The subpopulation of mesenchymal stem cells that differentiate toward cardiomyocytes is cardiac progenitor cells

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Research Article

The subpopulation of mesenchymal stem cells that differentiate toward cardiomyocytes is cardiac progenitor cells Feng Weia , Tingzhong Wang a , Juanjuan Liub , Yuan Dua , Aiqun Maa,⁎ a

Department of Cardiovascular Medicine, First Affiliated Hospital of Medical School of Xi'an Jiaotong University, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, No. 277 West Yanta Road, Xi'an, Shaanxi 710061, PR China b First Department of Medicine, Hospital of China Railway First Group, No. 319 East Section of South Second Ring Road, Xi'an, Shaanxi 710061, PR China

A R T I C L E I N F O R M A T I O N

A B S T R A C T

Article Chronology:

Mesenchymal stem cells (MSCs) are regarded as a promising source of cell-based therapy for heart

Received 21 May 2011

injury. In fact, less than 30% of MSCs contribute to cardiomyocytes differentiation, and the isolation

Revised version received 28 July 2011

procedure and biological characteristics of this population of cells remain unknown. Here we

Accepted 13 August 2011

isolate and investigate the biological characteristics of this subpopulation of MSCs. Twenty four

Available online 22 August 2011

MSC clones were randomly selected using single-cell monoclonal technology. After induced with 5-azacytidine, eight clones displayed cardiomyocyte-like morphologies, and highly (over 90%)

Keywords:

expressed cardiac-specific markers cTnT and α-actin, and displayed transient outward K+ current

Mesenchymal stem cell

(Ito), inwardly rectifying K+ current (IK1) and delayed rectifier K+ current (IKDR), which were

Cardiomyocyte

typical of cardiomocytes. Other clones merely showed Ito current, and the current densities were

Differentiation

different from those of cardiomyocytes. In contrast to the other clones, before induced with 5-

Biological characteristic

azacytidine, the eight clones expressed early cardiac markers GATA4 and NKX2.5, but not cTnT, α-

5-azacytidine

actin, CD44 and CD90, and had no potentials for adiopogenesis, osteogenesis or chondrogenesis after induction. Our data suggest that the subgroup of MSCs that contributes to cardiomyocytes differentiation is cardiac progenitor cells. Moreover, we show the preliminary purification of this population of cells with a high potential for cardiomyocytes differentiation using single-cell monoclonal technology. © 2011 Elsevier Inc. All rights reserved.

Introduction Ischemic heart diseases are main causes of morbidity and mortality worldwide, and the decrease or loss of functional cardiomyocytes is crucial in the pathogenesis of these diseases [1]. It has been shown that MSC transplantation helps improve the cardiac functions of injured heart [2–4] due to regeneration of cardiomyocytes [5–7] and

paracrine effects that promote angiogenesis [8–10], decrease cardiac fibrosis [11,12], and prevent host cardiomyocytes from apoptosis [13–15]. However, MSC transplantation only improves cardiac functions by less than 10% [2,3]. Many studies have revealed that MSCs are heterogeneous [16–18], which may be one of the factors that impair the treatment effect of transplantation [17,19]. Although only less than 30% of MSCs are committed to cardiomyocyte

⁎ Corresponding author at: Department of Cardiovascular Medicine, First Affiliated Hospital of Medical School of Xi'an Jiaotong University, No. 277 West Yanta Road, Xi'an, Shaanxi 710061, PR China. Fax: +86 29 8526 1809. E-mail address: [email protected] (A. Ma). Abbreviations: cTnT, cardiac troponin T; α-actin, cardiac actin; GATA4, transcription factor expressed in the early stage of the developing heart; NKX2.5, transcription factor expressed in the early stage of the developing heart. 0014-4827/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2011.08.011

