Human-derived decellularized extracellular matrix scaffold incorporating autologous bone marrow stem cells from patients with congenital heart disease for cardiac tissue engineering

Abstract

BACKGROUND:

Stem cells are used as an alternative treatment option for patients with congenital heart disease (CHD) due to their regenerative potential, but they are subject to low retention rate in the injured myocardium. Also, the diseased microenvironment in the injured myocardium may not provide healthy cues for optimal stem cell function.

OBJECTIVE:

In this study, we prepared a novel human-derived cardiac scaffold to improve the functional behaviors of stem cells.

METHODS:

Decellularized extracellular matrix (ECM) scaffolds were fabricated by removing cells of human-derived cardiac appendage tissues. Then, bone marrow c-kit+ progenitor cells from patients with congenital heart disease were seeded on the cardiac ECM scaffolds. Cell adhesion, survival, proliferation and cardiac differentiation on human cardiac decellularized ECM scaffold were evaluated in vitro. Label-free mass spectrometry was applied to analyze cardiac ECM proteins regulating cell behaviors.

RESULTS:

It was shown that cardiac ECM scaffolds promoted stem cell adhesion and proliferation. Importantly, bone marrow c-kit+ progenitor cells cultured on cardiac ECM scaffold for 14 days differentiated into cardiomyocyte-like cells without supplement with any inducible factors, as confirmed by the increased protein level of Gata4 and upregulated gene levels of Gata4, Nkx2.5, and cTnT. Proteomic analysis showed the proteins in cardiac ECM functioned in multiple biological activities, including regulation of cell proliferation, regulation of cell differentiation, and cardiovascular system development.

CONCLUSION:

The human-derived cardiac scaffold constructed in this study may help repair the damaged myocardium and hold great potential for tissue engineering application in pediatric patients with CHD.

Keywords

Extracellular matrix (ECM)c-kit cardiac differentiation tissue engineering congenital heart disease

1. Introduction

Congenital heart disease (CHD) is the most common group of congenital anomalies and the main cause of birth defect-related death [1,2]. Although surgery remains the gold standard for the treatment of CHD, it often leads to right ventricular (RV) hemodynamic changes and increased load to the heart, developing right ventricular dysfunction even RV failure in pediatric patients, especially in children with pulmonary stenosis, tetrology of fallot and hypoplastic left heart syndrome [3,4]. The restorative treatment for the patients with severe RV failure is heart transplantation, which suffers donor shortages and transplant rejection [5].

Due to the regenerative potential, stem cells have emerged as alternative treatment option for CHD patients [6]. A recent clinical trial showed that intracoronary administration of autologous cardiosphere-derived cells improved the right ventricular function and reduced the incidence of adverse events in children with CHD after surgery [7]. Multiple studies have also demonstrated that c-kit+ progenitor cells repaired injured myocardium and alleviated pediatric RV heart failure [8–10], suggesting a favorable choice for use in pediatric stem-cell therapy. Despite this encouraging outcome, similar problems were also found in pediatric CHD as have been shown previously in adult patients. Most of the injected c-kit+ progenitor cells were lost to the circulation, and only 0.1–10% of the cells was successfully grafted to the myocardium [11]. In addition, the diseased microenvironment in the injured myocardium may not provide healthy cues for optimal c-kit+ progenitor cell function [12].

To increase the retention rate and modify the local microenvironment of grafted cells, both synthetic and natural biomaterials have been used as scaffolds. However, most of synthetic materials, such as polylactic acid (PLA), polyglycolic acid (PGA), and polyglycerol sebacate (PGS), do not provide an ideal microenvironment to support cell growth and survival [13,14]. Recently, multiple ECM materials have been used in cardiac repair, including porcine-derived small intestine submucosa (SIS) [15], acellular bovine-derived pericardium (BP) [16], decellularized heart tissue [17], human amniotic membrane [18] and human placenta [19]. Among these natural materials, decellularized cardiac ECM was considered an ideal scaffold for stem cell delivery and cardiac repair in that it preserves microstructure and composition as well as providing the same microenvironment as the native heart. Although the treatment with c-kit+ stem cells delivered with scaffold has been associated with significant benefits in adults with cardiac diseases, only a few studies have been performed on children with CHD. Particularly, the functional behaviors such as retention, survival, proliferation, and differentiation of autologous bone marrow c-kit+ progenitor cells from CHD patients on cardiac decellularized ECM scaffold need to be fully investigated.

