Characterization of Rat Very Small Embryonic-Like Stem Cells and Cardiac Repair After Cell Transplantation for Myocardial Infarction

Cell isolation and sorting

Eight-week-old male Sprague-Dawley (SD) rats were purchased from Slac Laboratory Animal Corporation and housed in Department of Laboratory Animal Science of Fudan University, China. The investigation was permitted by the Law of the People's Republic of China on the Protection of Wildlife, and the protocol was approved by the Institutional Animal Care Committee of Fudan University, China. The femurs and tibias of rats were cut off under sterile conditions. The bone marrow was harvested by washing with phosphate-buffered saline (PBS) containing 800 U/mL heparin. Then, suspension of the bone marrow was added on Percoll (GE Healthcare) solution at the density of 1.073. After centrifugation (25 min, 1,750 rpm) at 4°C, the fraction of the mononuclear cells was collected.

For fluorescence-activated cell sorting, the mononuclear cells were blocked with 10% goat serum, and then, the cells were incubated with mouse anti-SSEA-1 antibody (1:200; Santa Cruz Biotechnology) for 50 min at 4°C. After rinsing with PBS, the cells were incubated with goat anti-mouse IgG conjugated with FITC (1:300; Chemicon Millipore) for 30 min. After washing, the cells were resuspended with Dulbecco's modified Eagle's medium (DMEM; Gibco Invitrogen) supplemented with 2% fetal bovine serum (FBS; Gibco Invitrogen), 500 U/mL penicillin, and 0.5 mg/mL streptomycin (Beyotime). The cells incubated only with second antibody were used as control. SSEA-1⁺ cells were sorted by a fluorescence-activated cell sorter (FACS Aria II; BD Biosciences), and collected in DMEM containing 20% FBS, 200 U/mL penicillin, and 0.2 mg/mL streptomycin [19]. Purity of SSEA-1⁺ cells was examined with the FACS again.

Detection of lineage markers

The sorted SSEA-1⁺ cells were incubated with CD45-PE antibody (Biolegend) and Ter-119-APC antibody (eBioscience) or Gr-1-PE antibody (eBioscience) and CD11b-APC antibody (Biolegend) for 50 min at 4°C. Incubation with PBS as control was performed. Then, expression of the lineage markers of the cells was detected by a FACS.

Cell proliferation and clone formation assays

To amplify the sorted cells, a feeder layer of MEFs was prepared. MEF line CF-1 (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai) was incubated in DMEM supplemented with 15% FBS. Then, the cells were collected and treated with 20 μg/mL mitomycin C (Hisun Pharmaceutical) at 37°C for 1 h. After mitomycin C solution had been removed, the cells were seeded in a culture dish at the density of 1×10⁶ cells/mL and incubated in a 37°C and 5% CO₂ incubator (Sanyon Electric). The cells were used as a feeder layer when they spread into a monolayer [20]. The sorted SSEA-1⁺ cells were seeded on a feeder layer of MEFs and incubated in DMEM supplemented with 10% FBS, 1% nonessential amino acids, 1,000 U/mL leukemia inhibitory factor (LIF; Chemicon Millipore), and 20 ng/mL basic fibroblast growth factor (bFGF; Gibco Invitrogen). LIF and bFGF were supplemented every 24 h, the medium was replaced every 2–3 days. At intervals of 4–7 days, colonies of SSEA-1⁺ cells were selected for passage. The SSEA-1⁺ colonies were mechanically detached into small clumps with a micropipette tip and seeded on a new MEF feeder layer. The cells seeded in a culture dish without feeder layer were used as a contrast experiment.

To evaluate the clone formation of SSEA-1⁺ cells, 0.6% agarose gel containing 1× DMEM, 20% FBS, 2,000 U/mL LIF, and 40 ng/mL bFGF was prepared in a 24-well plate. Density of SSEA-1⁺ cells was adjusted to 1×10³ cells/mL, 330 cells/mL, 110 cells/mL, and 37 cells/mL, respectively, with 0.3% agarose containing 1× DMEM and 20% FBS. Then, suspension of the cells (200 μL/well) was added on the agarose gel and incubated in an incubator for 2 weeks.

