- Open Access
Activation of Notch1 signalling promotes multi-lineage differentiation of c-KitPOS/NKX2.5POS bone marrow stem cells: implication in stem cell translational medicine
Stem Cell Research & Therapy volume 6, Article number: 91 (2015)
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-KitPOS/NKX2.5POS BMSCs into cardiomyocyte-like cells.
Total BMSCs were isolated from Sprague–Dawley rat femurs and c-KitPOS cells were purified. c-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS BMSCs were quantitated by flow cytometry. Following infection with an adenovirus over-expressing Notch1 intracellular domain (NICD), total BMSCs and c-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS 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.
c-KitPOS/NKX2.5POS cells were present among total BMSC populations, and these cells did not express markers of adult cardiomyocyte, SMC, or endothelial cell lineages. c-KitPOS/NKX2.5POS BMSCs exhibited a multi-lineage differentiation potential similar to total BMSCs. Following sorting, the c-Kit level in c-KitPOS/NKX2.5POS BMSCs was 84.4%. Flow cytometry revealed that Notch1 was the predominant Notch receptor present in total BMSCs and c-KitPOS/NKX2.5POS BMSCs. Total BMSCs and c-KitPOS/NKX2.5POS BMSCs overexpressing NICD had active Notch1 signalling accompanied by differentiation into cardiomyocyte, SMC, and endothelial cell lineages. Treatment of total BMSCs and c-KitPOS/NKX2.5POS BMSCs with exogenous Jagged1 activated Notch1 signalling and drove multi-lineage differentiation, with a tendency towards cardiac lineage differentiation in c-KitPOS/NKX2.5POS BMSCs.
c-KitPOS/NKX2.5POS cells exist in total BMSC pools. Activation of Notch1 signalling contributed to multi-lineage differentiation of c-KitPOS/NKX2.5POS BMSCs, favouring differentiation into cardiomyocytes. These findings suggest that modulation of Notch1 signalling may have potential utility in stem cell translational medicine.
Stem cell transplantation is emerging as a promising method to repair heart injuries [1-3]. Stem cells are self-replicating 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 . However, several recent clinical trials (SWISS-AMI, CELLWAVE, and C-CURE) for MI therapy involving BMCs have produced conflicting results [5-7], leading to debate concerning the efficacy of BMCs in treating heart disease .
The discovery of endogenous stem cells within heart tissue, termed cardiac stem cells (CSCs), offers great potential for stem cell research . CSCs have self-renewal and differentiation capacities that are necessary and sufficient for MI repair . 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 . 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 anti-inflammatory effects – but not differentiation . 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 . 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 . 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 . Notch signalling is involved with cell fate decisions including proliferation, lineage commitment, and terminal differentiation in many types of adult stem cells . In heart tissue-derived c-kitPOS CSCs, activation of Notch1 signalling directly upregulates Nkx2.5 expression, promoting cardiomyocyte commitment . Because Notch signalling has a remarkable cell-context dependency , we investigated whether Notch signalling affects c-KitPOS BMSCs.
In the present study, we assessed whether populations of BMSCs included c-KitPOS cells that displayed CSC differentiation properties and evaluated the effectiveness of Notch signalling activation for promoting differentiation of c-KitPOS BMSCs. We identified c-KitPOS/NKX2.5POS 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.
Isolation, morphology, and functional identification of rat BMSCs
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 × 103 cells/cm2) 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 . 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-KitPOS/NKX2.5POS BMSCs
Third-passage BMSCs were harvested and c-KitPOS cells isolated using a magnetic activated cell sorting (MACS) system (Miltenyi Biotec, Bergisch Gladbach, Germany) as described previously [22,23]. Diluted c-KitPOS 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-KitPOS/NKX2.5POS BMSCs. Differentiation of c-KitPOS/NKX2.5POS BMSCs into osteocytes, adipocytes, and SMCs was monitored as per the total BMSCs described above.
NICD overexpression by adenovirus-mediated transduction
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-KitPOS/NKX2.5POS 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.
Determination of Notch receptors and c-Kit expression by flow cytometry
Total BMSCs and c-KitPOS/NKX2.5POS BMSCs (2 × 106 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 isothiocyanate-conjugated 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-KitPOS/NKX2.5POS BMSC differentiation
Total BMSCs and c-KitPOS/NKX2.5POS 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-1,1-dimethylethyl 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.
RNA isolation, reverse transcription, and PCR
Total BMSCs and c-KitPOS/NKX2.5POS 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 ddH2O. 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−∆∆CT 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.