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differentiation [20–22], the therapeutical effects of transplantation using this subgroup of cells may improve significantly because of their high capacity in differentiation toward cardiomyocytes. Recently, markers for MSCs such as TM4SF1 [23], NOTCH3 [24] and CD146 [24,25] have been identified. The biological characteristics of MSCs that are able to repair different tissues, however, are quite different. For examples, PDGFRα-positive MSCs can regenerate skin epithial [26], and ITGA11-positive MSCs have potential for osteogenesis [24]. Although studies have shown that MSCs express GATA4 and NKX2.5 mRNAs at low levels before cardiac differentiation induction [21], the characteristics of the subpopulation of MSCs that contribute to cardiac differentiation is not clear, and an efficient procedure for the enrichment of this group of MSCs is still lacking. In this study, we show the purification of the specific MSCs that differentiate toward cardiomyocytes using single-cell monoclonal technology. Over 90% of the purified cells are potent in cardiomyocyte differentiation, and possess characteristics of cardiac progenitor cells. Therefore, these cells may be the most potent population of MSCs for cardiac therapy.

Differentiation–induction experiment All MSC clones were washed by phosphate-buffered saline (PBS) and then treated with complete medium containing 10 μmol/l of 5-azacytidine (Sigma-Aldrich, USA) for induction of cardiomyogenic differentiation. After being incubated for 24 h, cells were washed twice with PBS and the medium was changed with complete medium without 5-azacytidine. Then medium was changed every 3 days during the period of culture.

Immunocytochemistry of differentiated MSCs For immunofluorescence staining, cells were fixed for 20 min with 4% Paraformaldehyde and permeabilized for 30 min with 0.2% Triton X-100. Cells were blocked with goat serum and incubated with primary antibodies overnight at 4 °C. Primary antibodies were rabbit anti-rat polyclonal antibodies against cTnT (Santa Cruz, USA), αactin (Santa Cruz, USA) and GATA4 (Abcam, UK) at a dilution of 1:100. After being washed three times with PBS, cells were incubated with FITC/CY3-conjugated goat anti-rabbit IgG secondary antibodies at a dilution of 1:100 for 1 h at room temperature. Preparations were examined by fluorescence microscope.

Materials and methods RT-PCR and real-time quantitative PCR Animals and culture of MSCs Adult Sprague–Dawley (SD) rats (120 g–180 g) were obtained from Experimental Animal Center of Xi'an Jiaotong University. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). And all experimental procedures have been approved by the Care of Experimental Animals Committee of the First Affiliated Hospital of Medical School of Xi'an Jiaotong University. MSCs were prepared as we reported previously [27]. In brief, following isolating from rat bone marrow by density gradient separation of mononuclear cells, MSCs were seeded in culture flask containing Dulbecco's modified Eagle's medium–low glucose (DMEM-LG)(Gibco, USA) supplemented with 10% fetal bovine serum (FBS)(Gibco, USA) and cultured at 37 °C in a humidified atmosphere containing 5% CO2. When grown to 90% confluency, cells were harvested for passage by 0.25% trypsin (Amresco, USA).

Cloning of MSCs MSCs of the 3rd passage were suspended in DMEM at a density of 10 cells per milliliter, and 1.5 ml of this suspension was inoculated into a 60-mm tissue culture dish containing DMEM supplemented with 10% FBS. The cells were then cultured at 37 °C in a humidified atmosphere of 5% CO2. Sixteen hours later, cells were observed under an inverted phase contrast microscope, and each single cell was marked at the bottom of the dish. After approximately one week, single cell may proliferate and form a colony with 60–80 cells. The ones that grew well without other clones nearby were marked again with another color. To pick clones, cloning rings (Corning, USA) with sterile Vaseline were placed around the marked clones, and 0.25% trypsin was added into the cloning rings to trypsinize the cells. Then cells were re-suspended in DMEM and transferred into a 24-well plate to expand.