Electron microscopy

For scanning electron microscopy, the cells incubated on MEF feeder layer for 1 day and 7 days were fixed with 2.5% glutaraldehyde overnight at 4°C, respectively, and underwent a graded ethanol series and final twice rinsing in 100% ethanol for 15 min. Then, the cells went through another graded series to replace ethanol with hexamethyldisilazane. After being dried in air, the specimens were coated with carbon, and examined under a scanning electron microscope (SEM; FEI QUANTA200, Philips) [21].

For transmission electron microscopy, SSEA-1⁺ cells were fixed with 2.5% glutaraldehyde at 4°C, and then fixed with 1% osmium tetroxide. After being dehydrated with gradient alcohol, the cells were soaked with anhydrous acetone and Jpurr resin, and embedded with Jpurr resin. Ultrathin sections were gained with Reichert-ultracut E ultrathin microtome (Leica), and stained with 3% uranyl acetate and lead citrate. The ultrastructures of the cells were viewed by a CM120 transmission electron microscope [22].

Immunocytochemistry

For evaluating pluripotency of SSEA-1⁺ cells, the cells were added on coverslips and fixed with acetone and methanol (1:1) for 20 min at −20°C. After treatment with goat serum, rabbit anti-Oct4 antibody (1:200; Abcam), rabbit anti-Nanog antibody (1:200; Chemicon Millipore), and rabbit anti-Sox2 antibody (1:200; Sigma) were added on coverslips, respectively, and incubated at 4°C overnight. After incubation at 37°C for 2 h, the cells were incubated with goat anti-rabbit IgG conjugated with TRITC (1:300; Jackson ImmunoResearch Laboratories) for 30 min [23]. The nuclei were counterstained with 4', 6-Diamidino-2-phenylindole (DAPI, 1:1,000; Sigma). The cells were mounted with glycerophosphate and viewed under a fluorescent microscope (Olympus Corporation).

To examine expression of stem cell antigen-1 (Sca-1), SSEA-1⁺ cells were incubated with rabbit anti-Sca-1 antibody (1:200; eBioscience) at 37°C for 1 h. Subsequently, the cells were incubated with goat anti-rabbit IgG conjugated with TRITC (Jackson) at 37°C for 30 min. The nuclei were counterstained with DAPI (1:1,000; Sigma). The cells were viewed with an LSM 510 confocal lazer scanning microscope (Carl Zeiss MicroImaging GmbH) [24].

RT-PCR analysis

Expression of PSC transcriptional factor mRNAs Oct-4, Nanog, Rex1, Sox2, and Fgf4 in the cells was analyzed by RT-PCR. Total RNA of rat SSEA-1⁺ cells, mouse ESC line D3 cells (ES-D3 cells, Chinese Academy of Sciences, Shanghai), and rat marrow-derived mesenchymal stem cells (MMSCs) [25,26] was extracted, respectively. Primers (Sangon Biotech) used for amplification are listed in Table 1. The mRNA levels were normalized by using Gapdh as a housekeeping gene. PCR reactions were performed under the following conditions: 1 cycle at 45°C for 30 min and 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 60°C for 30 s and 72°C for 1kb/min, and 1 cycle at 72°C for 5 min. The PCR products were size-fractionated with 1.5% agarose (Takara Bio Inc.) gel electrophoresis [4]. The images were analyzed by software (Furi Science and Technology).

Tumorigenicity assay

For investigating possibility of tumorigenesis and teratoma formation, 0.5 mL suspension of SSEA-1⁺ cells at the density of 2×10⁶ cells/mL was subcutaneouly injected in the inguinal region on both sides of 10 mice (5 male and 5 female) with severe combined immunodeficiency (SCID; Shanghai Slac Laboratory Animal Corporation). The mice were sacrificed to examine formation of neoplasms at 4 weeks after the injection.