In the present study, we isolated c-kit+ progenitor cells from bone marrow aspirates of pediatric patients with CHD and characterized c-kit+ cell surface marker profile. To provide the similar local microenvironment as native heart, human cardiac decellularized ECM scaffolds were prepared from atrial appendage tissues supplied by cardiovascular surgery in hospital. Subsequently, cell adhesion, survival, proliferation and cardiac differentiation on human cardiac decellularized ECM scaffold were evaluated in vitro. Finally, the composition and proteins distribution of human cardiac ECM were also analyzed.

2. Materials and methods

2.1. Decellularization of human atrial appendage tissue

Human left atrial appendage tissues were obtained from 12 patients with severe valvular heart diseases and atrial fibrillation. The patients’ information was listed in Supplementary Table S1. The current study was performed following the rules of the Declaration of Helsinki. The use of the patients’ atrial appendage tissues was approved by the ethics committee of Renji Hospital, Shanghai (permit number: 2012027). All patients provided written informed consent before surgery. Human cardiac ECM scaffold was prepared as previously published protocols with minor modifications [20]. Briefly, human atrial appendage tissues were rinsed in ice-cold phosphate-buffered saline (PBS) to remove plasma and cut into 2 cm × 2 cm pieces. Then, tissue pieces were embedded in agarose (Sigma, USA) and were cut into 300 μm thick sections with a microtome (Leica VT-1200, Wetzlar, Germany). Subsequently, sections were decellularized using 0.5% sodium dodecyl sulfate (SDS; Sigma, USA) in deionized water and vortexed at 25 °C until the ECM was white. After washing with deionized water adequately, residual DNA was removed by incubation in 200 IU/ml DNase I solution (Roche, Basel, Switzerland). The samples were stored in deionized water containing 100 U/ml penicillin and 100 U/ml streptomycin (Gibco, New York, USA) at 4 °C until use.

2.2. Histological and immunohistochemical staining

Undecellularized and decellularized atrial appendage tissues were fixed in 4% paraformaldehyde at room temperature for 1 h, embedded in paraffin, and cut into slices with 5 μm thickness. Haematoxylin and eosin (H&E) staining, Masson’s trichrome staining, and Von Gieson (VG) staining were performed separately according to standard procedures. For IHC staining, the slices were immersed in citrate antigen retrieval buffer at 95 °C for 20 min. Then the slices were treated with 3% hydrogen peroxide solution at room temperature for 20 min, immediately transferred into a humidity chamber and incubated with 10% normal goat serum for 30 min. Hereafter, slices were incubated with anti-fibronectin or anti-laminin primary antibody (1:200, Abcam, Cambridge, UK) at 4 °C overnight, followed by rinsing with phosphate-buffered saline (PBS) three times. Slices were then incubated with biotinylaaketed secondary antibody for 30 min. After rinsed in PBS,samples were treated with diaminobenzidine solution (DAB). The slices were finally dehydrated, cover-slipped and imaged using a Nikon microscope (eclipse-80i, Japan).

2.3. Isolation, culture and characterization of c-kit+ bone marrow progenitor cells

This study was approved by the Institutional Review Board at Shanghai Children’s Medical Center (permit number: SCMCIRB-K2016025), and written informed consent was signed by parents of pediatric patients. The patients’ information was seen in Supplementary Table S2. 1–2 ml of bone marrow aspirate was obtained from pediatric patients with congenital heart disease. Human mononuclear cells were isolated from bone marrow aspirates by density gradient sedimentation with human lymphocyte separation medium (Biosera, England, UK). Subsequently, bone marrow mononuclear cells were subjected to magnetic activated cell sorting with anti-CD117 antibody conjugated with magnetic beads (Miltenyi Biotec, Germany) to obtain c-kit-positive bone marrow stem cells. The sorted c-kit+ cells were characterized by flow cytometry analysis for the following markers: CD29 (PE, Ebioscience, USA), CD31 (PE, Biolegend, USA), CD34 (PE, Biolegend, USA), CD44 (APC, Ebioscience, USA), CD45 (Percp/cy5.5, Ebioscience, USA), CD90 (Percp/cy5.5, Biolegend, USA). Bone marrow c-kit+ cells were seeded on cardiac ECM scaffold or fibronectin coated on culture plate and cultured in Iscove’s Modified Dulbecco’s Medium (Thermo Fisher Scientific, MA, USA) containing 10‰ fetal bovine serum (Biowest, MO, USA) without supplementation of any growth factor.