Total RNA was extracted using RNAfast200 Kit (Fastagen, China) according to the manufacturer's protocol. RNA was then reversetranscribed into cDNA using SYBR® RT-PCR Kit (Takara, Japan) for 15 min at 37 °C, 5 s at 85 °C. The endogenous ‘house-keeping’ gene (GAPDH) was used to evaluate the efficiency of reverse transcription. PCR was performed using 2 × Taq PCR MasterMix (Tiangen, China). Cycle conditions were as follows: 94 °C for 3 min followed by 30 cycles (94 °C denaturation for 30 s, 63.4 °C annealing for 30 s, 72 °C extension for 1 min), with a final incubation at 72 °C for 5 min. The following primers were employed: cTnT, forward 5′-CATGGAGAAGGACCTGAACGA-3′, reverse 5′-TCATTGCGAATACGCTGCTGT-3′ (product: 147 bp); α-actin, forward 5′-TATCACCAACTGGGACGACA-3′, reverse 5′-ATACATGGCAGGCACATTGA-3′ (product: 175 bp); GAPDH, forward 5′-AGAACATCATCCCTGCATCCA3′, reverse 5′-GCCTGCTTCACCACCTTCTTG-3′ (product: 184 bp). The PCR products were analyzed by electrophoresis on 2% agarose gel. Real-time quantitative PCR analysis was performed on a BioRad iQ5 (Bio-Rad, USA) using SYBR(R) Premix ExTaqTM(Perfect Real Time) Kit (Takara, Japan). Primers were used as follows: GATA4, forward 5′-CCAAGCAGGACTCTTGGAAC-3′, reverse 5′TCAGGAGTTGTTCCCACACA-3′ (product: 175 bp). NKX2.5, forward 5′-ACCCTCGGGCGGATAAGAA-3′, reverse 5′-GACAGGTA CCGCTGTTGCTTGA-3′ (product: 178 bp). A relative quantification method (2− ΔΔCt, where Ct is cycle threshold) was chosen for quantitative analysis. The relative quantitation value of target, normalized to the endogenous control GAPDH (house-keeping) gene and relative to a calibrator, is expressed as 2 − ΔΔCt (fold difference), where ΔCt = (Ct of target genes) − (Ct of endogenous control gene, GAPDH), and ΔΔCt = (ΔCt of samples for target gene) − ( ΔCt of calibrator for the target gene).

Electrophysiology The whole-cell patch-clamp technique was used as described previously [28]. Borosilicate glass electrodes (1.2 mm outer

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diameter) were pulled with a P-97 puller (Sutter Instrument, USA). The tip resistances were 2–5 MΩ when filled with pipette solution. The tip potentials were compensated before the pipette touched the cell. The GΩ-seal was obtained after a gentle negative suction. By gentle suction, cell membrane was ruptured to establish the wholecell configuration. Experimental data were acquired with a software pCLAMP 10.2 (Axon Instruments, USA). Data were stored on the computer for analysis offline. Tyrode's solution contained 140 mM NaCl, 5.4 mM KCl, 1.8 mM MgCl2, 1.8 mM CaCl2, 10 mM glucose and 10 mM HEPES, and pH was adjusted to 7.35 with NaOH. The pipette solution contained 20 mM KCl, 110 mM K-aspartate, 10 mM HEPES, 5.0 mM Na2-phosphocreatine, 5.0 mM Mg2-ATP, 1.0 mM MgCl2 and 0.05 mM EGTA, and pH was adjusted to 7.2 with KOH. K+ in pipette solutions and superfusion was replaced by equimolar Cs+ when sodium current or L-type Ca2+ current was recorded. All experiments were conducted at 22 °C–23 °C.

Flow cytometric analysis MSCs were trypsinized (0.25% trypsin) and washed twice with PBS. Then cells were stained according to the manufacturer's recommendations with the FITC/PE/PerCP-CY5.5/CY3-conjugated

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monoclonal antibodies against CD29 (Bioss, China), CD34 (Santa Cruz, USA), CD44 (Serotec, UK), CD45 (Serotec, UK), CD90 (Oxford Biotechnology, UK), CD105 (Bioss, China). Incubation was performed at room temperature for 20 min. Control group was incubated with FITC/PE/PerCP-CY5.5/CY3-conjugated IgG isotype control antibodies (Santa Cruz, USA). After incubation, cells were washed with PBS for two times. Quantitative analysis was performed using a flow cytometer (Beckman Coulter,USA).