Differentiation assay in vitro

To evaluate potency of differentiation of the cells in the embryoid-like bodies toward the cells from 3 germ layers, expression of ectodermal, mesodermal, and endodermal markers was examined by immunocytochemistry. Nestin, Brachyury, and E-cadherin were used for ectodermal, mesodermal, and endodermal markers, respectively. The cells in the embryoid-like bodies were picked out with a pipette and incubated in a 24-well plate with DMEM containing 5% FBS. At 4 weeks after incubation, the cells were collected and added on slides. The cells were labeled by rabbit anti-Nestin antibody (1:200; Chemicon Millipore), mouse anti-Brachyury antibody (1:200; Santa Cruz), and rabbit anti-E-cadherin antibody (1:200; Abcam), respectively. Subsequently, the cells were incubated with goat anti-mouse IgG conjugated with FITC (1:300; Chemicon Millipore) or goat anti-rabbit IgG conjugated with TRITC (1:300; Jackson) and viewed with a fluorescent microscope (Olympus Corporation).

For induction of differentiation toward cardiomyocytes, SSEA-1⁺ ELSCs of 2–3 passage were seeded on coverslips, and then incubated in DMEM supplemented with 10% FBS, 10 ng/mL bone morphogenetic protein 2 (BMP-2; PeproTech), 10 ng/mL insulin-like growth factor 1 (IGF-1; GeneWay, Biotech), and 10 ng/mL bFGF. The cytokines were supplemented daily, and the medium was replaced every 2–3 days. The cells without induction by cytokines were used as control. At 2 weeks after induction, expression of mRNAs of cardiac transcriptional factors GATA-4 (Gata4) and Nkx2-5 and mRNAs of cardiac specific proteins cTnI (Tnni3k), cTnT (Tnnt2), and Cx43 (Gja1) was analyzed by RT-PCR. The cardiomyocytes isolated from rat hearts were used as positive control. Cardiomyocytes were prepared according to the method described by Egorova et al. [27]. The primer sequences were listed in Table 1. PCR reactions were performed with the protocol as just described. At 4 weeks after induction, expression of cardiac specific proteins in the differentiated cells was examined with mouse anti-cTnT antibody (1:200; Santa Cruz) and rabbit anti-Cx43 antibody (1:200; Abcam) in double staining. After incubation with secondary antibodies conjugated with FITC and TRITC, the cells were viewed with a fluorescent microscope.

For inducing differentiation toward endothelial cells, ELSCs of 2–3 passage were seeded on coverslips, and incubated in DMEM supplemented with 10% FBS, 10 ng/mL vascular endothelial growth factor (VEGF; R&D Systems), 10 ng/mL bFGF, and 10 ng/mL transforming growth factor beta (TGF-β; R&D Systems). The cytokines were supplemented daily, and the medium was replaced every 2–3 days. The cells incubated with the medium without cytokines were used as control. At 2 weeks after induction, expression of Pecam1, Flt1, and Flk1 was analyzed by RT-PCR. The endothelial cells isolated from cerebral microvasculatures of rats by using the method previously described by Lu [28] were used as positive control. At 4 weeks after induction, the expression of endothelial markers in the differentiated cells was examined with mouse anti-Von Willebrand factor (vWF) antibody (1:200; Abcam) and rabbit anti-CD31 antibody (1:200; BD Biosciences) in double staining.

Establishing of MI models and cell transplantation

Sixty adult female SD rats (240–260 g) were anesthetized with ketamine (60 mg/kg) and xylazine (5–10 mg/kg) by a peritoneal injection. After endotracheal intubation and ventilation with a rodent ventilator (Harvard Apparatus), the heart was exposed through a 2 cm long left lateral thoracotomy. Left anterior descending coronary artery (LADCA) was ligated [4,29]. Successful MI was determined by observing a pale discoloration of the myocardium and a high T-wave on the electrocardiogram. After LADCA ligation, the chest of the rats was sutured. 2×10⁴ U penicillin G benzathine was intramuscularly injected daily for 3 days after the operation.

