was increased following the MSC treatment (Fig. 6). Fig. 6 shows the expression levels of the apoptosis-related proteins bcl-2 and bax in the different groups. The expression of the anti-apoptotic protein bcl-2 decreased by 28% in the DCM group compared with the control group. By contrast, following the MSC treatment, Bcl-2 expression increased by 34%. The MSC treatment also activated p-Akt, whereas the pro-apoptotic protein bax was down-regulated in this group. 3.11. Levels of VEGF, IGF-1 and hs-CRP following MSCs transplantation The serum level of hs-CRP was lower in the DCM + MSC group than in the non-treated group after one month (Fig. 7A, p < 0.05). The MSC-treated hearts had significantly increased levels of VEGF and IGF-1 (Fig. 7B and C, p < 0.01).

3.12. No evidence of multiorgan seeding following MSCs transplantation in DCM To examine the safety of MSC transplantation as a treatment for DCM, H&E stained heart, lung, liver and kidney sections were examined at one month after the MSC treatment, and no evidence of teratoma formation was found (Fig. 8). 4. Discussion Stem cell transplantation has been demonstrated to be beneficial in the treatment of cardiovascular diseases. Various types of stem cells have been tested as potential donor cells, but the optimal stem cell type remains largely unknown. Bone marrow-derived MSCs are the most frequently studied stem cell type. However, these cells have some limitations, including a low proliferative ability, rapid aging, and the requirement of an invasive procedure for harvesting. UCB-MSCs, which have wide potentials for cell differentiation, are easy to collect and have low immunogenicity; thus, they may be highly suitable for transplantation and show the most promise for use in xenogeneic settings (Jin et al., 2013). Currently, no data are available on the effects of UCB-MSCs in transgenic DCM mice; and previous DCM models have typically been drug-induced. In the present study, experimental DCM was induced via the cTnTR141W gene mutation in C57BL/6 mice. These mice are characterised as having thinner ventricular walls, enlarged ventricular chambers, myocardial hypertrophy, and interstitial fibrosis, which

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Fig. 4. Apoptosis and small arteriolar density analysis at one month after transplantation. (A) TUNEL assay of apoptotic nuclei (red arrows) in the myocardium; cells stained brown are TUNEL positive. 400×, scale bars = 12.5 ␮m. (B) Statistical analysis of cardiomyocyte apoptosis. The number of TUNEL-positive cells increased in the DCM and DCM + PBS groups and decreased following transplantation with MSCs. (C) a-smooth muscle actin (a-SMA) staining of small arterioles (diameters < 100 ␮m) showing a significantly higher number of small vessels (red arrows) in the DCM + MSC group. 200X, Scale bars = 100 ␮m. (D) Quantitative analysis of ␣-SMA+ vessels. HPF, high power field. * p < 0.05 vs DCM, DCM + PBS and control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

suggests that this is a useful animal model of human DCM (Juan et al., 2008). Thus, we evaluated the feasibility and efficacy of UCB-MSC transplantation in the cTnTR141W transgenic DCM mouse model. The major finding of this study is that transplantation of UCBMSCs in the transgenic model of DCM preserves cardiac function through multiple mechanisms, including the following: (1) inhibition of apoptosis of cardiomyocytes; (2) significantly attenuated cardiac fibrosis; (3) increased levels of the growth factors VEGF and IGF-1, which are required for angiogenesis; (4) increased angiogenesis; and (5) ultimately, improved cardiac function. Moreover, in vitro studies using MSC-conditioned medium further confirmed the paracrine effects of MSCs, which included protecting h9c2 cells from hypoxia-induced apoptosis. 4.1. MSCs transplantation improves cardiac function Our results showed that cardiac function was significantly preserved in the DCM + MSC group; the EF and FS were increased and dilation of the heart was reduced. Although the eGFP-labelled MSCs were detected in the host myocardium, no implanted MSCs differentiated into troponin-I positively stained cells, a phenotype of myogenic-like cells, thus, evidence of cardiomyocyte replacement was limited. These findings raise the question of whether direct differentiation of transplanted cells can account for the functional

