Cardioprotective effects of genetically engineered cardiac stem cells by spheroid formation on ischemic cardiomyocytes

Background Sca-1+ cardiac stem cells and their limited proliferative potential were major limiting factors for use in various studies. Methods Therefore, the effects of sphere genetically engineered cardiac stem cells (S-GECS) inserted with telomerase reverse transcriptase (TERT) were investigated to examine cardiomyocyte survival under hypoxic conditions. GECS was obtained from hTERT-immortalized Sca-1+ cardiac stem cell (CSC) lines, and S-GECS were generated using poly-HEMA. Results The optimal conditions for S-GECS was determined to be 1052 GECS cells/mm2 and a 48 h culture period to produce spheroids. Compared to adherent-GECS (A-GECS) and S-GECS showed significantly higher mRNA expression of SDF-1α and CXCR4. S-GECS conditioned medium (CM) significantly reduced the proportion of early and late apoptotic cardiomyoblasts during CoCl2-induced hypoxic injury; however, gene silencing via CXCR4 siRNA deteriorated the protective effects of S-GECS against hypoxic injury. As downstream pathways of SDF-1α/CXCR4, the Erk and Akt signaling pathways were stimulated in the presence of S-GECS CM. S-GECS transplantation into a rat acute myocardial infarction model improved cardiac function and reduced the fibrotic area. These cardioprotective effects were confirmed to be related with the SDF-1α/CXCR4 pathway. Conclusions Our findings suggest that paracrine factors secreted from transplanted cells may protect host cardiomyoblasts in the infarcted myocardium, contributing to beneficial left ventricle (LV) remodeling after acute myocardial infarction (AMI).


Background
Cardiac stem cells antigen-1 positive (Sca-1+) cells possess properties of cardiac and endothelial cell differentiation (Oh et al. 2003;Matsuura et al. 2004;Takamiya et al. 2011) and occur in adult murine hearts, which contain potential stem cells (Wang et al. 2014;Tateishi et al. 2007). For example, the knockdown of Sca-1 transcripts in cardiac stem cells (CSCs) significantly inhibited CSC proliferation and survival, resulting in decreased myocardial contractility (Tateishi et al. 2007;Bailey et al. 2012). Conversely, the expression of Sca-1+ CSCs significantly increased in the mouse heart after acute myocardial infarction (AMI) (Wang et al. 2006). Hypoxic injury such as AMI promoted Sca-1+ CSC migration to the infarcted zone to induce myocardial renewal (Liu et al. 2013).
Sca-1+ CSCs account for only 2% of all heart cells (Oh et al. 2003). Limited numbers of Sca-1+ CSCs and their limited proliferative potential were the major limitations for use in various studies. However, various genetically engineered stem cells inserted with human telomerase reverse transcriptase (TERT) gene showed immortalization without chromosomal aberrations or malignant transformation (Huang et al. 2008;Burk et al. 2019;Wolbank et al. 2009). Our previous study revealed that genetically engineered CSCs (GECS) maintained the stemness even after long-term culture (Park et al. 2016) .
In transitional two-dimensional (2D) cell systems, there were limitations in viability, proliferation, differentiation, and function. However, spheroids provided a three-dimensional environment that enabled intensive cell-to-cell contact and enhanced regenerative properties (Laschke and Menger 2017). To form spheroids, poly-2-hydroxyethyl methacrylate (poly-HEMA) is typically used as a non-adherent coating material for cell aggregation (Long et al. 2014). In our previous study, sphere formation of adipose stem cells was successfully engineered using poly-HEMA (Kim et al. 2015). Compared with adherent adipose stem cells, sphere formation could reduce antiapoptotic marker expression and increase that of hypoxic and growth factors.
Previous studies showed that CSCs secreted various cytokines and chemokines (Wollert and Drexler 2010;Tran and Damaser 2015). However, reports regarding paracrine factors secreted by sphere GECS (S-GECS) and their cardioprotective roles have been limited until now. This study aimed to establish S-GECS, investigate paracrine factors secreted by the S-GECS, and clarify their cardioprotective roles in in vitro and in vivo models.