Nine rats died after LADCA ligation, and 5 rats were kicked out because of the lack of an ideal echocardiogram. At 1 week after MI, the remaining 46 rats were randomly divided into control, MMSC, ELSC, and induced-ELSC groups. In induced-ELSC group, the cells were preinduced with 10 ng/mL BMP-2, 10 ng/mL IGF-1, and 10 ng/mL bFGF for 24 h before transplantation. The rats were anesthetized, and their hearts were exposed as just mentioned. The cells (1×10⁵) were suspended with 80 μL PBS and injected into the border of the infarcted myocardium through a micro-syringe at 4 spots (20 μL per spot). In control group, 80 μL PBS was injected [4,30]. 2×10⁴ U penicillin G benzathine was intramuscularly injected daily for 3 days after the operation.

Echocardiography

To evaluate the improvement of cardiac function, changes of echocardiograms were recorded with an ultrasonocardiograph (Visual Sonics) under anesthetization before MI, at 1 week after MI (baseline echocardiogram), and 4 weeks after cell transplantation. After adequate two-dimensional images were obtained, the M-mode cursor was positioned to the parasternal long axis view at the level papillary muscles. Left venticular (LV) end-diastolic diameter (LVEDD) and LV end-systolic diameter (LVESD) were measured from at least 3 consecutive cardiac cycles. LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV), Index of ejection fraction (EF, LVEDV-LVESV/LVEDV×100%), and fractional shortening (FS, LVEDD-LVESD/LVEDD×100%) were measured for examination of systolic function [4]. In ideal medels, EF was <50% at 1 week ligation of LADCA.

Morphological analysis

After echocardiography, the rats were sacrificed, and their hearts were rapidly excised. The tissue masses of upper, central, and lower infarcted regions were prepared and fixed in 4% paraformaldehyde for 36 h. After being dehydrated in sucrose solution, the masses were embedded in Tissue-Tek OCT Compound (Sakura Finetek) and frozen at −80°C. Frozen sections of 5 μm thickness were prepared by a Leica CM 1900 microtome (Leica Microsystems). Nine discontinuous sections were selected from each model and stained with Masson's trichrome stain (Sigma). The blue regions indicate scar tissues [25]. Scar area was defined as the percentage of blue region in whole myocardium of the cross-sectional area in LV wall by using computer-based planimetry with ImageJ analysis software (National Institutes of Health).

Survival and differentiation of the transplanted cells

For investigating the survival and differentiation of the transplanted cells within infarcted myocardium, Y chromosome specific gene of the cells was detected by fluorescence in situ hybridization [4,31]. The frozen sections were postfixed and immersed in 70% formamide to denature the fixed chromosome specimens. Then, the sections were dehydrated through gradient ethanol and dried in air. After being labeled with biotin, the Y chromosome specific probes (Shan Jing, Shanghai) were denatured in Hybrisol VII (Qbiogene) at 95°C for 5 min; then, the sections were incubated with the denatured probe at 42°C overnight. The sections were washed twice with 2× saline sodium citrate buffer and then incubated with streptavidin-FITC or streptavidin-Cy3 (Biolegend) at 37°C for 30 min. After treatment with blocking solution, the sections were incubated with rabbit anti-cTnT antibody (1:200; Santa Cruz), rabbit anti-Cx43 antibody, or mouse anti-CD31 antibody (1:200; BD Biosciences). Then, the sections were incubated with goat anti-rabbit IgG conjugated with TRITC or goat anti-mouse IgG conjugated with FITC. The nuclei were counterstained with DAPI. The Y chromosome-positive cells expressing cTnT, Cx43, or CD31 were counted under the fluorescence microscope, respectively.