2.4. Live/dead staining

After 7 days of culture, bone marrow c-kit+ cells on scaffolds were incubated with 5 μM Calcein AM and 8 μM propidium iodide (Thermo Fisher Scientific, MA, USA) at 37 °C for 20 min. After they were washed with PBS, c-kit+ cells in solution were imaged using a Nikon fluorescence microscope. Live cells were stained green and dead cells appeared red.

2.5. Scanning electron microscopy (SEM)

The surface of cardiac ECM scaffold was scanned using SEM. To observe cell adhesion to scaffold, c-kit+ cells seeded on cardiac ECM scaffold at day 7 were also examined by SEM. The samples were washed with PBS for three times, and then fixed in 2.5% glutaraldehyde diluted in PBS for 2 h at room temperature. Fixed samples were dehydrated in 50%, 70%, 90%, 95% and 100% ethanol followed by critical-point drying and sputter-coated with gold. Finally, samples examined under SEM (Quanta 200; FEI, OR, USA).

2.6. Cell proliferation detection

Cell proliferation assay (MTS) was performed to assess cell viability and proliferation according to the manufacturer’s instructions. Briefly, CellTite 96^® A Queous One Solution (Promega Corp, WI, USA) was added into the 96-well culture plate and incubated at 37 °C for 4 h on days 1, 3, 5, 7, 10 and 14, respectively. The absorbance was measured at 490 nm using Biotek Synergy^{^TM} HT Multi-Mode Microplate Reader (Biotek Instruments, VT, USA).

2.7. In vitro differentiation

After 7 days of culture, cells were fixed in 4% paraformaldehyde for 15 min, permeabilized using 0.5% Triton X-100 in PBS, and blocked with 5% normal donkey serum and 1% bovine serum albumin for 1 h. Subsequently, cells were incubated with primary antibody mouse anti-Ki67 (1:500, BD Biosciences, CA, USA), Rabbit anti-phosphohistone H3 (PH3, 1:500, Cell Signaling Technology, USA) or goat anti-Gata4 (1:200, Santa Cruz, USA) at 4 °C overnight, followed by incubation with secondary antibody donkey anti-mouse Alexa594 (1:1000, Life Technologies, USA), donkey anti-rabbit 488 (1:1000, Life Technologies, USA), or donkey anti-goat 594 (1:1000, Life Technologies, USA) for 1 h at room temperature. Finally, 4,6-diamidino-2 phenylindole (DAPI) (1:1000, Sigma, USA) was applied to counterstain cells nuclei. Fluorescent images were taken on a Nikon fluorescence microscope.

2.8. Reverse transcriptase-PCR

RNA was extracted from cultured cells at different time points (day 1, day 7, and day 14) using Quick-RNA TM Micro Prep (Zymo, USA). cDNA was obtained by means of reverse transcription using GoScript^{^TM} Reverse Transcription System (Promega, USA). PCR was performed using a standard procedure with denaturation at 94 °C for 15 s, annealing at 55–60 °C for 30 s and extension at 72 °C for 45 s. The number of cycles varied between 28 and 35 depending on the abundance of mRNA. The gene expression of Gata4, Nkx2.5, and cTnT was analyzed by β-actin as a housekeeping gene. The PCR products were subjected to electrophoresis on a 2% agarose gel containing 0.1% GelGreen (Genview, USA) and analyzed using Image lab system (Bio-Rad, USA). All samples were analyzed in triplicates.