Generation of GECS and characterization
GECS was obtained from hTERT-immortalized Sca-1+ CSC lines in our previous study (Fig. 1a) (Park et al. 2016). Sca-1+ CSCs sorted by magnetic-activated cell sorting were plated at 2 × 10 5 cells in 6-cm culture dishes in Dulbecco's modified Eagle's medium-low glucose (DMEM-LG) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/ streptomycin (P/S). Cells were infected with retroviruses harboring pLPCX-TERT-IRES-EGFP at 60% confluence for 3 days, and then selected in medium against 0.5 μg/mL puromycin in 10-cm culture dishes by repeated sub-culturing at a 1:3 ratio; this was performed three times per week for three weeks. For clonal analysis, the selected cells were plated in 96-well plates at one cell per 100 μL by limiting dilution in DMEM-LG supplemented with 10% FBS and 100 U/mL P/S. Briefly, wells containing one cell per well were selected by visual inspection alone 24 h after plating and then further cultured for 12 days. Among 20 single-cell-derived clones, two were finally selected based on microscopic examination of morphology, proliferation, green fluorescent protein expression, and hTERT expression.
To assess cardiac or endothelial differentiation, the cells were fixed with 4% PFA in PBS for 10 min, washed with PBST, and permeabilized with 0.1% Triton X-100 in PBS for 30 min. Cells were washed with PBST and blocked for nonspecific binding by incubation in PBST with 5% NGS for 30 min. Then, cells were incubated overnight at 4°C with the following primary antibodies: anti-cardiac troponin T (cTnT; Developmental Studies Hybridoma Bank, Iowa City, Iowa, USA) and anti-von Willebrand factor (vWF; DAKO, Carpinteria, CA, USA). After washing three times with PBST, the cells were stained with Alexa Fluor 594-conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA) for 30 min and washed three times in PBST. Nuclei were stained with DAPI. The cells were mounted with fluorescent mounting medium. Fluorescence images were obtained Adipogenic differentiation of GECS was induced by incubation in DMEM-LG supplemented with 5% FBS, 100 U/mL P/S, 1 μM dexamethasone, 10 μg/mL insulin, 100 μM indomethacin, and 0.5 μM methyl-isobutylxanthin (all from Sigma-Aldrich, St. Louis, MO, USA) for 10 days. Culture media were changed every 3 days. Adipogenic differentiation was assessed on day 10 using Oil Red O (Sigma-Aldrich, St. Louis, MO, USA) stain to indicate intracellular lipid accumulation. The cells were fixed with 4% PFA in PBS for 20 min, washed with 60% isopropanol, and stained with 0.3% Oil Red O solution in 60% isopropanol for 10 min. After washing three times with water, cells were de-stained in 100% isopropanol for 15 min. Osteogenic differentiation of GECS was induced by incubation in culture medium with 1 μM dexamethasone, 10 mM glycerophosphate, and 50 μM ascorbic acid (all from Sigma-Aldrich, St. Louis, MO, USA) for 21 days. Osteogenic differentiation was determined by Alizarin Red S (Sigma-Aldrich, St. Louis, MO, USA) staining.

Culture of sphere-genetically engineered cardiac stem cell formation
To produce S-GECS, 6-well tissue culture plates were coated with poly-HEMA (Sigma-Aldrich, St. Louis, MO, USA), which was dissolved in cell culture-tested ethanol at 12 mg/mL concentration (Merck Millipore, Burlington, MA, USA), and incubated at 40°C overnight. The S-GECS was seeded into each plate at a density of 1 × 10 6 cells/well and cultured up to 48 h in culture media to identify the ideal conditions for sphere formation. Phase-contrast images were obtained using the Leica DMI 3000B upright microscope (Leica, Wetzlar, Germany). S-GECS diameters were quantified using ImageJ v1.32 software (National Institutes of Health, Bethesda, MD, USA).