Statistical analysis

Data are expressed as the means±standard deviation. To statistically analyze the data, we performed Student's t-test and 1-way analysis of variance with Scheffe's post hoc multiple-comparison analysis. A value of P<0.05 was considered significant, and a value of P<0.01 was considered very significant.

Identification of Lin^-SSEA-1⁺ cells

After the procedures of FACS, the region R1 of lymphocytes was selected for gating of control sample. The same region was selected for gating of SSEA-1 labeling sample. The result of FACS shows that SSEA-1⁺ cells in the mononuclear cells were 0.015% (Fig. 1A, B). SSEA-1⁺ cells did not express lineage markers such as CD45, Ter-119, CD11b, and Gr-1 (Fig. 1C, D). In the sorted SSEA-1⁺ cells, percentage of the positive cells was 97.2±1.7.

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FIG. 1.

Sorting of SSEA-1⁺ cells from mononuclear cells of rat bone marrow by FACS. (A) Control sample. R1 region of lymphocytes was selected for gating. There are no positive cells in R2 region. (B) A sample of cells labeled with anti-SSEA-1 antibody. Positive cells are located in the right R2 region after gating. SSEA-1⁺ cells are 0.015% in the mononuclear cells. (C and D) FACS analysis of lineage markers on SSEA-1⁺ cells. Expression of CD45, Ter-119, Gr-1, and CD11b on SSEA-1⁺ cells is negative.

Proliferative ability and morphological features

Diameter of SSEA-1⁺ cells is 4–5 μm. The cell contains less cytoplasm, the nucleus is large, and the ratio of the nucleus to cytoplasm is very high. The organelles are not rich (Fig. 2A). At 1 day after incubation on MEF feeder layer, SSEA-1⁺ cells were round, and singly and dispersedly distributed. Microvilli on SSEA-1⁺ cells were rich. At 7 days, the cells proliferated into clusters, and the feeder cells under the cluster were pushed away (Fig. 2B). When SSEA-1⁺ cells were incubated in 0.3% agarose, the cells were dispersed at 1 day. At 1 week after incubation, the cells proliferated into clusters. The diameter of the most clusters is 30–50 μm. At 2 weeks after incubation, the cells are grown to embryoid-like bodies (Fig. 2C). In the culture dish without 0.3% agarose, proliferation of SSEA-1⁺ cells was not obvious.

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FIG. 2.

Ultrastructural features of SSEA-1⁺ cells and proliferation of the cells on MEF feeder. (A) Transmission electron microphotograph of a SSEA-1⁺ cell. The nucleus is large, and the chromatin in the nucleus is mainly euchromatin. The cytoplasm is less, and the organelles are not abundant. Scale bar=1 μm. (B) SSEA-1⁺ cells are singly distributed at 1 day after incubation on MEF feeder. There are rich microvilli on SSEA-1⁺ cells. At 7 days, the cells proliferated into clusters. The MEFs under the cluster are pushed away. White and black arrows indicate SSEA-1⁺ cells and MEFs, respectively. Scale bars=10 μm (phase contrast microphotographs) and 5 μm (scanning electron microphotographs). (C) Single SSEA-1⁺ cell proliferated into a cluster at 1 week and formed an embryoid-like body at 2 weeks after incubation in soft agarose. Scale bars=10 μm. MEF, mouse embryonic fibroblast.

Expression of PSC transcriptional factors

SSEA-1⁺ cells expressed PSC transcriptional factors Oct4, Nanog, and Sox2. These transcriptional factors were expressed mainly in the nuclei, while SSEA-1 was expressed mostly on the cell membrane (Fig. 3A). The SSEA-1⁺ cells also expressed Sca-1 (Fig. 3A, the lowest row of panels). RT-PCR analysis demonstrated that SSEA-1⁺ cells expressed PSC transcriptional factor mRNAs Oct4, Nanog, Rex1, Sox2, and Fgf4 (Fig. 3B). The expression levels of Oct4, Nanog, and Rex1 in ELSCs were lower than those in ES-D3 cells, but the expression levels of Sox2 and Fgf4 were higher than that in ES-D3 cells. The expression levels of Oct-4, Nanog, Rex1, Sox2, and Fgf4 in ELSCs and ES-D3 cells were significantly higher than those in MMSCs (Fig. 3C).

