Activation of Notch1 signalling promotes multi-lineage differentiation of c-KitPOS/NKX2.5POS bone marrow stem cells: implication in stem cell translational medicine

<jats:title>Abstract</jats:title><jats:sec>                         <jats:title>Introduction</jats:title>                         <jats:p>Transplantation of bone marrow mesenchymal stem cells (BMSCs) can repair injured hearts. However, whether BMSC populations contain cells with cardiac stem cell characteristics is ill-defined. We report here that Notch signalling can promote differentiation of c-Kit<jats:sup>POS</jats:sup>/NKX2.5<jats:sup>POS</jats:sup> BMSCs into cardiomyocyte-like cells.</jats:p>                      </jats:sec><jats:sec>                         <jats:title>Methods</jats:title>                         <jats:p>Total BMSCs were isolated from Sprague–Dawley rat femurs and c-Kit<jats:sup>POS</jats:sup> cells were purified. c-Kit<jats:sup>POS</jats:sup>/NKX2.5<jats:sup>POS</jats:sup> cells were isolated by single-cell cloning, and the presence of cardiomyocyte, smooth muscle cell (SMC), and endothelial cell differentiation markers assessed by immunofluorescence staining and semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis. Levels of c-Kit and Notch1–4 in total BMSCs and c-Kit<jats:sup>POS</jats:sup>/NKX2.5<jats:sup>POS</jats:sup> BMSCs were quantitated by flow cytometry. Following infection with an adenovirus over-expressing Notch1 intracellular domain (NICD), total BMSCs and c-Kit<jats:sup>POS</jats:sup>/NKX2.5<jats:sup>POS</jats:sup> cells were assessed for differentiation to cardiomyocyte, SMC, and endothelial cell lineages by immunofluorescence staining and real-time quantitative RT-PCR. Total BMSCs and c-Kit<jats:sup>POS</jats:sup>/NKX2.5<jats:sup>POS</jats:sup> cells were treated with the Notch1 ligand Jagged1 and markers of cardiomyocyte, SMC, and endothelial cell differentiation were examined by immunofluorescence staining and real-time quantitative RT-PCR analysis.</jats:p>                      </jats:sec><jats:sec>                         <jats:title>Results</jats:title>                         <jats:p>c-Kit<jats:sup>POS</jats:sup>/NKX2.5<jats:sup>POS</jats:sup> cells were present among total BMSC populations, and these cells did not express markers of adult cardiomyocyte, SMC, or endothelial cell lineages. c-Kit<jats:sup>POS</jats:sup>/NKX2.5<jats:sup>POS</jats:sup> BMSCs exhibited a multi-lineage differentiation potential similar to total BMSCs. Following sorting, the c-Kit level in c-Kit<jats:sup>POS</jats:sup>/NKX2.5<jats:sup>POS</jats:sup> BMSCs was 84.4%. Flow cytometry revealed that Notch1 was the predominant Notch receptor present in total BMSCs and c-Kit<jats:sup>POS</jats:sup>/NKX2.5<jats:sup>POS</jats:sup> BMSCs. Total BMSCs and c-Kit<jats:sup>POS</jats:sup>/NKX2.5<jats:sup>POS</jats:sup> BMSCs overexpressing NICD had active Notch1 signalling accompanied by differentiation into cardiomyocyte, SMC, and endothelial cell lineages. Treatment of total BMSCs and c-Kit<jats:sup>POS</jats:sup>/NKX2.5<jats:sup>POS</jats:sup> BMSCs with exogenous Jagged1 activated Notch1 signalling and drove multi-lineage differentiation, with a tendency towards cardiac lineage differentiation in c-Kit<jats:sup>POS</jats:sup>/NKX2.5<jats:sup>POS</jats:sup> BMSCs.</jats:p>                      </jats:sec><jats:sec>                         <jats:title>Conclusions</jats:title>                         <jats:p>c-Kit<jats:sup>POS</jats:sup>/NKX2.5<jats:sup>POS</jats:sup> cells exist in total BMSC pools. Activation of Notch1 signalling contributed to multi-lineage differentiation of c-Kit<jats:sup>POS</jats:sup>/NKX2.5<jats:sup>POS</jats:sup> BMSCs, favouring differentiation into cardiomyocytes. These findings suggest that modulation of Notch1 signalling may have potential utility in stem cell translational medicine.</jats:p>                      </jats:sec>

section_content: Stem cell transplantation is emerging as a promising method to repair heart injuries [1] [2] [3]. Stem cells are selfreplicating multipotent cells that can differentiate into a variety of cell types under certain conditions. Various types of stem cells, including bone marrow cells (BMCs), mesenchymal stem cells, haematopoietic stem cells, and adipose-derived stem cells, have been used in cellular therapies to repair damage following myocardial infarction (MI). Phase I and II clinical trials have shown that transplantation of adult BMCs in patients with ischaemic heart disease improves left ventricle function and infarct size even at long-term follow-up, compared with standard therapy [4]. However, several recent clinical trials (SWISS-AMI, CELLWAVE, and C-CURE) for MI therapy involving BMCs have produced conflicting results [5] [6] [7], leading to debate concerning the efficacy of BMCs in treating heart disease [8]. 