Apoptosis assay
To induce hypoxia via cobalt chloride (CoCl 2 ; Sigma-Aldrich, St. Louis, MO, USA), the cardiomyoblast cell line H9c2 was used. Then, 2 × 10 5 H9c2 cardiomyoblasts were seeded in six-well culture dishes and allowed to reach 80% confluence in DMEM-LG supplemented with 10% FBS and 100 U/mL P/S in a humidified incubator at 37°C and 5% CO 2 . Cells were treated with or without 150 μM CoCl 2 for 24 h in Mesencult MSC Basal Medium (STEM-CELL Technologies, Vancouver, Canada) supplemented with 2% FBS and 100 U/mL P/S or in S-GECS CM. Annexin V (AV) and propidium iodide (PI) staining were performed using an FITC Annexin V Apoptosis Detection Kit II (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions, analyzed on a FACS CantoII flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed using FlowJo v10 software.

Acute myocardial infarction model and cell transplantation
A total of 18 rats were sorted randomly at a 1: 1: 1 ratio into 3 groups: the sham control, A-GECS, and S-GECS. Female SD rats weighing 180-200 g were anesthetized by intraperitoneal (IP) injection with a mixture of ketamine (60 mg/kg) and xylazine hydrochloride (7.5 mg/kg). An 18-gauge angiocatheter (BD Biosciences, San Jose, CA, USA) was utilized as an intubation tube throughout the procedure. The left coronary artery of the heart was ligated with a 6-0 silk suture located 5 mm from the left coronary atrial appendage. After confirming the presence of AMI, A-GECS (1 × 10 6 ) or S-GECS prepared in 100 μL culture medium were injected at three periinfarct areas. S-GECS were harvested after 48 h of plating 1 × 10 6 GECS/well on poly-HEMA-coated 6-well plates. Larger 25-gauge syringes were used to deliver S-GECS (Cho et al. 2017). The A-GECS seeding confluency was 90% (Dong et al. 2012). For the control treatment, 100 μL culture medium was injected. After cell transplantation, the chest wall, muscle layers, and skin were closed with 3-0 silk sutures. All AMI-induced rats were continuously monitored from surgery to recovery. For sacrifice, rats were euthanized by IP injection with a mixture of ketamine (60 mg/kg) and xylazine hydrochloride (7.5 mg/kg).

Echocardiographic analysis
Echocardiography was performed at 1, 7, and 28 days after cell transplantation using a Vivid 7 Echocardiography System (GE Healthcare, Chicago, IL, USA) with a 10 MHz small linear array transducer for animal research. Parasternal long-and short-axis views were obtained. Rats were anesthetized with a mixture of ketamine (60 mg/kg) and xylazine hydrochloride (7.5 mg/kg). The posterior wall thickness in diastole and systole (PWTd and PWTs, respectively), left ventricular end of diastolic and systolic volumes (LVEDV and LVESV, respectively), and left ventricular anterior wall thickening were measured using a 2-dimensional Mmode view. The LV volume and ejection fraction (EF) were calculated using the modified Simpson's method. The fractional shortening (FS) percentage was also computed. All parameters were assessed over 3 consecutive cardiac cycles, and each value was averaged from two measurements. Echocardiography was performed by an experienced cardiologist who was blind to the study group.

Immunohistochemical analysis of tissue sections
The heart tissues were fixed in 4% PFA and embedded in paraffin. Then, 5 or 10 μm thick sections were divided from the infarcted LV wall and septum of paraffinembedded hearts.
Masson's trichrome (MT) staining was performed using the Trichrome Stain Kit (Sigma-Aldrich, St. Louis, MO, USA) with the following modifications: nuclei were stained with Celestine Blue solution followed by Gill's hematoxylin stain (both from Sigma-Aldrich, St. Louis, MO, USA), and tissue was incubated for 1 h in Bouin's solution before muscle staining with Biebrich scarlet-acid fuchsin (Sigma-Aldrich, St. Louis, MO, USA). Stains were quantified using Image-Pro 7.0 software (Media Cybernetics, Rockville, MD, USA).
Hematoxylin and eosin (H&E) staining was conducted by the conventional method. For immunohistochemical staining, the heart tissue sections were treated with proteinase K (Merck Millipore, Burlington, MA, USA) incubated, blocked with 5% NGS, and washed with PBST. Then, the tissue sections were incubated with primary antibodies against CXCR4 (Abcam, Cambridge, UK) and CD31 (BD Biosciences, San Jose, CA, USA). The tissue sections were subsequently stained with Alexa Fluor 594-conjugated antirabbit or anti-rat antibodies (all from Molecular Probes, Eugene, OR, USA) and then incubated with DAPI. Fluorescence images were obtained using a TE-FM Epi-Fluorescence System attached to an Olympus BX61 inverted microscope. CXCR4 cells were quantified using Image-Pro 7.0 software.