Trilineage differentiation Adipogenic, osteogenic and chondrogenic differentiation were performed as described previously [17]. MSC clones were expanded for 3 days in 6-well microplates until 50% confluency. Adipogenesis was induced with a differentiation medium which consists of DMEM supplemented with 10% FBS, 0.5 mM isobutylmethylxanthine, 0.5 μM dexamethasone and 50 μM indomethacin (Sigma-Aldrich, USA). Osteogenesis was induced with a differentiation medium which consists of DMEM supplemented with 10% FBS, 10 mM βglycerophosphate, 200 nM dexamethasone and 50 μM L-ascorbic acid 2-phosphate (Sigma-Aldrich, USA). Chondrogenesis was induced with a differentiation medium which consists of DMEM

Fig. 1 – Characteristics of MSCs and the formation of MSC clones. (A) The third passage of MSCs show fibroblast-like morphology; (B) single MSC produced its first offspring cell 24 h after being seeded on dish; (C) single MSC formed a colony consisted of 20–40 cells after five days of growth; (D) expression of cell surface antigens on MSCs. Scale bar = 50 μm.

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supplemented with 10 ng/ml of transforming growth factor-β3, 100 ng/ml of bone morphogenetic protein-2 (Protec, Germany), 200 nM dexamethasone, 50 μg/ml of L-ascorbic acid 2-phosphate, 100 μg/ml of pyruvate, 40 μg/ml of proline (Sigma-Aldrich, USA). After 3 weeks of cultivation in differentiation medium, MSCs were fixed in 4% paraformaldehyde for 25 min and stained with 1% Alizarin Red S (Sigma-Aldrich, USA) for 30 min to detect calcified extracellular matrix. To detect lipid accumulation, MSCs were stained with 0.3% Oil Red O (Sigma-Aldrich, USA) in 0.6% isopropanol

for 1 h. To detect matrix deposition of sulfated glycosaminoglycans, cultures were stained overnight at 25 °C with 1% Alcian Blue 8-GX (Sigma-Aldrich, USA).

Statistical analysis Data are presented as mean± SD. Statistical analysis was performed by Student's unpaired t-test or one-way ANOVA followed by the Student–Newman–Keuls test for multiple comparisons among the

Fig. 2 – Differentiation of MSCs toward cardiomyocytes. (A) Before being treated with 5-azacytidine, MSCs displayed fibroblast-like shapes; (B) after two weeks of treatment, MSCs significantly increased in sizes and connected to the adjacent cells; (C) four weeks after treatment, MSCs displayed cardiomyocyte-like morphologies [scale bar = 50 μm]; (D) representative images showing protein expressions of the cardiac markers cTnT and α-actin in different clones of MSCs after induction. Some clones were positive for cTnT and α-actin after four or six weeks of induction (clones 1–4 represented), others were negative (clone-5 represented) [scale bar = 50 μm]; (E) detection of mRNA expressions of the cardiac markers cTnT and α-actin in different clones of MSCs after induction. Eight out of 24 MSC clones were positive for cTnT and α-actin at mRNA level after 4 or 6 weeks of induction [DNA Marker (500 bp)].

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three groups. P < 0.05 was considered to be statistically significant differences.

Results Characteristics of MSCs and the formation of MSC clones MSCs of the 3rd passage distributed uniformly and were adherent cells with typical fibroblast-like shapes (Fig. 1A). They had great proliferation potential and maintained their morphologies throughout the culture period. Flow cytometric analysis showed that they expressed CD29, CD44, CD90 and CD105, but not CD34 and CD45 (Fig. 1D). MSCs of the 3rd passage were prepared for selecting single-cell clones. Single cell retained its fibroblast-like morphology after being seeded on dish. Twenty-four hours later, an offspring cell was produced and was round in shape. Then the cell gradually regained its typical fibroblast-like morphology (Fig. 1B). After five days, single cell formed a colony that consisted of 20–40 cells with the same size and morphology (Fig. 1C). Finally, 24 clones successfully formed colonies and were used to complete the whole experiment.

Part of the MSC clones differentiated toward cardiomyocytes after induction Clones of MSCs displayed uniform fibroblast-like shapes before induction (Fig. 2A). After being treated with 5-azacytidine for two weeks, some clones of MSCs significantly increased in sizes and connected to the adjacent cells (Fig. 2B). After four weeks of treatment, MSCs increased further in sizes, showed rod shapes, and integrated with the surrounding cells, which were similar to the morphology of cardiomyocytes (Fig. 2C). After four weeks of treatment, eight out of the 24 MSC clones expressed proteins (Fig. 2D) and mRNAs (Fig. 2E) of the cardiac marker cTnT, but marker α-actin was not expressed. Six weeks after treatment, α-actin was significantly expressed at both protein level (Fig. 2D) and mRNA level (Fig. 2E) in the same MSC clones that were positive for cTnT. In other clones, the expressions of cTnT and α-actin were not detectable after four and six weeks of induction (Figs. 2D and E). This suggested that only part of the MSCs had the potentials to acquire cardiac phenotypes after induction.