The discovery of endogenous stem cells within heart tissue, termed cardiac stem cells (CSCs), offers great potential for stem cell research [9]. CSCs have selfrenewal and differentiation capacities that are necessary and sufficient for MI repair [10]. The phase I clinical trials SCIPIO (ClinicalTrials. gov NCT00474461) and CADUCEUS (ClinicalTrials. gov NCT00893360) have been conducted using autologous CSCs [11, 12]. The feasibility, safety, and effectiveness of autologous CSC injection were assessed in these trials, with encouraging preliminary results evidenced by a reduction in the myocardial scar mass or improvement in the left ventricular ejection fraction following cell treatment. However, a major obstacle limiting the clinical application of endogenous CSCs is the requirement for heart tissues as a cellular source, which increases the risk of injury and complications. Furthermore, obtaining the desired cell numbers for in vivo transplantation is time consuming because heart tissue-derived CSCs grow slowly. There is therefore a need for an alternative and easily accessible cell source that can be substituted for endogenous CSCs. 

Mesenchymal stem cells are multipotent stem cells that can be easily obtained and handled, and which exhibit multilineage differentiation potential [13]. As ideal seed cells, mesenchymal stem cells have been widely used in tissue engineering, cell transplantation, and gene therapy. Mesenchymal stem cell transplantation contributes to the recovery of heart injuries, including those caused by MI, mainly through angiogenesis, paracrine signalling, activation of endogenous CSCs, and antiinflammatory effectsbut not differentiation [14]. In the C-CURE trial, bone marrow mesenchymal stem cells (BMSCs) were exposed to a cocktail of cardiogenic growth factors prior to cell transplantation, which promoted in vivo functions post transplantation [6]. This effect demonstrates that activating certain signalling pathways in stem cells can promote desirable biofunctions in vivo. The heterogeneity of BMSCs may account for their low efficiency of cardiomyocyte differentiation in vivo, thus limiting their long-term potential to repair heart injuries. The isolation of a specific subpopulation of MSCs with a tendency for cardiomyocyte differentiation under the influence of specific signalling would therefore be of great value to stem cell translational medicine. 

The Notch signalling pathway is highly conserved evolutionarily and operates in both vertebrates and invertebrates. Notch signalling pathways are activated by interactions between adjacent cells, and influence the regulation of cell, tissue, and organ differentiation and development [15]. The Notch signalling pathway includes receptors, ligands, DNA-binding proteins, and effector molecules. There are four receptors (Notch1 to Notch4) and five ligands (DII1, DII3, DII4, Jaggedl, and Jagged2) in mammalian cells. Notch signalling occurs close to the cell surface receptor upon Notch ligand binding, with the receptor releasing a notch receptor intracellular domain (NICD) from the cell membrane. This NICD translocates to the nucleus to regulate gene transcription, thereby influencing cell proliferation and differentiation [15]. Notch signalling is involved with cell fate decisions including proliferation, lineage commitment, and terminal differentiation in many types of adult stem cells [16]. In heart tissue-derived c-kit POS CSCs, activation of Notch1 signalling directly upregulates Nkx2. 5 expression, promoting cardiomyocyte commitment [17]. Because Notch signalling has a remarkable cell-context dependency [18], we investigated whether Notch signalling affects c-Kit POS BMSCs. 

In the present study, we assessed whether populations of BMSCs included c-Kit POS cells that displayed CSC differentiation properties and evaluated the effectiveness of Notch signalling activation for promoting differentiation of c-Kit POS BMSCs. We identified c-Kit POS /NKX2. 5 POS BMSCs in total BMSC populations, and activation of Notch1 promoted multilineage differentiation of these cells. These findings suggest that targeted modulation of Notch1 signalling may be useful in stem cell translational medicine.
section_name: Introduction

section_name: Methods

section_content: Sprague-Dawley rats (6 weeks old, both genders) were purchased from the Experimental Animal Center of Guangdong Medical University, Zhanjiang, China. All animal procedures were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of Guangdong Medical University. Unsorted total BMSCs were isolated from rat femurs according to a previously described protocol [19, 20]. Briefly, rats were sacrificed with chloroform anaesthesia, and femurs were aseptically removed and soaked in 75% ethanol for 10 minutes. Bone marrow was then flushed out using Dulbecco's modified Eagle's medium (DMEM; Hyclone, Beijing, China) supplemented with 2% (v/v) foetal bovine serum (FBS; Gibco, Grand Island, NY, USA). The released cells were collected, and the red blood cells in collected bone marrow-derived primary cells were removed using red blood cell lysis buffer (Sigma-Aldrich, Shanghai, China). Cell pellets were washed three times with phosphate-buffered saline (PBS) and cells were resuspended in DMEM supplemented with 10% FBS, 100 U/ml recombinant rat leukaemia inhibitory factor (LIF3010; EMD Millipore, Billerica, MA, USA), 100 U/ml penicillin, and 0. 1 μg/ml streptomycin, and were maintained at 37°C in saturated humidity with 5% carbon dioxide. After 2 days, nonadherent cells were removed, and medium changes were performed every 3 days thereafter. Cell morphology was monitored under an inverted microscope. Fourth passage cells were used for differentiation experiments. Total BMSCs (3 × 10 3 cells/cm 2 ) were seeded in six-well plates. For osteogenesis, cells were treated with inducing medium (RASMX-90021; Cyagen Biosciences, Guangzhou, China) for 3 weeks. Cells were then fixed with 4% formaldehyde for 30 minutes and stained with Alizarin Red. For adipocyte differentiation, cells were maintained in adipocyte genesis medium (RASMX-90031; Cyagen Biosciences) for 3 weeks. Cells were then stained with Oil Red O [21]. For smooth muscle cell (SMC) differentiation, cells grown on coverslips were treated with medium containing 10 ng/ml transforming growth factor-β1 (100-21; PeproTech, Rocky Hill, NJ, USA) for 8 days and then subjected to immunofluorescence staining for the SMC marker smooth muscle myosin heavy chain. 