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 isothiocyanate-conjugated or phycoerythrin-conjugated IgG (H + L; Proteintech, Wuhan, China) at room temperature for 30 minutes. Nuclei were counterstained with 4′,6-diamidino-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.
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.
Isolation of c-KitPOS/NKX2.5POS cells from total BMSCs and assessment of cellular morphology
Total BMSCs are a heterogeneous cell population including various phenotypical cell subpopulations . 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 . Furthermore, c-Kit is a relatively stable stem cell marker  which can be reliably detected in heart tissue-derived CSCs even after 40 passages . We employed MACS to isolate c-KitPOS cells from total BMSCs. Single-cell cloning was then used to establish c-KitPOS and NKX2.5POS 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-KitPOS/NKX2.5POS BMSCs, and single c-KitPOS/NKX2.5POS BMSCs could form clones well (see Additional file 3). c-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS BMSCs
We performed flow cytometry to analyse the expression of c-Kit and Notch1 to Notch4 receptors on total BMSCs and c-KitPOS/NKX2.5POS BMSCs. Before MACS sorting, 7.3% of total BMSCs were c-Kit-positive, 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-KitPOS/NKX2.5POS BMSCs (Figure 2D). Notch1 was the predominantly expressed form of receptor in both total BMSC and c-KitPOS/NKX2.5POS BMSC populations, Following sorting, Notch1-positive cells increased from 67.2% in total BMSCs to 77.8% in c-KitPOS/NKX2.5POS BMSCs (Figure 2D). These results suggest that Notch1 signalling plays a crucial role in the biology of c-KitPOS/NKX2.5POS BMSCs, which supports our rationale for investigating Notch1.
Forced Notch1-NICD expression leads to Notch1 signalling activation and multilineage differentiation in total BMSCs and c-KitPOS/NKX2.5POS BMSCs
Total BMSCs and c-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS 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 . We constructed an adenovirus to overexpress NICD (NICD-Ad) independent of Notch1 activation. At 8 days post infection with NICD-Ad, we observed enhanced Hes1 mRNA expression in both total BMSCs and c-KitPOS/NKX2.5POS BMSCs, compared with expression in controls (NC-Ad and MOCK; Figure 4). These results suggest that forced expression of Notch1-NICD in total BMSCs and c-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS BMSCs (Figure 5). These data indicate that forced expression of NICD in total BMSCs and c-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS BMSCs led to enhanced osteogenic and adipogenic differentiation (see Additional file 5). This probably reflects the complexity of Notch1 signalling in stem cell differentiation.
Jagged1-triggered Notch1 signalling activation induces multi-lineage differentiation
We treated total BMSCs and c-KitPOS/NKX2.5POS BMSCs with Jagged1 to activate Notch1 signalling, as Notch1 was the predominant Notch receptor expressed by these cells. Exogenous recombinant Jagged1 activated Notch1 signalling in both total BMSCs and c-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS BMSCs into cardiomyocyte, SMC, and endothelial cell lineages.
Jagged1-activated Notch1 signalling predominantly induces cardiomyocyte differentiation in c-KitPOS/NKX2.5POS BMSCs
Following exposure of c-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS BMSCs are more responsive to Notch1 signalling stimuli than total BMSCs. We further evaluated the tendency of Jagged1-treated c-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS BMSCs facilitated predominately cardiomyocyte differentiation, while differentiation into endothelial and SMC lineages was much weaker (Figure 8).
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 tissue-derived 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 . A potential explanation for the inconsistent results of these clinical trials may be the variability of cell types used . Ellison and colleagues have highlighted the necessity of heart tissue-derived CSCs for MI repair . 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 . Furthermore, their phenotype is not influenced by confluency during culture expansion . Additionally, a meta-analysis concluded that BMSCs have promising prospects for MI treatment . BMSCs exhibit a multilineage in vitro differentiation potential, including cardiomyocytes. However, the functions of BMSCs in vivo are mainly mediated in a paracrine manner  and these cells do not directly differentiate into functional cardiomyocytes . 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-KitPOS/NKX2.5POS subtype of BMSCs. We successfully isolated c-KitPOS 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-KitPOS cells. Finally, c-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS 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-KitPOS BMSCs in MI treatment has been proposed by previous reports [36,37]; however, we further investigated the c-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS BMSCs. c-KitPOS/NKX2.5POS BMSCs proliferated slower than total BMSCs, but faster than c-KitPOS CSCs derived from rat hearts, indicating that c-KitPOS/NKX2.5POS BMSCs can be expanded more easily than heart tissue-derived c-KitPOS CSCs.