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Electrophysiological properties of the induced MSC clones For further experiments, clones of MSCs after induction were pooled and divided into two groups based on whether they could differentiate toward cardiomyocytes after 5-azacytidine treatment. The differentiated group, which was positive for cardiac markers cTnT and α-actin after induction, had the potential to differentiate toward cardiomyocytes; the undifferentiated group, which did not express cardiac markers after induction, had no capacity to differentiate toward cardiomyocytes. To understand the electrophysiological function of the induced MSCs, patch-clamp technology was used to detect the functional current (INa, ICa,L, IK) expressions of MSCs. In the differentiated group, three types of membrane current were observed in MSCs. One type of current, activated by voltage steps between − 140 mV and 40 mV in 10 mV increments from a holding potential of − 40 mV, was typical of the inwardly rectifying K + current (IK1) (Fig. 3). IK1 was observed in 66.67% (8/12) of the cells. The second type of current, elicited by voltage steps between − 60 mV and 60 mV in 10 mV increments from a holding potential of − 80 mV, was an outward delayed rectifier current (Fig. 4). This current was activated at − 20 mV, and could be significantly suppressed by 20 mM tetraethylammonium. These features suggested that this current was likely a delayed rectifier K + current (IKDR). This current was observed in 81.82% (9/11) of the cells. The third type of current was recorded when voltage steps between − 40 mV and 70 mV from holding potential of − 90 mV were applied, and displayed properties of the transient outward K+ current (Ito) (Fig. 5A). It was significantly inhibited by 5 mM 4-aminopyridine (4-AP). Furthermore, the average current density of Ito (32.08 ± 3.12 pA/pF) was similar to that in adult rat ventricular cardiomyocytes (31.16 ±2.78 pA/pF) at 60 mV, although the membrane capacitance (9.21 ± 1.13 pF) was much lower than that of mature cardiomyocyte (118.56± 5.48 pF) (Fig. 5C). This type of current was observed in 91.67% (11/12) of MSCs. In the undifferentiated group, only one type of potassium current (Ito) was recorded in 61.54% (8/13) of MSCs, but the average current density (54.86 ± 4.26 pA/pF) was different from that of the cells in the differentiated group (32.08 ± 3.12 pA/pF) and cardiomyocytes (31.16 ± 2.78 pA/pF). Moreover, the average membrane capacitance (2.18 ± 0.14 pF) was also much lower than that of the cells in the differentiated group (9.21 ± 1.13 pF) and cardiomyocytes (118.56 ± 5.48 pF).

Fig. 3 – Inwardly rectifying K+ current (IK1) in differentiated MSCs. IK1 current was recorded in a representative cell with the protocol shown in the inset. The I–V curve showed a strong inward rectification typical of IK1.

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Fig. 4 – Delayed rectifier K+ current (IKDR) in differentiated MSCs. IKDR was recorded in a representative MSC with the voltage protocol shown in the inset in the absence (control) and presence of 20 mM TEA. The I–V relationship was shown in the absence and presence of 20 mM TEA. IKDR was substantially inhibited by TEA (p < 0.05 at 10 to 60 mV vs. control), and the effect was reversed after washout.

Fig. 5 – Transient outward K+ current (Ito) in MSCs and mature cardiomyocytes. (A) Ito was recorded in a representative MSC with a voltage step protocol shown in the inset in the absence (control) and presence of 5 mM 4-AP. The I–V relationship was shown in the absence and presence of 5 mM 4-AP. Ito was significantly suppressed by 4-AP (p < 0.05 at −20 to 60 mV vs. control), and the effect was reversed after washout; (B) Ito was recorded in a representative adult rat ventricular cardiomyocyte with the same protocol used in differentiated MSCs; (C) comparisons of membrane capacitance and Ito current density among undifferentiated MSCs, differentiated MSCs, and adult rat ventricular cardiomyocytes at 60 mV (★p < 0.05, ★★p < 0.01).