Isolation, cloning, morphology, and identification of c-Kit POS /NKX2. 5 POS BMSCs Third-passage BMSCs were harvested and c-Kit POS cells isolated using a magnetic activated cell sorting (MACS) system (Miltenyi Biotec, Bergisch Gladbach, Germany) as described previously [22, 23]. Diluted c-Kit POS cells were seeded in six-well plates and maintained in DMEM supplemented with 10% FBS, 0. 2 mmol/l glutathione (G5763; Sigma Aldrich), 2. 5 U/ml erythropoietin (287-TC-500; R&D Systems, Minneapolis, MN, USA), and 10 ng/ml recombinant fibroblast growth factor (400-29; PeproTech). Cellular morphology of single cell-derived clones was monitored at 3, 7, 14, 21, and 28 days, and confluent cells were used to assess marker expression by immunofluorescence staining and RT-PCR analysis. Cells that were positive for c-Kit and early cardiac marker NKX2. 5, but negative for markers of mature cardiomyocytes (α-sarcomeric actin, cardiac troponin T (cTnT)), SMCs (SM22α), and endothelial cells (von Willebrand factor (vWF)), were used in subsequent experiments. These cells were classed as c-Kit POS /NKX2. 5 POS BMSCs. Differentiation of c-Kit POS /NKX2. 5 POS BMSCs into osteocytes, adipocytes, and SMCs was monitored as per the total BMSCs described above.
section_name: Isolation, morphology, and functional identification of rat BMSCs

section_content: NICD cDNA (5,468 to 7,820 nucleotides; NM_001105721) was obtained from the cDNA library of Genechem (Shanghai, China) with the following primers: 5′-AGG TCG ACT CTA GAG GAT CCC GCC ACC ATG CGC AAG CGC AGG CGG CA-3′ and 5′-TCC TTG TAG TCC ATA CCC TTA AAT GCC TCT GGA ATG TGG G-3′. The 2,399 base pair PCR product was cloned into a linearised adenovirus plasmid GV314 (Genechem) with T4 DNA ligase and transfected into competent Escherichia coli cells. Positive clones were selected by ampicillin resistance and then sequenced by ABI3730 sequencing analysis (Invitrogen, Shanghai, China). The NICD overexpression adenovirus (NICD-Ad) was packaged in HEK293T cells and purified with an Adeno-X™ Virus Purification Kit (BD Biosciences, San Jose, CA, USA). The endpoint dilution method was used to determine the viral titre. Adenovirus particles expressing a scrambled sequence (NC-Ad; purchased from Genechem) served as a negative control. 

For cell infection, total BMSCs and c-Kit POS /NKX2. 5 POS BMSCs were seeded in six-well plates with or without coverslips. After 24 hours, the NICD-Ad or NC-Ad virus was added to the cells at a multiplicity of infection of 100 in the presence of 4 μg/ml polybrene. Cells not transfected with virus were classified MOCK. Twenty-four hours after infection, medium was changed to complete DMEM and then changed every 3 days. After 72 hours, the viral infection efficiency was assessed under fluorescence microscopy. Cells were harvested for experiments at day 8 after infection.
section_name: NICD overexpression by adenovirus-mediated transduction

section_content: Total BMSCs and c-Kit POS /NKX2. 5 POS BMSCs (2 × 10 6 cells) were fixed in cold 70% ethanol overnight. After washing with PBS, cells were incubated with primary antibodies against Notch1 to Notch4 and c-Kit at room temperature for 1 hour. Following three washes with PBS, cells were incubated with fluorescein isothiocyanateconjugated or phycoerythrin-conjugated IgG in the dark at room temperature for 30 minutes. Samples were then subjected to flow cytometric analysis using a BD FACSCanto II (BD Biosciences). Data were analysed with FACSDiva software (BD Biosciences). 

Exogenous Jagged-1-induced total BMSC and c-Kit POS / NKX2. 5 POS BMSC differentiation Total BMSCs and c-Kit POS /NKX2. 5 POS BMSCs were maintained in complete DMEM and seeded in six-well plates with coverslips or 6 cm dishes. For induced differentiation, five treatment groups were established and cultured in DMEM + 10% FBS: Jagged1, with 2. 5 μg/ml Jagged1 Fc Chimera (99-JG; R&D Systems); IgG, with 2. 5 μg/ml IgG (14131; Sigma Aldrich); Notch1 inhibitor N-(2S-(3,5-difluorophenyl)acetyl)-L-alanyl-2-phenyl-1,1dimethylethyl ester-glycine (DAPT), with 2. 5 μg/ml Jagged1 Fc Chimera and 10 μmol/ml DAPT (D5942; Sigma Aldrich) dissolved in dimethyl sulfoxide; DMSO, with ≤0. 03% dimethyl sulfoxide (v/v); and MOCK, with medium alone. Medium was changed every 3 days. After 8 days, cells grown on coverslips were subjected to immunofluorescence staining, while cells grown in dishes were subjected to mRNA expression analysis.
section_name: Determination of Notch receptors and c-Kit expression by flow cytometry