and structural benefits identified here. Unidentified factors may contribute to the preservation of heart function in DCM. 4.2. Anti-apoptotic and anti-inflammatory effects of UCB-MSCs Apoptosis plays an important role in the pathogenesis of cardiovascular diseases, including ischaemic heart disease, cardiomyopathy, and heart failure (Takemura et al., 2013; Tucka et al., 2012; Yaoita and Maruyama, 2008). Many studies have reported that apoptosis is also a significant contributor to the development of DCM. Consistent with previous investigations, an increase in cardiac myocyte apoptosis was observed in the cTnTR141W transgenic DCM model. MSC treatment ameliorated this increased apoptosis, which may have contributed to the improvement of heart function. Serum hs-CRP, a nonspecific biochemical marker of inflammation, is closely associated with cardiovascular diseases (Mendall et al., 2000; Sesso et al., 2003), Serum hs-CRP can induce myocardial cell apoptosis, thus causing ventricular damage or dysfunction (Clark et al., 2001). Of importance in this study, DCM mice with depressed LVEF and LVFS and an increased left ventricular diameter also had a substantially elevated hs-CRP level. Interestingly, this level was markedly suppressed by the MSC therapy. Furthermore, consistent with our results, other groups have recently shown that MSCs can regulate inflammation and rescue injured tissues in various diseases (Prockop and Oh, 2012; Zhu et al., 2014), Taken

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Fig. 5. Anti-apoptotic effect of UCM on h9c2 cells after hypoxic injury. (A) Apoptosis was quantified by FACS analysis after staining with Annexin V/propidine iodine (PI). (B) The percentage of apoptotic cells (Annexin V+/PI− cells and Annexin V+/PI+ cells) increases when cells are subjected to hypoxia for 24 h (33.8% ± 4.8% vs 6.5% ± 1.2%). However, when media were supplemented with UCM-N, the percentage of apoptotic cells decreased significantly (UCM-N 16.7% ± 2.7%). Importantly, UCM-H further decreased the percentage of apoptotic cells (UCM-H 12.2% ± 1.9%). The Akt inhibitor LY294002 abrogated the protective effect of UCM-N. Data are from 3 independent experiments; * p < 0.05 vs Hypoxia +UCM-N, ** p < 0.01 vs Hypoxia, *** p < 0.001 vs Control.

Fig. 6. MSCs regulate the Akt signalling pathway in DCM. (A) Western blot expression of p-Akt, Akt, Bcl-2, Bax and GAPDH. (B) Quantitative analysis of p-Akt regulation. (C) Quantitative analysis of the Bcl-2/Bax ratio. Data are shown as the mean ± SD of at least three independent experiments. * p < 0.05, # p < 0.01 vs DCM and DCM + PBS.

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Fig. 7. Effects of MSC treatment on the expression of VEGF and IGF-1 in heart tissue and hs-CRP in serum. As shown by ELISA, the levels of VEGF and IGF-1 increased significantly after MSC treatment. However, the level of hs-CRP level decreased; * p < 0.01 vs control, DCM, DCM + PBS, # p < 0.05 vs DCM and DCM + PBS.

Fig. 8. Representative photomicrographs of HE staining from the DCM + MSC group show no evidence of teratoma formation in the heart, lung, kidney, liver and spleen. 10×, scale bars = 100 ␮m.

together, these data provide evidence that UCB-MSC transplantation can attenuate the inflammatory response and inhibit cell apoptosis. 4.3. MSCs transplantation up-regulates p-Akt and bcl-2 protein Next, to elucidate the mechanisms by which MSCs inhibit apoptosis and inflammation in DCM, we explored the role of the PI3K/Akt signalling pathway. which is a powerful survival signalling pathway in many systems (Franke et al., 1997), especially in the regulation of physiological heart growth (Walsh, 2006), myocyte survival, energy production and apoptosis, and it has been shown to have powerful protective effects in a variety of cardiovascular diseases (Sussman et al., 2011). In the present study, we found a significantly reduced p-Akt level in the cTnTR141W mice; this reduction was reversed following treatment with MSCs. Many studies have suggested that Akt signalling confers cardioprotection in vivo against pathological processes; for example, Mangi et al. (2003) demonstrated that MSCs modified with Akt prevented remodelling and restored the functioning of an infarcted heart, Our results are in agreement with these previous findings. Bcl-2 is a critical inhibitor of apoptotic cell death in ventricular myocytes. A role of the Bcl-2 protein in stem cell therapy has been widely reported; Li et al. found that MSCs engineered to express Bcl-2 inhibited apoptosis and improved heart function in a rat left anterior descending ligation model (Li et al., 2007). In addition,

many drugs may improve the survival of MSCs and the effect of transplantation by increasing bcl-2 expression, such as trimetazidine (Wisel et al., 2009) and rosuvastatin (Zhang et al., 2013). Our in vivo results showed that bcl-2 expression was significantly increased following the MSC treatment in the DCM mice, suggesting that the MSCs were responsible for this elevated expression.