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MSCs in the differentiated group had a high differentiation potential toward cardiomyocytes To evaluate the differentiation efficiency of MSCs in the differentiated group, the percent of cells that expressed cTnT and αactin were investigated. The cTnT and α-actin positive cells were both over 90% when observed under a fluorescence microscope (Figs. 6A and D). Flow cytometric analyses showed that the expression of cTnT and α-actin was 95.02% (Figs. 6B and C) and 96.24% (Figs. 6E and F), respectively. These results demonstrated that the subgroup of MSCs purified using single-cell monoclonal technology had a high differentiation potential toward cardiomyocytes.

The subpopulation of MSCs that contributed to differentiation toward cardiomyocytes was cardiac progenitor cells To understand the biological characteristics of MSCs that contributed to differentiation toward cardiomyocytes, MSC clones without induction were also pooled and divided into two groups based on whether they could differentiate toward cardiomyocytes after 5azacytidine treatment. The differentiated group, which was positive for cardiac markers cTnT and α-actin after induction, has the potential to differentiate toward cardiomyocytes. The undifferentiated group, which did not express cardiac markers after induction, has no capacity to differentiate toward cardiomyocytes. Before induction, by qualitative analysis using gel electrophoresis, MSCs in the differentiated group expressed GATA4 and

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NKX2.5, and no cTnT and α-actin transcripts were detected. None of the four cardiac markers (GATA4, NKX2.5, cTnT, α-actin) was expressed in MSCs in the undifferentiated group (Fig. 7A). However, by quantitative analysis using 2− ΔΔCt method, the expression of GATA4 and NKX2.5 in the differentiated group was 16.50 times and 6.12 times higher than that in the undifferentiated group, respectively (Fig. 7B). Immunostaining showed that GATA4 was expressed in MSCs in the differentiated group, but not in the undifferentiated group (Fig. 7C). These results indicated that the specific cells from MSCs that contributed to differentiation toward cardiomyocytes expressed the early cardiac markers GATA4 and NKX2.5 before induction. Surface antigen analyses showed that MSCs in the differentiated group did not express CD34, CD44 or CD90; while MSCs in the undifferentiated group expressed CD44 and CD90, but not CD34 (Fig. 7D). Furthermore, MSCs in the undifferentiated group had adipogenesis, osteogenesis and chondrogenesis capabilities when cultivated in differentiation media (Fig. 7E). However, MSCs in the differentiated group lost these abilities (Fig. 7E). These results suggested that the specific cells in the differentiated group might have lost the typical characteristics of MSCs.

Discussion Here we demonstrate that 5-azacytidine induces cardiomyocytelike morphology in only a small portion of MSCs, and these cells express the cardiac markers cTnT and α-actin, as well as

Fig. 6 – Evaluation of differentiation efficiency of MSCs in the differentiated group. (A) Immunostaining for cTnT; (B) analysis of cTnT-positive cells by flow cytometry; (C) isotype control for analysis of cTnT-positive cells by flow cytometry; (D) immunostaining for α-actin; (E) analysis of α-actin-positive cells by flow cytometry; (F) isotype control for analysis of α-actin-positive cells by flow cytometry. Scale bar = 50 μm.

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Fig. 7 – Biological characteristics of the specific MSCs that had potentials to differentiate toward cardiomyocytes. (A) Electrophoresis demonstrated that GATA4 and NKX2.5 were expressed at low levels in MSCs in the differentiated group, but not in MSCs in the undifferentiated group. The expressions of cTnT and α-actin were not detected in both groups; (B) the relative mRNA expressions of the cardiac markers in MSCs between the differentiated group and the undifferentiated group. Values are mean ± SD. ★p < 0.05 vs. undifferentiated group; (C) immunostaining showed that GATA4 was detected in MSCs in the differentiated group, but not in MSCs in the undifferentiated group; (D) analyses of surface antigens revealed that MSCs in the differentiated group did not express CD34, CD44 or CD90; MSCs in the undifferentiated group expressed CD44 and CD90, but not CD34; (E) MSCs in the undifferentiated group had the abilities for adipogenesis, osteogenesis and chondrogenesis when cultivated in differentiation media. In contrast, MSCs in the differentiated group lack the capacity for trilineage differentiation. Scale bar = 50 μm.