Statistical analysis
All statistical values are expressed as the mean ± standard deviation (SD). Significant differences between means were determined using Student's t-test or analysis of variance (ANOVA) followed by the Tukey test. Statistical significance was set at p < 0.05. All statistical analyses were performed using Sigma STAT software v4.0 (IBM, San Jose, CA, USA).
S-GECS was successfully formed on poly-HEMAcoated plates after seeding. The sphere diameter increased along with culture time, and S-GECS morphological changes were observed with phase contrast imaging ( Fig. 1c and d). Regarding the cell number per mm 2 , sphere diameter, and morphology via phase contrast imaging, the optimal conditions for S-GECS was considered to be 1052 GECS cells/mm 2 and 48 h culture time (Fig. 1c). Total cell numbers in each sphere were higher in the sample with 1052 GECS cells/mm 2 than in that with 526 GECS cells/mm 2 at 48 h (165.65 ± 24.09 vs. 641.18 ± 60.47).
HIF-1α and HIF-2α, known as hypoxia-induced survival factors, showed significantly increased mRNA expression, which was increased 1.75-fold and 1.89-fold, respectively, after the 48 h culture compared to 24 h (Additional file 1: Figure S1B). To confirm this condition, qPCR was performed. The 48 h culture showed a similar ratio of the mRNA expression of pro-apoptotic factor BAX/antiapoptotic factor Bcl-2, which was 1.10-fold higher than that in the 24 h culture. To compare the expression of growth factors between the 24 and 48 h cultures, SDF-1α, VEGF-A, IGF-1, and MCP-1 were analyzed by qPCR. The 48 h S-GECS culture showed significantly higher mRNA expression of SDF-1α (2.02-fold), VEGF-A (1.30-fold), IGF-1 (1.4-fold), and MCP-1 (1.19-fold). SDF-1α and IGF-1 were significantly increased in the 48 h culture compared to the 24 h culture.
To clarify which paracrine factors were secreted by A-GECS and S-GECS, growth factor expression was compared by ELISA (Fig. 2c). Compared with A-GECS and S-GECS showed quantitatively higher expression of SDF-1α (2.89-fold) and MCP-1 (1.24-fold), but no differences were found in IGF-1.

S-GECS CM protects H9c2 cardiomyocytes from CoCl 2induced hypoxic injury
CoCl 2 treatment induced apoptotic death of cardiomyocytes through HIF-1α-dependent stabilization of p53 protein. Therefore, CoCl 2 was used to produce a hypoxia-mimicking environment and to determine whether paracrine factors secreted from S-GECS protect against CoCl 2 -induced cardiomyocyte death. The effect of S-GECS CM on early and late apoptosis in CoCl 2 -treated cardiomyocytes is shown in Fig. 3a. At the baseline, the rates of early and late apoptosis were 3.90% and 1.36%, respectively. S-GECS CM significantly reduced the proportion of early apoptotic (AV+/PI-) cardiomyocytes during CoCl 2 -induced hypoxic injury from 21.63 to 3.43% (Fig. 3b); furthermore, the percentage of late apoptotic (AV+/PI+) cardiomyocytes was also reduced from 9.72 to 1.63% in the same conditions.