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FIG. 3.

Expression of PSC transcriptional factors in SSEA-1⁺ cells and 3 germ layer markers on the cells of embryoid-like bodies. (A) In immunostaining, PSC transcriptional factors Oct4, Nanog, and Sox2 are expressed in SSEA-1⁺ cells. Oct4, Nanog, and Sox2 are located in the nucleus, SSEA-1 is mainly distributed on the cell membrane. SSEA-1⁺ cells also expressed Sca-1. Scale bars=5 μm. (B) SSEA-1⁺ cells express PSC transcriptional factor mRNAs Oct-4, Sox2, Nanog, Rex1, and Fgf4. (C) In statistical analysis of PSC transcriptional factor mRNA expression, the levels of Oct4, Nanog, and Rex1 expression are lower in SSEA-1⁺ cells than those in ES-D3 cells, and the levels of Sox2 and Fgf4 expression are also higher. ^# P<0.05. Comparing with MMSCs, mRNA expression of PSC transcriptional factors in SSEA-1⁺ and ES-D3 cells are stronger. *P<0.01. (D) The cells of embryoid-like bodies express Nestin, Brachyury, and E-cadherin after incubation in Dulbecco's modified Eagle's medium without leukemia inhibitory factor and bFGF for 4 weeks. Nuclei were stained with DAPI. Scale bars=50 μm. PSC, pluripotent stem cell; bFGF, basic fibroblast growth factor; MMSC, marrow-derived mesenchymal stem cell; DAPI, 4', 6-diamidino-2-phenylindole. Color images available online at www.liebertonline.com/scd

Expression of 3 germ layer markers

At 1 week after incubation in DMEM without LIF and bFGF, morphological changes of the cells of the embryoid-like bodies were not obvious. At 3–4 weeks after incubation, the cells became flat, oval, or spindle. Expression of ectodermal marker Nestin, mesoderm marker Brachyury, and endoderm marker E-cadherin on the cells could be detected by immunocytochemistry (Fig. 3D). Neither tumor nor teratoma was observed in the inguinal region of SCID mice at 4 weeks after transplantation of ELSCs.

Differentiation toward cardiomyocytes and endothelial cells in vitro

After induction with BMP-2, IGF-1, and bFGF for 2 weeks, SSEA-1⁺ cells became spindle and arranged in parallel. At 3–4 weeks after induction, the cells changed into short columnar shape and conjuncted each other. RT-PCR analysis revealed that the cells expressed Gata4, Nkx2-5, Tnni3k, Tnnt2, and Gja1 at 2 weeks after induction (Fig. 4A). Statistical analysis showed that the expression levels of cardiac early transcriptional factor mRNAs Gata4 and Nkx2-5, and cardiac specific protein mRNAs Tnni3k, Tnnt2, and Gja1 in the induced cells were lower than the levels of the rat cardiomyocytes (Fig. 4B). At 4 weeks after induction, the differentiated cells expressed cTnT and Cx43 (Fig. 4C). In control group, shape of the cells did not obviously change, and expression of cTnT and Cx43 was not observed (Fig. 4C). After induction with VEGF, bFGF, and TGF-β for 2 weeks, RT-PCR analysis demonstrated that the cells expressed Pecam1, Flt1, and Flk1. The expression level of Pecam1 in the induced cells is lower than that in microvascular endothelial cells of rats, but the expression levels of Flt1 and Flk1 in the cells after induction are higher (Fig. 4D, E). At 4 weeks after induction, the differentiated cells became polygon and expressed vWF and CD31 (Fig. 4F). Expression of vWF and CD31 was not observed in the cells without induction by cytokines (Fig. 4F).