section_content: Total BMSCs and c-Kit POS /NKX2. 5 POS BMSCs were subjected to mRNA expression analysis. Total RNA was extracted with RNAiso Plus (D9108B; TaKaRa, Dalian, China). To generate cDNA, total RNA (1 μg) was reverse transcribed using an RT kit (DRR047A; TaKaRa) with an oligo(dT18) primer. Quantitative PCR was conducted using a LightCycler480 II instrument (Roche China, Guangzhou, China), and semi-quantitative PCR was performed using a Mastercycler® Gradient Thermal cycler (Eppendorf, Wesseling-Berzdorf, Germany). Primers were synthesised by Sangon Biotech (Shanghai, China) and are listed in Additional file 1. For quantitative PCR, reaction volumes of 20 μl included 10 μl SYBR® Premix Ex Taq™ (catalogue number DRR820A; Takara), 0. 8 μl each primer (10 μM), 2 μl cDNA, and 6. 4 μl ddH 2 O. PCR amplification conditions were as follows: denaturation at 95°C for 30 seconds, followed by 45 cycles of 95°C for 5 seconds, and 60°C for 20 seconds. The relative amount of target mRNAs was determined using cycle threshold values and plotted as the fold change compared with control groups (2 -ΔΔCT method). Semi-quantitative PCR conditions were 95°C for 5 minutes, followed by 31 cycles at 95°C for 45 seconds, 58°C for 45 seconds, and 72°C for 1 minute. PCR products were separated by 1. 5% agarose gel electrophoresis and visualised under ultraviolet light using a gel documentation system (Bio-Rad, Hercules, CA, USA). For all PCR analyses, the expression level of β-actin served as a loading control.
section_name: RNA isolation, reverse transcription, and PCR

section_content: Cells grown on coverslips were subjected to indirect immunofluorescence to identify the expression and location of cleaved Notch1 (NICD) and differentiation markers. Following overnight incubation with primary antibodies at 4°C, cells were washed three times with PBS. Cells were then incubated with fluorescein isothiocyanateconjugated or phycoerythrin-conjugated IgG (H + L; Proteintech, Wuhan, China) at room temperature for 30 minutes. Nuclei were counterstained with 4′,6diamidino-2-phenylindole (5 μg/ml) at room temperature for 15 minutes. Images were captured under a laser scanning confocal microscope (TCS SP5 II; Leica, Wetzlar, Germany). Primary antibodies used are listed in Additional file 2.
section_name: Immunofluorescence staining

section_content: Statistical analyses were performed using PRISM Software (Version 5; GraphPad Software, San Diego, CA, USA). Data are expressed as the mean ± standard deviation. Student's t test was used to analyse differences between two groups. For multiple group comparisons, analysis of variance was carried out followed by the Student-Newman-Keuls test. P <0. 05 was considered statistically significant.
section_name: Statistical analysis

section_content: Isolation of c-Kit POS /NKX2. 5 POS cells from total BMSCs and assessment of cellular morphology Total BMSCs are a heterogeneous cell population including various phenotypical cell subpopulations [24]. We successfully isolated BMSCs from rat femurs according to a protocol described previously [19, 20]. Unsorted total BMSCs displayed a typical fibroblast-like morphology (Figure 1A, B, C ). c-Kit is a receptor tyrosine kinase which is bound by stem cell factor to activate signalling pathways crucial for stem cell functions such as heart tissue-derived CSC migration [25]. Furthermore, c-Kit is a relatively stable stem cell marker [26] which can be reliably detected in heart tissue-derived CSCs even after 40 passages [27]. We employed MACS to isolate c-Kit POS cells from total BMSCs. Single-cell cloning was then used to establish c-Kit POS and NKX2. 5 POS cells that were negative for GATA-4, cTnT, SM22α, and vWF expression, as determined by immunofluorescence staining and semi-quantitative RT-PCR (Figure 2A, C ). These cells were defined as c-Kit POS /NKX2. 5 POS BMSCs, and single c-Kit POS /NKX2. 5 POS BMSCs could form clones well (see Additional file 3). c-Kit POS /NKX2. 5 POS BMSCs exhibited a fibroblast-like morphology similar to that of total BMSCs, and there were no obvious morphological differences between the two cell types (Figure 1 ). However, c-Kit POS /NKX2. 5 POS BMSCs grew slower than the total BMSCs, but faster than heart tissue-derived CSCs (see Additional file 4). c-Kit and Notch receptor expression by total BMSCs and c-Kit POS /NKX2. 5 POS
section_name: Results