Cardioprotection by SDF-1α secreted from S-GECS
Since SDF-1α was significantly increased in various analyses, CXCR4 expression was investigated. Western blot analysis showed that CXCR4 expression was significantly increased in H9c2 cardiomyocytes treated with CoCl 2 for 24 h (1.30-fold) as well as in those treated with S-GECS CM (1.20-fold). Co-treatment with CoCl 2 and S-GECS CM showed the highest expression of CXCR4 (1.78-fold) (Fig. 3c).
Gene silencing via CXCR4 siRNA was performed to investigate whether the protective effects of S-GECS was abolished (Fig. 3d). The percentage of apoptosis in H9c2 cardiomyocytes treated with CoCl 2 and NC siRNA for 24 h was significantly decreased from 13.77 ± 1.29% to 6.02 ± 0.59% in the presence of S-GECS CM (Fig. 3e). In H9c2 cardiomyocytes treated with CoCl 2 and CXCR4 siRNA for 24 h, the presence of S-GECS CM significantly decreased the percentage of apoptosis from 16.83 ± 1.55% to 10.08 ± 1.45%. In addition, the percentage of apoptosis was significantly increased after treatment with CXCR4 siRNA compared to NC siRNA in CoCl 2 -and S-GECS CM-treated cells (6.02 ± 0.59% vs. 10.08 ± 1.45%, p < 0.05).
To investigate the downstream pathway of SDF-1α/ CXCR4, western blot analysis of the Akt and Erk signaling pathways was performed. Interestingly, the p-Akt level did not increase with CoCl 2 treatment (Fig. 3f), but was significantly increased following S-GECS CM treatment (1.55-fold). Furthermore, CoCl 2 and S-GECS CM treatment significantly increased p-Erk levels (4.84-fold and 5.93-fold, respectively) compared with non-treated condition (Fig. 3g). The co-treatment of CoCl 2 and S-GECS CM increased the p-Erk level up to 13.11-fold.

Cardioprotection after S-GECS transplantation into infarcted myocardium
S-GECS was injected into the peri-infarct area of a rat model with AMI. Rats in the sham group were injected with an equivalent volume of medium with no cells for use as a control (Fig. 4a).
In the S-GECS group, CXCR4 expression in the border zone was significantly increased compared to that in the sham or A-GECS groups (6.83 ± 1.17 vs. 2.67 ± 0.58 or 4.50 ± 0.71 cells/× 1000 field, respectively; p < 0.05) ( Fig.  4g and h). As GECS were tagged with eGFP, we detected eGFP signals after 28 days of transplantation to reveal their retention in the transplanted sites. The S-GECS group showed better cellular engraftment in the border zone than the A-GECS group (Fig. 4i). In addition, the expression of mCD31, which is known as an endothelial marker, was increased in the border zone compared to the sham or A-GECS groups (Fig. 4j).