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FIG. 4.

Differentiation of ELSCs toward cardiomyocytes and endothelial cells after induction with cytokines in vitro. (A) The cells express Gata4, Nkx2-5, Tnni3k, Tnnt2, and Gja1 at 2 weeks after induction with 10 ng/mL bone morphogenetic protein 2, 10 ng/mL insulin-like growth factor 1, and 10 ng/mL bFGF. (B) The levels of Gata4, Nkx2-5, Tnni3k, Tnnt2, and Gja1 expression in the induced cells were lower than those in rat cardiomyocytes. *P<0.05, **P<0.01. (C) At 4 weeks after induction, the differentiated cells express cTnT and Cx43. Expression of cTnT and Cx43 in the control group was not observed. Scale bars=10 μm. (D) The cells express Pecam1, Flt1, and Flk1 at 2 weeks after induction with 10 ng/mL vascular endothelial growth factor, 10 ng/mL bFGF and 10 ng/mL transforming growth factor beta. (E) The level of Pecam1 expression in cells after induction is lower than that in microvascular endothelial cells of rats, but the levels of Flt1 and Flk1 expression in the induced cells are higher. *P<0.05, **P<0.01. (F) At 4 weeks after induction, the differentiated cells express vWF and CD31. Expression of vWF and CD31 in the control group was not observed. Nuclei were stained with DAPI. Scale bars=10 μm. ELSC, embryonic-like stem cell; vWF, Von Willebrand factor. Color images available online at www.liebertonline.com/scd

Improvement of LV function after cell transplantation

Serial echocardiographic analysis indicated that heart function in all rats was severely compromised after LADCA occlusion for 1 week. Significant functional loss continued to the fourth week in control group. The hearts transplanted stem cells demonstrated an increase in heart function during the same time interval (Fig. 5A). The echocardiograms showed that LVEDD and LVESD in induced-ELSC group were smaller than that in baseline, and control, MMSC and ELSC groups. LVESD in MMSC and ELSC groups was also smaller than that in control group (Fig. 5B, C). In control group, LVEDV and LVESV obviously increased at 4 weeks after MI. Comparing with baseline, and control, MMSC and ELSC groups, LVEDV and LVESV in induced-ELSC group significantly decreased. In MMSC and ELSC groups, LVEDV and LVESV also decreased as compared with control group (Fig. 5D, E). EF and FS significantly increased in MMSC, ELSC, and induced-ELSC groups comparing with control group, and EF in induced-ELSC group also increased comparing with MMSC group (Fig. 5F, G).

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FIG. 5.

Improvement of cardiac function at 4 weeks after cell transplantation. (A) Cardiac function of female rats with myocardial infarction was examined by echocardiography at 4 weeks after transplantation. (B and C) LVEDD and LVESD in induced-ELSC group are smaller than that in baseline, and control, MMSC, and ELSC groups. LVESD in MMSC and ELSC groups is also smaller comparing with control group. (D and E) LVEDV and LVESV in induced-ELSC group significantly decrease comparing with baseline, and control, MMSC, and ELSC groups. Comparing with the control group, LVEDV and LVESV in MMSC and ELSC groups also decrease. (F and G) EF and FS significantly increase in MMSC, ELSC, and induced-ELSC groups comparing with the control group. EF in induced-ELSC group is greater than that in MMSC group. *, #, &, and § indicated that the group is compared with baseline, control, MMSC, and ELSC groups, respectively. *P, ^# P, ^& P, and ^§ P<0.01. n=8 (control group), 10 (MMSC group), 9 (ELSC group), and 11 (induced-ELSC group). LVEDD, LV end-diastolic diameter; LVESD, left venticular end-systolic diameter; LVEDV, LV end-diastolic volume; LVESV, LV end-systolic volume; EF, ejection fraction; FS, fractional shortening.