Notch signalling contributes to stem cell quiescence, proliferation, and differentiation depending on the context in which the signal is activated . Boni and colleagues reported that Notchl directly regulates NKX2.5 promoter activity in mouse heart-derived CSCs, thereby positively regulating cardiomyocyte differentiation . Chen and colleagues demonstrated that activation of Notch signalling in mouse cardiosphere cells – a heterogeneous progenitor cell population – promotes SMC differentiation through a J kappa-recombining binding protein-dependent signalling pathway in vitro . However, activation of Notch signalling in embryonic stem cells has been reported to produce opposite results . 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 . These inconsistencies highlight the need for further efforts to clarify the exact role of Notch1 signalling in c-KitPOS/NKX2.5POS BMSC differentiation.
We found that Notch1 was the predominant Notch receptor form expressed by both total BMSCs and c-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS BMSCs into cardiomyocyte, SMC, and endothelial cell lineages. Furthermore, expression levels of cardiomyocyte and endothelial cell lineage markers in cells derived from c-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS BMSCs. This suggests that c-KitPOS/NKX2.5POS 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 . Activation of Notch1 signalling therefore promotes c-KitPOS/NKX2.5POS 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 [5-7]. 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 . 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 . We aimed to identify these alternative cells from bone marrow that possess properties of heart-derived CSCs.
We found that c-KitPOS/NKX2.5POS 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 marrow-derived CSCs that express c-Kit and NKX2.5 . Furthermore, these bone marrow-derived CSCs could repair injured hearts following transplantation to MI-model animals. Whether our current c-KitPOS/NKX2.5POS BMSCs 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 heart-derived CSCs . We found that c-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS BMSCs and c-KitPOS 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-KitPOS/NKX2.5POS BMSCs. However, the effects of Notch1 signalling activation in c-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS BMSCs. The role of crosstalk between Notch and other signalling pathways should also be considered.
Our study demonstrates the existence of c-KitPOS/NKX2.5POS cells within total BMSC populations. These cells exhibit unique properties in colonic, proliferative, and multilineage differentiation potentials. c-KitPOS/NKX2.5POS BMSCs are predisposed to Notch1-induced cell differentiation when compared with total BMSCs. Targeted activation of Notch1 signalling in c-KitPOS/NKX2.5POS BMSCs leads to multilineage differentiation, suggesting that Notch1 signalling is a potential target for stem cell translational medicine research.
bone marrow cell
bone marrow mesenchymal stem cell
cardiac stem cell
cardiac troponin T
Dulbecco’s modified Eagle’s medium
foetal bovine serum
magnetic activated cell sorting
negative control-expressing adenovirus
notch receptor intracellular domain
smooth muscle cell
von Willebrand factor
Malliaras K, Marban E. Cardiac cell therapy: where we’ve been, where we are, and where we should be headed. Br Med Bull. 2011;98:161–85.
Leri A, Kajstura J, Anversa P, Frishman WH. Myocardial regeneration and stem cell repair. Curr Probl Cardiol. 2008;33:91–153.
Jakob P, Landmesser U. Current status of cell-based therapy for heart failure. Curr Heart Fail Rep. 2013;10:165–76.
Jeevanantham V, Butler M, Saad A, Abdel-Latif A, Zuba-Surma EK, Dawn B. Adult bone marrow cell therapy improves survival and induces long-term improvement in cardiac parameters: a systematic review and meta-analysis. Circulation. 2012;126:551–68.
Surder D, Manka R, Lo Cicero V, Moccetti T, Rufibach K, Soncin S, et al. Intracoronary injection of bone marrow-derived mononuclear cells early or late after acute myocardial infarction: effects on global left ventricular function. Circulation. 2013;127:1968–79.
Bartunek J, Behfar A, Dolatabadi D, Vanderheyden M, Ostojic M, Dens J, et al. Cardiopoietic stem cell therapy in heart failure: the C-CURE (Cardiopoietic stem Cell therapy in heart failURE) multicenter randomized trial with lineage-specified biologics. J Am Coll Cardiol. 2013;61:2329–38.
Assmus B, Walter DH, Seeger FH, Leistner DM, Steiner J, Ziegler I, et al. Effect of shock wave-facilitated intracoronary cell therapy on LVEF in patients with chronic heart failure: the CELLWAVE randomized clinical trial. JAMA. 2013;309:1622–31.
Leri A, Anversa P. Stem cells: bone-marrow-derived cells and heart failure – the debate goes on. Nat Rev Cardiol. 2013;10:372–3.
Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S, et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell. 2003;114:763–76.