2.9. Label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS)

The frozen decellularized cardiac appendage tissues were grinded and homogenized in a protein lysis buffer containinig 8 mM urea, 100 mM Tris-HCl, 10 mM dithiothreitol (DTT), and protease inhibitor. Then samples were centrifuged at 10,000× g for 30 min, and the supernatants were collected and transferred into new microcentrifuge tubes. The protein concentration in supernatant was determined using Bradford Assay (Thermo Fisher Scientific, MA, USA). Next, the proteins were digested with trypsin at 1:100 enzyme-to-protein ratios at 37 °C for at least 12 h. The peptide mixture was loaded into a C18 trap column (3 μm, 0.10 × 20 mm), and subsequently loaded onto an C18 analytical column (1.9 μm, 0.15 × 120 mm) with two solvent buffer (A: 99.9% water and 0.1% formic acid; B: 79.9% acetonitrile, 20% water, and 0.1% formic acid) for 90 min at a flow rate of 250 nL/min. Samples were analyzed in triplicate by using a Q-Exactive mass spectrometer (Thermo Fisher Scientific, MA, USA). Proteins were identified using Proteome Discoverer 1.4 (Thermo Fisher Scientific, USA) and Mascot search engine version 2.2 (Matrix Science, London, UK) based on the human Swissprot proteome database. The protein false discovery rate (FDR) was set at <1%, and identified proteins were required to be derived by at least two independent peptides.

2.10. Statistical analysis

Statistical analysis were performed using GraphPad prism 5.0 software (GraphPad Software, Inc., CA, USA). Results were expressed as mean ± standard deviation. Mann–Whitney U test was performed for the analysis of data resulting from cell proliferation, mRNA expression, and cardiogenic differentiation. P < 0.05 was set to indicate statistical significance.

3. Results

3.1. Characterization of human cardiac ECM scaffold

To observe the structure and components of decellularized human cardiac scaffold, native atrial appendage tissue and cardiac ECM scaffold were compared by histological and immunohistochemical staining, as shown in Fig. 1. H&E staining showed that cardiac myocytes with red cytoplasm and blue nuclei could be seen in native cardiac appendage tissue, while only a network of ECM fibers existed in cardiac ECM scaffold (Fig. 1A), suggesting the successful decellularization. Masson staining and VG staining indicated that collagen fibers were still well preserved in cardiac ECM scaffold (Fig. 1B,C). In addition, the results showed that fibronectin and laminin of human cardiac appendage were also preserved after decellularization (Fig. 1D,E).

Fig. 1.

Characterization of the decellularized cardiac ECM scaffold. (a) H&E staining indicates removal of cell nuclei in decellularized cardiac appendage tissue. (b) Masson staining shows the distribution of collagen fibers (blue) and myocardial fibers (red) before and after decellularization. (c) Van Gieson staining shows collagen (red) distribution in decellularized cardiac appendage tissue. Immunohistochemical staining shows retention of (d) fibronectin and (e) laminin after decellularization. Scale bar: 100 μm.

3.2. Phenotype of human bone marrow c-kit+ stem cells

The surface marker-expression pattern of human c-kit+ stem cells was analyzed using flow-cytometry on day 1 (Fig. 2). The flow-cytometry data showed that human bone marrow stem cells were positive for hematopoietic markers CD34 (18.3%) and CD45 (70.9%), and also positive for mesenchymal markers CD29 (8.2%), CD44 (43.3%), and CD90 (2.0%). In addition, based on the flow-cytometry results, endothelial marker CD31 (17.2%) was also expressed on the surface of c-kit+ stem cells. These results suggested that human bone marrow c-kit+ stem cells are heterogeneous cell populations.

Fig. 2.

Characterization of bone marrow c-kit+ stem cells. Flow cytometry analysis reveals that c-kit+ stem cells are positive for hematopoietic markers (CD34, CD45), mesenchymal markers (CD29, CD44, and CD90), and endothelial marker (CD31).

3.3. Cell attachment and viability

The SEM image of (Fig. 3a) demonstrated three-dimensional network architectures of the ECM scaffold, confirming the removal of cell. The cells adhesion to ECM scaffold after incubation for 5 days was also observed using SEM. As shown in Fig. 3b, c-kit+ stem cells attached to the surface of ECM scaffolds and exhibited the spindle-like or polygonal shapes, presenting multiple pseudopodium. In Fig. 3c, almost all the cells were stained positively for calcein AM (green), indicating that they are viable on the surface of ECM scaffold or within the scaffold.