section_content: We performed flow cytometry to analyse the expression of c-Kit and Notch1 to Notch4 receptors on total BMSCs and c-Kit POS /NKX2. 5 POS BMSCs. Before MACS sorting, 7. 3% of total BMSCs were c-Kitpositive, while 84. 4% of cells were c-Kit-positive following a single round of sorting (Figure 2B ). This result indicates that MACS is a suitable method for enriching c-Kit-positive cell populations from BMSCs. Varied expression levels of Notch receptors were detected in total BMSCs and c-Kit POS /NKX2. 5 POS BMSCs (Figure 2D ). Notch1 was the predominantly expressed form of receptor in both total BMSC and c-Kit POS /NKX2. 5 POS BMSC populations, Following sorting, Notch1-positive cells increased from 67. 2% in total BMSCs to 77. 8% in c-Kit POS /NKX2. 5 POS BMSCs (Figure 2D ). These results suggest that Notch1 signalling plays a crucial role in the biology of c-Kit POS / NKX2. 5 POS BMSCs, which supports our rationale for investigating Notch1. Total BMSCs and c-Kit POS /NKX2. 5 POS BMSCs were treated with specific agents to induce osteogenic, adipogenic, and SMC differentiation, and differentiated cells expressed typical markers of differentiation (Figure 3 ). Both total BMSCs and c-Kit POS /NKX2. 5 POS BMSCs therefore displayed multilineage differentiation potential, suggesting that these cells could be induced to differentiate into desired cell types by other cues. 

Binding of ligand Jagged1 to the Notch1 receptor releases NICD to the nucleus. NICD forms a protein complex with CSL(CBF1/Su(H)/Lag-1), which then turns the co-repressor into a co-activator, subsequently activating transcription of target genes [15]. We constructed an adenovirus to overexpress NICD (NICD-Ad) independent Figure 4 ). These results suggest that forced expression of Notch1-NICD in total BMSCs and c-Kit POS /NKX2. 5 POS BMSCs led to activation of Notch1 signalling. 

We next evaluated the expression of markers of cardiomyocyte (NKX2. 5 and cTnT), SMC (SM22α), and endothelial cell (vWF) differentiation. Protein and mRNA levels of NKX2. 5, cTnT, SM22α, and vWF were upregulated in NICD-overexpressing total BMSCs and c-Kit POS / NKX2. 5 POS BMSCs (Figure 5 ). These data indicate that forced expression of NICD in total BMSCs and c-Kit POS / NKX2. 5 POS BMSCs activated Notch1 signalling and induced multilineage differentiation, supporting a role for Notch1 signalling in the differentiation of BMSCs. Interestingly, we also found that forced activation of Notch1 in c-Kit POS /NKX2. 5 POS BMSCs led to enhanced osteogenic and adipogenic differentiation (see Additional file 5). This probably reflects the complexity of Notch1 signalling in stem cell differentiation.
section_name: BMSCs

section_content: We treated total BMSCs and c-Kit POS /NKX2. 5 POS BMSCs with Jagged1 to activate Notch1 signalling, as Notch1 was the predominant Notch receptor expressed by these cells. 

(See figure on previous page. ) Figure 2 Phenotypic identification of c-Kit POS /NKX2. 5 POS bone marrow mesenchymal stem cells. (A) Confluent, c-Kit POS /NKX2. 5 POS bone marrow mesenchymal stem cells (BMSCs) were passaged and third-passage cells were subjected to immunofluorescence staining for c-Kit, NKX2. 5, and α-sarcomeric actin (α-SA). Unsorted total BMSCs served as control. (B) Assessment of c-Kit expression in total BMSCs before and after magnetic activated cell sorting by flow cytometric analysis. (C) Semi-quantitative RT-PCR analysis of markers for stem cells (c-Kit), cardiomyocytes (NKX2. 5, GATA-4, and cardiac troponin T (cTnT)), smooth muscle cells ( SM22α) and endothelial cells (von Willebrand factor (vWF)). A sample from a neonatal Sprague-Dawley rat heart was used as positive control. (D) Flow cytometry was performed to detect the presence of Notch1 to Notch4 receptors. For immunofluorescence staining, target proteins were detected with fluorescein isothiocyanate (FITC)-conjugated or phycoerythrin (PE)-conjugated IgG. Images were captured by fluorescence microscopy. Scale bar: 50 μm. For semi-quantitative RT-PCR analysis, β-actin mRNA was used as an internal control. For flow cytometric analysis, isotype control IgG was used to set the threshold. Exogenous recombinant Jagged1 activated Notch1 signalling in both total BMSCs and c-Kit POS /NKX2. 5 POS BMSCs as evidenced by upregulation of Hes1 and NICD, while the Notch1 inhibitor DAPT attenuated this effect (Figure 6 ). Jagged1-induced Notch1 activation in c-Kit POS /NKX2. 5 POS BMSCs was stronger than that in total BMSCs, as indicated by increased immunofluorescence signals and Hes1 levels in Jagged1-treated cells (Figure 6 ). 

We next assessed the differentiation of total BMSCs and c-Kit POS /NKX2. 5 POS BMSCs exposed to Jagged1. Jagged1-induced Notch1 signalling led to enhanced NKX2. 5, cTnT, SM22α, and vWF expression, while inhibition of Notch1 signalling by DAPT reduced their expression at both mRNA and protein levels (Figure 7 ). These findings demonstrate that Jagged1-triggered Notch1 signalling induced differentiation of total BMSCs and c-Kit POS /NKX2. 5 POS BMSCs into cardiomyocyte, SMC, and endothelial cell lineages. 