Histological changes after cell transplantation

Masson's trichrome staining showed that myocardial tissue in the infracted region was mainly replaced by fibrous tissue in control group at 4 weeks after MI. In groups of cell transplantation, the regenerated myocardium in the infarcted region could be observed. Cardiomyocytes in the fibrous tissue obviously increased in induced-ELSC group (Fig. 6A). The percentage of scar area in control, MMSC, ELSC, and induced-ELSC groups was 20.12±2.91, 15.81±1.82, 13.53±1.59, and 8.89±1.25, respectively. Scar area in induced-ELSC group significantly decreased comparing with control, MMSC, and ELSC groups. Scar area in MMSC and ELSC groups also decreased comparing with control (Fig. 7).

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FIG. 6.

Scar area, collagen deposition, and survival and differentiation of the cells at 4 weeks after transplantation. (A) Masson's trichrome staining. In the infracted region, myocardium (red) is significantly replaced by fibrous tissue (blue, arrows) in control group. Myocardium was slightly repaired in MMSC and ELSC groups. In induced-ELSC group, cardiomyocytes in the fibrous tissue obviously increased. (B) Survival and differentiation of the cells. Survival and differentiation of the transplanted cells were examined with fluorescence in situ hybridization and immunofluorescent staining. Y chromosome-positive cells (green, arrows) are located in the peri-infarcted and infarcted regions. There are more Y chromosome-positive cells in induced-ELSC group than MMSC and ELSC groups. Most Y chromosome-positive cells express cTnT and Cx43 (red, arrowheads). The differentiated cells are parallel in arrangement with recipient cardiomyocytes. In induced-ELSC group, Cx43 expression was observed at conjunction of Y chromosome-positive cells and recipient cardiomyocytes (indicated with arrows and arrowheads). In the lowest row of panels, some Y chromosome-positive cells (red, arrows) in the peri-infarcted and infarcted regions express CD31 (green, arrowheads). In induced-ELSC group, Y chromosome-positive cells expressing CD31 are situated on the wall of the microvessels. Large squares are high magnification of the selected areas (small squares). Scale bars=20 μm. Color images available online at www.liebertonline.com/scd

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FIG. 7.

Quantity analysis of scar areas after cell transplantation. Scar area in induced-ELSC group significantly decreases comparing with control, MMSC, and ELSC groups. Scar areas in MMSC and ELSC groups also decrease comparing with the control group. ^# P, ^& P, and ^§ P<0.01. n=8 (control group), 10 (MMSC group), 9 (ELSC group), and 11 (induced-ELSC group).

Differentiation of the transplanted cells in vivo

At 4 weeks after cell transplantation, Y chromosome fluorescence in situ hybridization demonstrated that Y chromosome-positive cells were observed in the peri-infarcted and infarcted regions. There are more Y chromosome-positive cells in induced-ELSC group comparing with MMSC and ELSC groups, and most Y chromosome-positive cells express cTnT and Cx43 (Fig. 6B). Expression of cTnT and Cx43 in the Y chromosome-positive cells indicates that transplanted ELSCs undergo cardiomyocyte differentiation in the peri-infarcted and infarcted regions. The differentiated cells are parallel in arrangement with recipient cardiomyocytes. Cx43 expression was observed at conjunction of Y chromosome-positive cells and recipient cardiomyocytes in induced-ELSC group (Fig. 6B). The result indicates that gap junction may be established between the differentiated cells and recipient cardiomyocytes.

Angiogenesis in the peri-infarcted and infarcted regions was determined by the number of CD31⁺ microvessels at 4 weeks after transplantation. Y chromosome fluorescence in situ hybridization demonstrated that some transplanted cells expressed CD31, and density of microvessels significantly increase in induced-ELSC group comparing with MMSC and ELSC groups. Y chromosome-positive cells expressing CD31 were observed on the wall of some microvessels in induced-ELSC group (Fig. 6B, the lowest row of panels). The results imply that the transplanted stem cells undergo endothelial differentiation and directly participate in angiogenesis in the peri-infarcted and infarcted regions.