functional potassium currents. These results suggest that only a subpopulation of MSCs have the capacities to differentiate toward cardiomyocytes. Importantly, in contrast to other MSCs, this subpopulation of MSCs expresses the early cardiac markers GATA4 and NKX2.5, but not cTnT, α-actin, CD44 and CD90 before induction, and they do not have the potential for adiopogenesis, osteogenesis or chondrogenesis after induction. These indicate that this subpopulation of cells are different from typical MSCs and may be cardiac progenitor cells. It has been demonstrated that MSCs can differentiate into cardiomyocytes in vivo and in vitro [29–33], and transplanted MSCs contribute to the function of injured heart. However, the percentage of MSCs that differentiate toward cardiomyocytes is always lower than 30% [20–22], because MSCs derived from bone marrow are usually collected using traditional method involving

density gradient separation of mononuclear cells and subsequent tissue cultures, and are largely heterogeneous [16–18]. Transplantation using the purified subgroup of MSCs may extensively enhance the differentiation efficiency and treatment effect. But an effective isolation protocol of this subgroup of MSCs is still lacking. Using single-cell monoclonal technology, we isolate a pure subgroup of MSCs, which have high potential for differentiation toward cardiomyocytes. Using the purified cells for transplantation would not only improve the treatment effect, but also avoid proarrhythmia caused by contaminations from cells with no cardiomyocytes differentiation potential. Although immunoselection maybe a better method for the purification of MSCs with high cardiomyocyte differentiation potential, a specific cell surface marker useful for immunoselection is not available so far, due to

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the unknown biological characteristics of this subpopulation of MSCs. Although it was reported previously that MSCs isolated with traditional methods have low levels of GATA4 and NKX2.5 mRNA expressions before induction [21], so far, it has not identified which subpopulation of MSCs contributes to cardiomyocyte differentiation and what characteristics they have. Our data demonstrate that the specific cells in MSCs involved in differentiation toward cardiomyocytes are different from typical MSCs, and may be cardiac progenitor cells based on their biological characteristics. For these results, we speculate that specific cell surface markers for cardiac progenitor cells such as Sca-1 [34], c-kit [35] and MDR-1 [36], may be used as the specific cell surface marker for this population of cells. Unfortunately, specific cell surface marker for these cells remains unknown. Although GATA4 and NKX2.5 are specific for this subpopulation of cells, they are not cell surface markers. Traditional MSC markers CD44 and CD90 are not suitable to be used as competent surface markers to isolate the subpopulation of MSCs that contribute to cardiomyocytes differentiation. Nevertheless, it is interesting why a subpopulation of MSCs exist in bone marrow (BM) and express early cardiac markers and cardiac-specific proteins upon exposure to 5-azacytidine. One might speculate that these potential cardiac progenitors in bone marrow may be responsible for cardiac repair after cardiac injury. These cells reside in the BM, are capable of mobilizing into the peripheral circulation by G-CSF released by ischemic myocardium after MI [37]. The mobilized cardiac progenitors can be potentially chemo-attracted to injured myocardium in an SDF-1-, HGF-, and LIF-dependent manner [38] to protect against myocardial infarction through regeneration and paracrine effects by serving as carriers of cytoprotective proteins [37]. In conclusion, for the first time, our study demonstrates that the specific MSCs that contribute to cardiomyocytes differentiation have the characteristics of cardiac progenitor cells. Moreover, after purification by single-cell monoclonal technology, these potential cardiac progenitor cells retain a much higher potential for cardiomyocytes differentiation. However, further studies are necessary to identify and confirm the specific cell surface markers for these cells in order to effectively enrich them for future clinical applications including heart injury treatment.

Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (NSFC, No. 81000063 and No. 30800455) and Research Fund for the Doctoral Program of Higher Education of China (No. 200806981027). We thank Prof. Zhiquan Liu and Dr. Wenhui Jiang for their helpful suggestions in study design.

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