Jagged1-activated Notch1 signalling predominantly induces cardiomyocyte differentiation in c-Kit POS /NKX2. 5 POS
section_name: Jagged1-triggered Notch1 signalling activation induces multi-lineage differentiation

section_content: Following exposure of c-Kit POS /NKX2. 5 POS BMSCs to exogenous Jagged1, the increases in expression of cardiomyocyte and endothelial cell lineage markers were much stronger than those in total BMSCs (Figure 7A ). There was no obvious difference in expression of the SMC differentiation marker SM22α (Figure 7A ). These findings suggest that c-Kit POS /NKX2. 5 POS BMSCs are more responsive to Notch1 signalling stimuli than total BMSCs. We further evaluated the tendency of Jagged1-treated c-Kit POS /NKX2. 5 POS BMSCs to differentiate into cardiomyocytes, SMCs, and endothelial cells by counting cTnT-positive, SM22α-positive, and vWF-positive cells in 10 representative fields of view to determine the percentages of positive cells in each lineage. Jagged1- induced activation of Notch1 signalling in c-Kit POS / NKX2. 5 POS BMSCs facilitated predominately cardiomyocyte differentiation, while differentiation into endothelial and SMC lineages was much weaker (Figure 8 ).
section_name: BMSCs

section_content: Stem cell therapy is emerging as a promising treatment strategy for MI. Current investigations are endeavouring to identify the most suitable type of stem cell for MI treatment [28, 29]. The feasibility, safety, and effectiveness of stem cell transplantation based on heart tissuederived CSCs have been demonstrated in animal models of MI. Recent results of several phase I clinical trials have also have provided encouraging results [11, 12]. However, the SWISS-AMI trial has generated conflicting results [5]. A potential explanation for the inconsistent results of these clinical trials may be the variability of cell types used [8]. Ellison and colleagues have highlighted the necessity of heart tissue-derived CSCs for MI repair [10]. However, considering the potential for damage caused by the acquisition of heart tissue for CSCs, there is a need for an alternative cell source to substitute for heart tissue-derived CSCs. 

Compared with heart tissue-derived CSCs, BMSCs are able to be handled and expanded more easily [14]. Furthermore, their phenotype is not influenced by confluency during culture expansion [30]. Additionally, a meta-analysis concluded that BMSCs have promising prospects for MI treatment [31]. BMSCs exhibit a multilineage in vitro differentiation potential, including cardiomyocytes. However, the functions of BMSCs in vivo are mainly mediated in a paracrine manner [32] and these cells do not directly differentiate into functional cardiomyocytes [33]. A possible reason for this indirect effect may be the multiple subpopulations of cells with different differentiation potentials that constitute total BMSCs. Screening purified BMSCs for highly-efficient differentiation to cardiomyocytes could therefore be of great benefit to stem cell-based cardiac repair. 

Because Notch signalling plays a critical role in stem cell differentiation into cardiomyocytes [17, 34, 35], we examined whether forced activation of Notch1 signalling could efficiently induce cardiac differentiation of BMSCs. Therefore, we infected unsorted total BMSCs with a recombinant NICD-expressing adenovirus. Forced expression of NICD in BMSCs promoted multilineage differentiation, including to cardiomyocytes, suggesting that activation of the Notch1 signalling pathway is functionally relevant in BMSC differentiation. 

We next examined the function of Notch1 signalling on the c-Kit POS /NKX2. 5 POS subtype of BMSCs. We successfully isolated c-Kit POS cells from total BMSCs using MACS as described previously [22, 23, 25]. Cell clones positive for NKX2. 5 were then successfully derived from single c-Kit POS cells. Finally, c-Kit POS /NKX2. 5 POS cells were found to be negative for markers of adult cardiomyocyte (α-sarcomeric actin, cTnT), SMC (SM22α), and endothelial cell (vWF) differentiation. The purity of c-Kit POS /NKX2. 5 POS BMSCs after a single MACS reached 84. 4%, significantly higher than that of the 7. 3% in total BMSCs before sorting. 

The use of c-Kit POS BMSCs in MI treatment has been proposed by previous reports [36, 37] ; however, we further investigated the c-Kit POS /NKX2. 5 POS subpopulation and showed that, of the four known Notch receptors, these cells predominantly expressed Notch1. This observation led us to examine the effects of ligand-induced activation of Notch1 signalling on differentiation of c-Kit POS /NKX2. 5 POS BMSCs. c-Kit POS /NKX2. 5 POS BMSCs proliferated slower than total BMSCs, but faster than c-Kit POS CSCs derived from rat hearts, indicating that c-Kit POS /NKX2. 5 POS BMSCs can be expanded more easily than heart tissue-derived c-Kit POS CSCs. 

Notch signalling contributes to stem cell quiescence, proliferation, and differentiation depending on the context in which the signal is activated [38]. Boni and colleagues reported that Notchl directly regulates NKX2. 5 promoter activity in mouse heart-derived CSCs, thereby positively regulating cardiomyocyte differentiation [17]. Chen and colleagues demonstrated that activation of Notch signalling in mouse cardiosphere cellsa heterogeneous progenitor cell populationpromotes SMC differentiation through a J kapparecombining binding protein-dependent signalling pathway in vitro [39]. However, activation of Notch signalling in embryonic stem cells has been reported to produce opposite results [40]. A recent study by Matsuda and colleagues proposed that the cell density of heart tissue-derived CSC cultures influences Notch1 activation status and differentiation potential, influencing in vivo CSC function and potential utility for MI therapy [41]. These inconsistencies highlight the need for further efforts to clarify the exact role of Notch1 signalling in c-Kit POS / NKX2. 5 POS BMSC differentiation. 