Fig. 3.

Seeding of human bone marrow c-kit+ stem cells on cardiac ECM scaffold. (a) Representative SEM image showing the 3D structure of the decellularized cardiac ECM scaffold. (b) SEM images indicating cell adhesion to the aortic ECM scaffold. (c) Bone marrow c-kit+ stem cells are stained green (live) or red (dead) at day 7 after seeding on the cardiac ECM scaffold. Scale bar: 100 μm.

3.4. Cardiac ECM scaffold promoted the proliferation of c-kit+ stem cells in vitro

To evaluate the proliferation of c-kit+ stem cells on scaffolds, MTT assay and fluorescence immunostaining were performed. Observation under light microscope showed that the number of c-kit+ stem cells on ECM scaffold increased after cultured for 5 days (Fig. 4a,b). The MTT assay demonstrated that OD values of c-kit+ stem cells in both groups gradually increased from day 1 to day 7, reach the maximum level on day 7, and then declined (Fig. 4e). However, the proliferation rate of c-kit+ stem cells that were seeded on ECM scaffolds was significantly higher than that of the control group from the beginning to the end of the experimental period (14 days) (Fig. 4e). To further confirm these results, c-kit+ stem cells were immunostained with markers PH3 and Ki67, which are indicators of cell proliferation. As shown in Fig. 4c,d, the number of PH3 and (or) Ki67 positive cells in ECM scaffold group was much higher than that in control group (15.7 ± 1.5% vs. 5.7% ± 0.7%, P < 0.01), indicating that ECM scaffold significantly promoted cell proliferation. The above results disclose the stimulated effect of human cardiac appendage-derived ECM scaffold on bone marrow c-kit+ stem cells in vitro.

Fig. 4.

Proliferation of c-kit+ stem cells on cardiac ECM scaffold. (a,b) Optical microscope images of c-kit+ cell proliferation on cardiac ECM scaffold at day 1 (a), and day 7 (b). (c,d) Proliferation of c-kit+ cells on cardiac ECM scaffolds is assessed by double immunofluorescence staining of protein Ki67 and pH3. Proliferative ratio (%) = (number of Ki67-positive cells + number of PH3-positive cells)/total cell number × 100%. Proliferative ratios are calculated from 5 different random view fields. Scale bar: 50 μm. (e) MTS assay is performed to detect cell proliferation on days 1, 3, 5, 7, 10 and 14, n = 3 for each group.

3.5. Cardiomyogenic differentiation of c-kit+ stem cells on ECM scaffold

The effect of the ECM scaffold on the differentiation of bone marrow c-kit+ stem cells was investigated using PCR and immunocytochemistry. Reverse-transcription PCR analysis revealed that the gene expressions of Gata4 and Nkx2.5, the early markers of cardiac differentiation, were up-regulated in cells on ECM scaffold on day 7 of culture, and were further increased on day 14. Although the expression of Gata4 and Nkx2.5 genes were also detected in cells on day 14 in control group, their levels were much lower compared to that in cells on ECM scaffold (P < 0.05, Fig. 5b,c). In addition, cTnT gene level in cells on ECM scaffold was much higher in comparison to that in control group (P < 0.01, Fig. 5d). Immunofluorescence staining was also performed to detect the expression of Gata4 protein in differentiated c-kit+ stem cells in both groups (Fig. 5e). The staining results showed that some cells (account 6.9 ± 1.2%) cultured on cardiac ECM scaffold expressed Gata4 protein but only very few cells (0.7 ± 0.2%) in control group on day 14 (P < 0.01, Fig. 5f). These data demonstrated that ECM scaffold promoted cardiac differentiation of bone marrow c-kit+ stem cells.

Fig. 5.

Cardiogenic differentiation of bone marrow c-kit+ stem cells in vitro. (a–d) RT-PCR indicating up-regulated gene expression levels of cardiac markers (including Gata4, Nkx2.5, and cTnT) in scaffold+c-kit+ group compared to c-kit+ group at different time point. (e,f) Immunofluorescence staining showing that scaffold+c-kit+ group contains greater number of Gata4-positive cells than c-kit+ group (P < 0.01) at day 14. Percentages of Gata4-positive cells are calculated from 3 different random view fields. Scale bar: 50 μm.