4.4. MSC implantation increased the expression of VEGF and IGF-1—Paracrine mechanism VEGF and IGF-1, two important paracrine factors, are related to cardiac remodelling (Koudstaal et al., 2014; Moon et al., 2014; Troncoso et al., 2014). Surprisingly, we observed significantly increased levels of the growth factors VEGF and IGF-1 in MSCtreated hearts. Interestingly, a previous study has demonstrated that hearts treated with embryonic stem cells have significantly increased levels of the growth factors HGF and IGF-1 (Singla et al., 2012). Mu et al. have also found evidence that bone marrowderived MSCs improve the cardiac function of rabbits with DCM via up-regulation of VEGF and its receptors (Mu et al., 2011), Taken together, these results suggest that released factors may activate endogenous cardiac progenitors to protect and repair damaged myocardium, and our findings further corroborate this hypothesis. Therefore, a paracrine mechanism may play an important role in stem cell therapy. For example, VEGF could promote the continuous angiogenesis of ischemic myocardium; vessel growth caused

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by MSCs transplantation contributes to further improvement of cardiac function. Further studies are required to determine whether other growth factors participate in inhibiting the pathomechanisms of DCM. Whether stem cell transplantation can stimulate the host to produce more paracrine factors is an important research aspect that should be addressed in the future. Potential paracrine mechanisms involved in MSC-mediated cardiac protection were further evaluated using MSC-conditioned medium in vitro. This conditioned medium reduced the hypoxiainduced apoptosis of h9c2 cells. Furthermore, hypoxic preconditioning of the conditioned media caused an even more pronounced anti-apoptotic effect. Many studies have shown that MSCs exhibit increased paracrine effects under hypoxic conditions, allowing for the improved treatment of various diseases (Chang et al., 2013; Chen et al., 2014; Haneef et al., 2014; Hwang et al., 2012); These results suggest that maintaining the prolonged and adequate release of factors is a potential method to increase the beneficial effects of stem cell therapy. 4.5. Angiogenesis/vasculogenesis The development of angiogenesis following MSC implantation in ischaemic myocardial tissue has been extensively investigated (Psaltis et al., 2008; Yoon et al., 2010). However, little is known about the impact on DCM, and recent studies have demonstrated the vascular potential of UCB-MSCs (Roura et al., 2012). Moreover, Roura, et al found that a 3D engineered fibrin patch composed of UCBMSCs attenuated infarct-derived cardiac dysfunction when transplanted locally over a myocardial wound (Roura et al., 2015). Further, marked vascular derangements and impaired vasculogenic and angiogenic responses have been reported in patients with DCM (Roura and Bayes-Genis, 2009). In the present study, we found that the number of small vessels (␣-sma stained) and CD31positively stained cells, a surface marker of endothelial cells, were remarkably higher in the DCM + MSC group. In addition, VEGF, an important active protein involved in the induction of angiogenesis and improvement of cardiac function, was increased in the MSCtreated hearts. In summary, the MSCs promoted angiogenesis in the DCM mice, thereby attenuating LV remodelling processes while also promoting cardioprotective effects. The success of stem cell therapy largely depends on the recipient’s myocardial environment. In contrast with ischaemic cardiomyopathy, cTnTR141W transgenic DCM mice have normal coronary circulation, which potentially allows the animals to receive the nutrients necessary for the survival of transplanted MSCs. Hence, we detected eGFP-labelled MSCs in the hearts at one month after transplantation. 4.6. MSC transplantation limits fibrosis and hypertrophy Previous studies have suggested that myocardial fibrosis is an independent and incremental predictor of mortality and sudden cardiac death in DCM (Gulati et al., 2013). Therefore, we evaluated myocardial fibrosis and cytoplasmic vacuolisation in the DCM transgenic mice and assessed whether UCB-MSCs can inhibit cardiac fibrosis. The results showed that treatment with MSCs significantly inhibited cytoplasmic vacuolisation and fibrosis in the hearts of the DCM mice. These mice also exhibited reduced gene expression of the profibrotic marker Col1␣1. Furthermore, MSC transplantation altered the synthesis and degradation of collagen through the down-regulation of collagen I, effectively reversing myocardial fibrosis and the increased collagen remodelling and contributing to improved cardiac structure and function in the DCM mice.