Ellison GM, Vicinanza C, Smith AJ, Aquila I, Leone A, Waring CD, et al. Adult c-kit(pos) cardiac stem cells are necessary and sufficient for functional cardiac regeneration and repair. Cell. 2013;154:827–42.
Bolli R, Chugh AR, D’Amario D, Loughran JH, Stoddard MF, Ikram S, et al. Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial. Lancet. 2011;378:1847–57.
Makkar RR, Smith RR, Cheng K, Malliaras K, Thomson LE, Berman D, et al. Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised phase 1 trial. Lancet. 2012;379:895–904.
Kan I, Melamed E, Offen D. Integral therapeutic potential of bone marrow mesenchymal stem cells. Curr Drug Targets. 2005;6:31–41.
Williams AR, Hare JM. Mesenchymal stem cells: biology, pathophysiology, translational findings, and therapeutic implications for cardiac disease. Circ Res. 2011;109:923–40.
Kopan R, Ilagan MX. The canonical Notch signaling pathway: unfolding the activation mechanism. Cell. 2009;137:216–33.
Koch U, Lehal R, Radtke F. Stem cells living with a Notch. Development. 2013;140:689–704.
Boni A, Urbanek K, Nascimbene A, Hosoda T, Zheng H, Delucchi F, et al. Notch1 regulates the fate of cardiac progenitor cells. Proc Natl Acad Sci U S A. 2008;105:15529–34.
Schwanbeck R, Martini S, Bernoth K, Just U. The Notch signaling pathway: molecular basis of cell context dependency. Eur J Cell Biol. 2011;90:572–81.
Yu J, Li M, Qu Z, Yan D, Li D, Ruan Q. SDF-1/CXCR4-mediated migration of transplanted bone marrow stromal cells toward areas of heart myocardial infarction through activation of PI3K/Akt. J Cardiovasc Pharmacol. 2010;55:496–505.
Grassel S, Stockl S, Jenei-Lanzl Z. Isolation, culture, and osteogenic/chondrogenic differentiation of bone marrow-derived mesenchymal stem cells. Methods Mol Biol. 2012;879:203–67.
Zheng YH, Xiong W, Su K, Kuang SJ, Zhang ZG. Multilineage differentiation of human bone marrow mesenchymal stem cells and. Exp Ther Med. 2013;5:1576–80.
Guo J, Jie W, Kuang D, Ni J, Chen D, Ao Q, et al. Ischaemia/reperfusion induced cardiac stem cell homing to the injured myocardium by stimulating stem cell factor expression via NF-kappaB pathway. Int J Exp Pathol. 2009;90:355–64.
Guo J, Jie W, Shen Z, Li M, Lan Y, Kong Y, et al. SCF increases cardiac stem cell migration through PI3K/AKT and MMP2/9 signaling. Int J Mol Med. 2014;34:112–8.
Prins HJ, Braat AK, Gawlitta D, Dhert WJ, Egan DA, Tijssen-Slump E, et al. In vitro induction of alkaline phosphatase levels predicts in vivo bone forming capacity of human bone marrow stromal cells. Stem Cell Res. 2014;12:428–40.
Kuang D, Zhao X, Xiao G, Ni J, Feng Y, Wu R, et al. Stem cell factor/c-kit signaling mediated cardiac stem cell migration via activation of p38 MAPK. Basic Res Cardiol. 2008;103:265–73.
He JQ, Vu DM, Hunt G, Chugh A, Bhatnagar A, Bolli R. Human cardiac stem cells isolated from atrial appendages stably express c-kit. PLoS One. 2011;6, e27719.
Miyamoto S, Kawaguchi N, Ellison GM, Matsuoka R, Shin’oka T, Kurosawa H. Characterization of long-term cultured c-kit + cardiac stem cells derived from adult rat hearts. Stem Cells Dev. 2010;19:105–16.
Doppler SA, Deutsch MA, Lange R, Krane M. Cardiac regeneration: current therapies-future concepts. J Thorac Dis. 2013;5:683–97.
Quijada P, Sussman MA. Making it stick: chasing the optimal stem cells for cardiac regeneration. Expert Rev Cardiovasc Ther. 2014;12:1275–88.
Haack-Sorensen M, Hansen SK, Hansen L, Gaster M, Hyttel P, Ekblond A, et al. Mesenchymal stromal cell phenotype is not influenced by confluence during culture expansion. Stem Cell Rev. 2013;9:44–58.
Martin-Rendon E, Brunskill S, Doree C, Hyde C, Watt S, Mathur A, et al. Stem cell treatment for acute myocardial infarction. Cochrane Database Syst Rev. 2008;4, CD006536.