3.6. Proteomic analysis of cardiac appendage ECM

The decellularized human cardiac appendage was subjected to label-free proteomic analysis. A total of 211 ECM proteins were identified. These proteins were further classified into core ECM proteins and ECM-associated proteins. Core ECM proteins include glycoproteins, collagens and proteoglycans. ECM-associated proteins can be classified into ECM regulators, ECM-affiliated proteins, and secreted factors.

The ECM proteins were further analyzed using Gene ontology (GO) annotation. As shown in Fig. 6b, the annotation was divided into three major GO branches, including biological processes, cellular components and molecular functions. It was shown that 211 ECM proteins were involved in many cell activities, such as cell adhesion, integrin binding, and extracellular structure organization. Data of Venn diagram showed that 23 proteins were related to cardiovascular system development, and 21 proteins were associated with regulation of cell proliferation (Fig. 6C). In addition, total 39 ECM proteins participated in regulation of cell differentiation (Fig. 6C).

Fig. 6.

Proteomic characterization of human cardiac ECM. (a) SDS-PAGE pattern of cardiac ECM. Rat tail type Icollagen is used as control. (b) GO analysis of cardiac ECM proteins. (c) Venn diagram of cardiac ECM proteins function in regulation of cell differentiation and proliferation. (d) Top 10 KEGG pathways that cardiac ECM proteins participate in.

4. Discussion

Most children with CHD require invasive surgical intervention. Despite significant progress in operation, patients with complex heart malformations usually suffer ventricular dysfunction after staged palliative surgery and most of them ultimately develop heart failure. Stem cell therapies showed great promise in preclinical and clinical studies to improve ventricular function in patients with CHD. However, the therapeutic efficacy was greatly compromised by low cell retention and survival following implantation [21]. To overcome these deficiencies, scaffold was commonly used to provide a suitable microenvironment for transplanted cells, thus promoting cell growth.

An ideal cardiac tissue engineering scaffold should not only provide physical support for the seeded cells, but also should imitate physical and biochemical cues in natural extracellular matrix for guiding the cellular behaviors such as cell attachment, proliferation, and differentiation [22]. Because of the intricate native structure and composition, decellularized cardiac ECM scaffolds have shown great promise for the repair of damaged myocardium, as well as for the delivery of stem cells [23–25]. Undoubtedly, human-derived cardiac ECM would be the best scaffold for cardiac tissue engineering because of the extremely approximate structure and characteristics with native heart. Once implanted in the defective heart, cardiac ECM scaffolds would be expected to promote stem cell retention and guide cell differentiation, thus strengthening the ventricular function in children with heart failure secondary to CHD.

In this study, the personalized cardiac scaffolds were prepared from human atrial appendage tissue of patients with atrial fibrillation. In vitro experiments showed that the cardiac ECM scaffolds possess good biocompatibility and promote cell adhesion and proliferation. Furthermore, cardiac ECM scaffolds directed the differentiation of bone marrow c-kit+ progenitor cells towards cardiomyocytes without supplement with any growth factors or cytokines. Further, results from proteomic analysis suggested that several ECM proteins may function in regulating the cardiogenic differentiation of c-kit+ progenitor cells. These outcomes demonstrated that decellularized cardiac ECM scaffolds show great potential for stem cell delivery and cardiac tissue engineering.

Stem cells are a major focus in regenerative medicine, since they promise to provide unlimited amounts of cells for transplantation. Multiple types of stem cell populations, including c-kit+ cells, were used to improve cardiac function which has been impaired in patients with CHD [8,9]. While some researcher claimed that c-kit+ progenitor cells exist endogenously in the adult mammalian heart and regenerate heart tissue [26,27], other investigators refuted this view [28,29]. Furthermore, it is difficult to obtain sufficient c-kit+ cells from heart tissues for tissue engineering application. In our research, c-kit+ cells were separated from bone marrow of pediatric patients with CHD. There are multiple advantages for autologous bone marrow c-kit+ cells when applied in pediatric cardiac tissue engineering, such as easy accessibility, repeatable harvest and eliminating the possibility of immune response [30]. Furthermore, it was shown that c-kit+ cells were more abundantly detected in children with CHD in comparison to age-matched healthy children and adult patients with heart disease [31]. We found that bone marrow c-kit+ cells account for about 7.8 ± 0.6% of total bone marrow mononuclear cells in CHD patients with age from 1 to 5 years old (data not shown).