We found that Notch1 was the predominant Notch receptor form expressed by both total BMSCs and c-Kit POS /NKX2. 5 POS BMSCs. We therefore treated these two types of cells with exogenous Jagged1 to mimic the binding of ligand and receptor. As shown in our pilot investigation, 2. 5 μg/ml exogenous Jagged1 maximised Hes1 mRNA expression (see Additional file 6); we therefore treated the cells with 2. 5 μg/ml Jagged1 in the subsequent experiments. Jagged1 activated Notch1 signalling and promoted differentiation of both total BMSCs and c-Kit POS /NKX2. 5 POS BMSCs into cardiomyocyte, SMC, and endothelial cell lineages. Furthermore, expression levels of cardiomyocyte and endothelial cell lineage markers in cells derived from c-Kit POS /NKX2. 5 POS BMSCs were greater than those of cells derived from total BMSCs. Moreover, differentiation of the cardiomyocyte lineage was favoured over differentiation of endothelial cell and SMC lineages in c-Kit POS /NKX2. 5 POS BMSCs. This suggests that c-Kit POS /NKX2. 5 POS BMSCs preferentially differentiate into cardiac myocytes following activation of Notch1 signalling. These in vitro results support a previous report which found that Notch1 signalling in BM-derived cells is critical for cardiac repair [42]. Activation of Notch1 signalling therefore promotes c-Kit POS /NKX2. 5 POS BMSC differentiation, suggesting that targeting Notch1 signalling may be beneficial for stem cell translational medicine in MI repair. 

At least two phase I clinical trials have demonstrated that transplantation of homogeneous heart-derived CSCs to patients is beneficial for heart function recovery [5] [6] [7]. However, the isolation of CSCs from heart tissue is potentially damaging and in vitro maintenance of cell populations is complex and time consuming. An improved understanding of the origin of CSCs should aid progress towards effective, viable treatment options. Presently, little is known concerning the origin of heart-derived CSCs. Heart resident CSCs may be either cells that have resided there from foetal life onwards or could come from the bone marrow or circulation [43]. An early report demonstrated that sex-mismatched heart transplantation patients possessed recipient-derived Y chromosomes in the nuclei of some cardiomyocytes, suggesting that bone marrow or circulation-derived stem cells had participated in the regenerative process [44]. We aimed to identify these alternative cells from bone marrow that possess properties of heart-derived CSCs. 

We found that c-Kit POS /NKX2. 5 POS cells within total BMSC populations display some properties of CSCs, being positive for NKX2. 5 but negative for markers of adult cardiomyocytes, SMCs, and endothelial cells. Guo and colleagues proposed the existence of bone marrowderived CSCs that express c-Kit and NKX2. 5 [45]. Furthermore, these bone marrow-derived CSCs could repair injured hearts following transplantation to MI-model animals. Whether our current c-Kit POS /NKX2. 5 POS BMSCs (See figure on previous page. ) Figure 7 Jagged1 activation of Notch1 signalling induces multilineage differentiation of total and c-Kit POS /NKX2. 5 POS bone marrow mesenchymal stem cells. Total bone marrow mesenchymal stem cells (BMSCs) and c-Kit POS /NKX2. 5 POS BMSCs were classified into Jagged1, IgG, N-(2S-(3,5-difluorophenyl)acetyl)-L-alanyl-2-phenyl-1,1-dimethylethyl ester-glycine (DAPT), dimethyl sulfoxide (DMSO), and MOCK treatment groups (see Methods). After 8 days, the expression of differentiation markers for cardiomyocytes (NKX2. 5 and cardiac troponin T (cTnT)), smooth muscle cells (SM22α), and endothelial cells (von Willebrand factor (vWF)) were examined by (A) real-time quantitative RT-PCR and (B) immunofluorescence staining. Triplicate experiments were performed for real-time quantitative RT-PCR analysis (n = 3). *P <0. 05, **P <0. 01, ## P <0. 01, ### P <0. 001 versus other associated groups. For immunofluorescence staining, target proteins were detected with phycoerythrin-conjugated or fluorescein isothiocyanate-conjugated IgG. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Representative images were captured by confocal microscopy. Scale bar: 50 μm. represent the same cell population as those identified by Guo and colleagues is interesting and warrants further study. 

Notch1 signalling plays a pivotal role in the fate of heartderived CSCs [17]. We found that c-Kit POS /NKX2. 5 POS BMSCs were predisposed to Notch1 signalling-induced multilineage differentiation, with a preference for cardiac differentiation when compared with total BMSCs. Further clarification of the differences between c-Kit POS / NKX2. 5 POS BMSCs and c-Kit POS heart-derived CSCs may facilitate the clinical use of BMSCs. 