Figeac F, Lesault PF, Le Coz O, Damy T, Souktani R, Trebeau C, et al. Nanotubular crosstalk with distressed cardiomyocytes stimulates the paracrine repair function of mesenchymal stem cells. Stem Cells. 2014;32:216–30.
Siegel G, Krause P, Wohrle S, Nowak P, Ayturan M, Kluba T, et al. Bone marrow-derived human mesenchymal stem cells express cardiomyogenic proteins but do not exhibit functional cardiomyogenic differentiation potential. Stem Cells Dev. 2012;21:2457–70.
Urbanek K, Cabral-da-Silva MC, Ide-Iwata N, Maestroni S, Delucchi F, Zheng H, et al. Inhibition of notch1-dependent cardiomyogenesis leads to a dilated myopathy in the neonatal heart. Circ Res. 2010;107:429–41.
Li H, Yu B, Zhang Y, Pan Z, Xu W. Jagged1 protein enhances the differentiation of mesenchymal stem cells into cardiomyocytes. Biochem Biophys Res Commun. 2006;341:320–5.
Kubo H, Berretta RM, Jaleel N, Angert D, Houser SR. c-Kit + bone marrow stem cells differentiate into functional cardiac myocytes. Clin Transl Sci. 2009;2:26–32.
Khan M, Mohsin S, Khan S, Riazuddin S. Lin-c-kit(+) BM-derived stem cells repair Infarcted Heart. J Stem Cells Regen Med. 2010;6:15–25.
Gude N, Sussman M. Notch signaling and cardiac repair. J Mol Cell Cardiol. 2012;52:1226–32.
Chen L, Ashraf M, Wang Y, Zhou M, Zhang J, Qin G, et al. The role of notch 1 activation in cardiosphere derived cell differentiation. Stem Cells Dev. 2012;21:2122–9.
Schroeder T, Meier-Stiegen F, Schwanbeck R, Eilken H, Nishikawa S, Hasler R, et al. Activated Notch1 alters differentiation of embryonic stem cells into mesodermal cell lineages at multiple stages of development. Mech Dev. 2006;123:570–9.
Matsuda T, Miyagawa S, Fukushima S, Kitagawa-Sakakida S, Akimaru H, Horii-Komatsu M, et al. Human cardiac stem cells with reduced notch signaling show enhanced therapeutic potential in a rat acute infarction model. Circ J. 2013;78:222–31.
Li Y, Hiroi Y, Ngoy S, Okamoto R, Noma K, Wang CY, et al. Notch1 in bone marrow-derived cells mediates cardiac repair after myocardial infarction. Circulation. 2011;123:866–76.
Smits AM, van Vliet P, Hassink RJ, Goumans MJ, Doevendans PA. The role of stem cells in cardiac regeneration. J Cell Mol Med. 2005;9:25–36.
Quaini F, Urbanek K, Beltrami AP, Finato N, Beltrami CA, Nadal-Ginard B, et al. Chimerism of the transplanted heart. N Engl J Med. 2002;346:5–15.
Guo HD, Wang HJ, Tan YZ, Wu JH. Transplantation of marrow-derived cardiac stem cells carried in fibrin improves cardiac function after myocardial infarction. Tissue Eng Part A. 2011;17:45–58.
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).
The authors declare that they have no competing interests.
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.
Ranran Ding and Xiaofan Jiang contributed equally to this work.
is Table S1 presenting the primers used for PCR. bp, base pairs.
is Table S2 presenting the primary antibodies.
is Figure S1 showing representative images of clones derived from cell of c-Kit POS /NKX2.5 POS BMSCs. c-KitPOS/NKX2.5POS 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.
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-KitPOS/NKX2.5POS BMSCs and cardiac c-KitPOS 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-KitPOS cells sorted from collagenase and trypsin-treated neonatal rat heart tissues by MACS were deemed c-KitPOS CSCs. ***P <0.001 versus other groups. Each cell line was repeated in three wells on every time point (n = 3).
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-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS 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.
is Figure S4 showing the effects of Jagged1 on Hes1 expression. c-KitPOS/NKX2.5POS 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.
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Ding, R., Jiang, X., Ha, Y. et al. Activation of Notch1 signalling promotes multi-lineage differentiation of c-KitPOS/NKX2.5POS bone marrow stem cells: implication in stem cell translational medicine. Stem Cell Res Ther 6, 91 (2015) doi:10.1186/s13287-015-0085-2
- Notch1 Signalling
- Notch Receptor
- Bone Marrow Mesenchymal Stem Cell
- Cardiac Stem Cell