In addition, many investigators reported that c-kit+ cells differentiate not only into cardiovascular cells but also into osteoblasts, adipocytes and skeletal muscle cells [32,33]. In these studies, cardiac committed differentiation of c-kit+ cells were achieved by means of adding chemical reagents, such as growth factors [34], 5-azacytidine [35], or dexamethasone [36]. However, these reagents usually have more or less side effects on human body, and it is hard to determine the optimal concentration for c-kit+ cell differentiation with minimum side effects. In our study, the seeded c-kit+ stem cells on cardiac ECM scaffold differentiated towards cardiomyocytes in the absence of any chemical reagent or cytokine. Immunofluorescence staining showed that bone marrow c-kit+ stem cells expressed Gata4 protein in the nuclei after seeded on cardiac ECM scaffold at day 14. Furthermore, RT-PCR results indicated that the levels of three genes, including Gata4, Nkx2.5, and cTnT, were increased in c-kit+ stem cells. These results demonstrated the in vitro transition of c-kit+ stem cells into cardiomyocytes. Obviously, this is beneficial to improve the impaired cardiac function in children with CHD.

In addition to providing tissue support, the cardiac ECM also acts as a signal transducer for intercellular communication. Specific ECM proteins interact with cells and play an important role in cell-cell signal transmission to control cell behavior [37,38]. We speculate that ECM component may be responsible for the cardiogenic differentiation of bone marrow c-kit+ progenitor cells. Thus proteomic detection of the patient-derived cardiac ECM composition was performed using label-free MS/MS. The proteomic analysis indicated that several ECM proteins were closely related to cardiac differentiation of c-kit+ cells in pediatric patients with CHD, including fibronectin, thrombospondin-1, vascular endothelial growth factor receptor 2, Alpha2-antiplasmin, and apolipoprotein E. It is well established that fibronectin plays a pivotal role in cell behavior, such as cell growth, survival and proliferation through interacting with integrin receptors and downstream signaling pathways [39,40]. In addition, thrombospondin-1, a matricellular protein, binds to cell surface receptors CD36 or CD47, modulating cell-ECM interactions, including focal adhesions. Also, thrombospondin-1 activates the pro-survival AKT signaling pathway through inhibiting matrix metalloproteinase activity, thus facilitating cell survival [41]. Nevertheless, more information regarding these ECM proteins and their roles in cardiac differentiation is needed to advance this field of functional scaffold. Future studies should attempt to elucidate the precise mechanisms responsible for the cardiac differentiation of c-kit+ cells guided by cardiac ECM scaffold.

5. Conclusion

We developed human cardiac tissue-derived scaffold using decellularization method. It was found that cardiac ECM scaffold improved functional behavior of bone marrow c-kit+ cells from patients with CHD, including promoting cell adhesion, survival and proliferation. More importantly, cardiac ECM scaffold directed cardiogenic differentiation of bone marrow c-kit+ cells by providing favorable microenvironment. Subsequent experiments using cardiac ECM scaffold to repair heart defects in animals are under way. The human derived cardiac ECM scaffold may be further applied to bone marrow c-kit+ cell-based cardiac tissue repair. Also, the appropriate microenvironment provided by the cardiac ECM may produce a new strategy for the study of cardiogenic differentiation of bone marrow c-kit+ cells. Furthermore, deep understanding on how the ECM regulates cell microenvironment may contribute to create intelligent scaffolds to repair damaged myocardium.

Conflict of interest

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This research was funded by the Science and Technology Foundation of Xuzhou (KC20155), the Outstanding Talent Research Funding of Xuzhou Medical University (D2016021).

Supplementary materials

The supplementary materials are available from https://dx-doi-org-s.web.bisu.edu.cn/10.3233/BME-211368.

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