Some limitations were present in our investigation. We provide in vitro evidence concerning Notch1 signalling in the differentiation of c-Kit POS /NKX2. 5 POS BMSCs. However, the effects of Notch1 signalling activation in c-Kit POS /NKX2. 5 POS BMSCs in an in vivo setting mimicking transplantation for repair of injured heart tissue post MI should be assessed in future studies. Additionally, more differentiation markers could be examined to better identify cell differentiation commitment and changes to cellular electrophysiological functions. Future studies should also focus on the optimal timing and degree of Notch signalling activation to promote cardiomyocyte differentiation of c-Kit POS /NKX2. 5 POS BMSCs. The role of crosstalk between Notch and other signalling pathways should also be considered.
section_name: Discussion

section_content: Our study demonstrates the existence of c-Kit POS / NKX2. 5 POS cells within total BMSC populations. These cells exhibit unique properties in colonic, proliferative, and multilineage differentiation potentials. c-Kit POS / NKX2. 5 POS BMSCs are predisposed to Notch1-induced cell differentiation when compared with total BMSCs. Targeted activation of Notch1 signalling in c-Kit POS / NKX2. 5 POS BMSCs leads to multilineage differentiation, suggesting that Notch1 signalling is a potential target for stem cell translational medicine research.
section_name: Conclusions

section_name: Acknowledgements

section_content: The authors thank RJ Li for her contributions of some reagents/materials to the research. They thank WQ Wu for technical support in laser scanning confocal microscopy. They are also grateful to XG Wang for technical support in flow cytometry. The authors thank YZ Lu for critical comments of the manuscript. This study was supported by grants from the National Natural Science Foundation of China ( 81160020 and 81170121 ), the Key project of Chinese Ministry of Education ( 212137 ), the Technology Innovation Project of Department of Education of Guangdong Province ( 2013KJCX0088 ), the grants of Hainan provincial Special Fund for Social Development ( SF201417 ), Hainan International Cooperation Project ( HJHZ2013-06 ), and the Excellent Graduate Student Training Program of Guangdong Medical University ( YS2014013 ).
section_name: Acknowledgements

section_name: Additional files

section_content: Additional file 1: is Table S1 presenting the primers used for PCR. bp, base pairs. Additional file 2: is Table S2 presenting the primary antibodies. 

Additional file 3: is Figure S1 showing representative images of clones derived from cell of c-Kit POS /NKX2. 5 POS BMSCs. c-Kit POS / NKX2. 5 POS BMSCs were diluted into single-cell suspension and plated in six-well plates allowing it to form clones. Left panel, a small clone (the number of cells is <50), magnification: ×200; Right panel, two adjacent larger clones (the number of cells is >50), magnification: ×100. 

Additional file 4: is Figure S2 showing the growth curve of total BMSCs, c-Kit POS /NKX2. 5 POS BMSC and cardiac c-Kit POS CSC. Total BMSCs, c-Kit POS /NKX2. 5 POS BMSCs and cardiac c-Kit POS CSCs were plated in six-well plates allowing cells to proliferate, and then total cell numbers were counted every day for consecutive 7 days. For isolation of cardiac CSCs, hearts form neonatal rat were used, and c-Kit POS cells sorted from collagenase and trypsin-treated neonatal rat heart tissues by MACS were deemed c-Kit POS CSCs. ***P <0. 001 versus other groups. Each cell line was repeated in three wells on every time point (n = 3). 

Additional file 5: is Figure S3 showing the adipogenic and osteogenic differentiation in total BMSCs and c-Kit POS /NKX2. 5 POS BMSCs. (A) Total BMSCs and c-Kit POS /NKX2. 5 POS BMSCs were induced to adipogenic and osteogenic differentiation using special inducing media for 3 weeks, and then cells were subjected to quantitative RT-PCR analysis of markers for adipocyte lineage (PPARγ2, FABP2) and osteoblast lineage (ALP, osteopontin). Data depicted as mean ± standard deviation from three independent experiments. (B) c-Kit POS /NKX2. 5 POS BMSCs were infected with NICD-Ad and NC-Ad at multiplicity of infection = 100; cells without adenovirus infection was set as MOCK controls. Eight days post adenovirus infection, samples were used to quantitative RT-PCR analysis of marker for osteogenic differentiation (ALP) and adipogenic differentiation (PPARγ2) expression. Data depicted as mean ± standard deviation from three independent experiments. **P <0. 01 versus other groups; ### P <0. 001 versus other groups. 

Additional file 6: is Figure S4 showing the effects of Jagged1 on Hes1 expression. c-Kit POS /NKX2. 5 POS BMSCs were seeded onto six-well plates. After cell recovery for a whole night, completed media containing recombinant Jagged1 (final concentration, 0, 1. 25, 2. 5 and 5. 0 μg/ml) was added. After 8 days post Jagged1 treatment, samples were used to quantitative RT-PCR analysis Hes1 (target for judgement of Notch1 activation) expression. Data depicted as mean ± standard deviation from three independent experiments. *** P <0. 001 versus other groups.
section_name: Additional files

section_content: The authors declare that they have no competing interests. 

Authors' contributions WJ, JG, and ZS conceived and coordinated the study. RD, XJ, YH, ZW, and WJ carried out the experiments. HJ and SZ collected and analysed the data. XJ, RD, and SZ contributed reagents/materials. WJ and ZS wrote the manuscript. All authors read and approved the final manuscript.
section_name: Competing interests