Patients with DCM exhibit not only myocardial fibrosis but also myocardial hypertrophy (Jefferies and Towbin, 2010). MSC transplantation ameliorated cardiomyocyte hypertrophy in the transgenic mouse model of DCM, as indicated by a decreased HW/BW ratio and down-regulated expression of the hypertrophic marker genes Acta1 and Col1␣1. In summary, to our knowledge, this is the first study to propose that xenogeneic UCB-MSCs improve cardiac function in a transgenic model of DCM. Mechanistically, this effect may be attributed to the suppression of multiple pathological processes, including cardiomyocyte apoptosis, inflammation, fibrosis, and Akt pathway signalling, as well as to up-regulation of VEGF and IGF-1 and enhanced angiogenesis. The benefits of MSC treatment are most likely caused by paracrine effects. For these reasons, UCB-MSCs are a promising candidate for adjunct therapy in patients suffering from familial DCM. 5. Funding sources This work was supported by grants from the Specialised Research Fund for the Doctoral Program of Higher Education (2012110611045). The Ethics Committee for Animal Research of Fuwai Hospital approved our study. 6. Disclosures None. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejcb.2015.11.003. References Carmeliet, P., 2000. VEGF gene therapy: stimulating angiogenesis or angioma-genesis? Nat. Med. 6, 1102–1103. Chang, C.P., Chio, C.C., Cheong, C.U., Chao, C.M., Cheng, B.C., Lin, M.T., 2013. Hypoxic preconditioning enhances the therapeutic potential of the secretome from cultured human mesenchymal stem cells in experimental traumatic brain injury. Clin. Sci. 124, 165–176. Chen, L., Xu, Y., Zhao, J., Zhang, Z., Yang, R., Xie, J., Liu, X., Qi, S., 2014. Conditioned medium from hypoxic bone marrow-derived mesenchymal stem cells enhances wound healing in mice. PLoS ONE 9, e96161. Clark, D.J., Cleman, M.W., Pfau, S.E., Rollins, S.A., Ramahi, T.M., Mayer, C., Caulin-Glaser, T., Daher, E., Kosiborod, M., Bell, L., Setaro, J.F., 2001. Serum complement activation in congestive heart failure. Am. Heart J. 141, 684–690. Cui, B., Li, E., Yang, B., Wang, B., 2014. Human umbilical cord blood-derived mesenchymal stem cell transplantation for the treatment of spinal cord injury. Exp. Ther. Med. 7, 1233–1236. Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., Krause, D., Deans, R., Keating, A., Prockop, D., Horwitz, E., 2006. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8, 315–317. Franke, T.F., Kaplan, D.R., Cantley, L.C., 1997. PI3 K: downstream AKTion blocks apoptosis. Cell 88, 435–437. Gulati, A., Jabbour, A., Ismail, T.F., Guha, K., Khwaja, J., Raza, S., Morarji, K., Brown, T.D., Ismail, N.A., Dweck, M.R., Di Pietro, E., Roughton, M., Wage, R., Daryani, Y., O’Hanlon, R., Sheppard, M.N., Alpendurada, F., Lyon, A.R., Cook, S.A., Cowie, M.R., Assomull, R.G., Pennell, D.J., Prasad, S.K., 2013. Association of fibrosis with mortality and sudden cardiac death in patients with nonischemic dilated cardiomyopathy. JAMA 309, 896–908 (the journal of the American Medical Association). Haneef, K., Naeem, N., Khan, I., Iqbal, H., Kabir, N., Jamall, S., Zahid, M., Salim, A., 2014. Conditioned medium enhances the fusion capability of rat bone marrow mesenchymal stem cells and cardiomyocytes. Mol. Biol. Rep. 41, 3099–3112. Hwang, H.J., Chang, W., Song, B.W., Song, H., Cha, M.J., Kim, I.K., Lim, S., Choi, E.J., Ham, O., Lee, S.Y., Shim, J., Joung, B., Pak, H.N., Kim, S.S., Choi, B.R., Jang, Y., Lee, M.H., Hwang, K.C., 2012. Antiarrhythmic potential of mesenchymal stem cell is modulated by hypoxic environment. J. Am. Coll. Cardiol. 60, 1698–1706. Jang, H.R., Park, J.H., Kwon, G.Y., Lee, J.E., Huh, W., Jin, H.J., Choi, S.J., Oh, W., Oh, H.Y., Kim, Y.G., 2008. Effect of preemptive treatment with human umbilical cord blood-derived mesenchymal stem cells on the development of renal ischemia-reperfusion injury in mice. Am. J. Physiol. Renal Physiol. 128 (1), 83–90.

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