Discussion
This study showed that poly-HEMA could produce unattached and floating S-GECS. S-GECS showed significantly reduced antiapoptotic marker levels and increased levels of hypoxic and growth factors compared with A-GECS. These cardioprotective effects were demonstrated to be related with the CXCR4 and SDF-1α pathways. S-GECS transplantation into infarcted hearts could reduce the infarct size and improve cardiac function.
Cell therapy using stem cells remains a highlighted option for neovascularization in many cardiovascular ischemic diseases (Leeper et al. 2010). Among several stem cell types, cardiac stem cells have been investigated and established for use in myocardial regeneration (Beltrami et al. 2003). However, Sca-1+ CSCs account for only 2% of all heart cells, limiting their use for in vitro and in vivo studies. In the previous study, Sca-1+ CSCs were immortalized by the hTERT gene to reduce genetic unsteadiness and sustain similar phenotypic characteristics and multi-differentiation (Park et al. 2016). TERT activity is associated with stem cell function (Blasco 2007). TERT gene insertion into stem cells resulted in immortalization with no evidence of malignant transformation (Bentzon et al. 2005). The established Sca-1+ CSCs possessed a long-term proliferation capacity and multipotent differentiation potential. In addition, cardioprotective effects against hypoxic injury were identified.
Nevertheless, the 2D environment of the cell culture is another limiting factor. In 2D cultures, essential signaling pathways may be lost or compromised Fig. 4 S-GECS transplantation into infarcted myocardium and cardiac regeneration. a Schematic diagram of S-GECS transplantation into infarcted myocardium. b Representative echocardiographic images at 1, 7, and 28 days following cell transplantation. c and d Cardiac function assessed by EF and FS at 1, 7, and 28 days following cell transplantation among three groups. n = 4, 5, and 5 rats in each group, *p < 0.05. e and f Masson's trichrome staining on tissue sections after transplantation to determine the degree of fibrosis in A-GECS and S-GECS groups. *p < 0.05. g and h CXCR4 expression in the border zone in the S-GECS group. *p < 0.05. Nuclei were stained with DAPI (blue). i Representative images showing eGFP-positive transplanted cells in the border zone at 28 days of transplantation. Nuclei were stained with DAPI (blue). j Endothelial marker mCD31 expression in the border zone in the S-GECS group. Nuclei were stained with DAPI (blue). All quantification analyzed using images from n = 4, 5, and 5 rats in each group, each measured in triplicate. All results are representative; scale bars represent 100 μm (Debnath and Brugge 2005). Cell morphology, receptor expression, or ECM interactions may also differ. As a result, 3D culture systems have been increasingly adopted. One relatively simple method for obtaining 3D spheroids is to generate forced-floating cells using poly-HEMA (Lin and Chang 2008). Poly-HEMA is neutrally charged and interferes with cell adhesion proteins, including integrins and cadherins, thereby contributing to the formation of spheres. In our study, sphere formation with poly-HEMA was successful, and this method was beneficial in various ways. The process was simple and reproducible, and equal cell numbers could be seeded in each well. Spheroid size was adjustable if needed. In addition, morphologically homogenous spheroids could be easily produced in large quantities. These morphologic characteristics corresponded to their proliferative potential and differentiation (Kern et al. 2006) as well as the survivability and behavior of cardiac stem cells in ischemic conditions (Li et al. 2007). Therefore, hypoxic factors and growth factors were significantly increased in S-GECS compared to A-GECS under the optimal conditions of 1052 GECS cells/mm 2 and a 48 h culture period. As shown in Fig. 4i, the S-GECS group showed better cellular engraftment in infarcted regions than the A-GECS group. Increased cellular retention in the S-GECS group induced the improvement of cardiac function via secreting more SDF-1α compared with the A-GECS group. In accordance with our results, (Cho et al. 2013;Cho et al. 2012) reported that transplantation of cardiospheres to AMI animal models increased engraftment, differentiation, and paracrine effects in vivo compared with the same cells cultured in a 2D environment. Although all cardiac stem cells expressing c-kit, MDR1, Sca-1, Flk-1, or islet-1 have growth potential, the patterns of secreted growth factors differ slightly (Barile et al. 2007). Particularly, cardiac Sca-1+ cells were enriched with various growth factors and cytokines that might be involved in cardiac repair (Oh et al. 2003). Although the cardioprotective effects of CSCs were evidenced by MCP-1, these effects were confirmed to be related with the CXCR4 and SDF-1α pathways in our study. SDF-1α was expressed in cardiomyocytes and fibroblasts and upregulated in myocardial ischemia (Hu et al. 2007). These results were consistent with previous findings that SDF-1α plays an important role in CSC migration, proliferation, and differentiation (Abbott et al. 2004). As a downstream pathway of SDF-1α, PI3K/Akt signaling was related with cell growth, survival, and protein synthesis (Cain and Ridley 2009). Similarly, the MEK/Erk pathway could transduce extracellular information into intracellular responses, owing to cell chemotactic responses (Tarcic et al. 2012). In fact, SDF-1α stimulated both p-Erk and p-Akt levels (Chen et al. 2015). These findings were consistent with our results that S-GECS treatment increased the levels of p-Erk and p-Akt via the SDF-1α/CXCR4 pathway. These secreted factors from S-GECS would be cardioprotective due to a paracrine mechanism. Hypoxic stress increased the production of various factors (Kinnaird et al. 2004). In fact, the ERK pathway was upregulated in hypoxic conditions (Minet et al. 2000). As shown in our study, hypoxic injury induced by CoCl 2 treatment increased only the level of p-Erk. The administration of S-GECS CM was able to induce beneficial effects even after hypoxic injury, which strongly suggests the involvement of the paracrine mechanism.
After S-GECS was injected into the infarcted area, infarct size was observed to decrease and cardiac function improved compared to controls. Cardiac function evaluated by EF and FS showed significant improvement in the S-GECS group compared to A-GECS or sham groups at 28 days follow-up. The fibrotic area determined by Masson's trichrome staining showed consistent results due to CXCR4 and mCD31 expression in the border zone. The paracrine factors might influence nearby cells and decrease inflammation and fibrosis after AMI by promoting cardiac regeneration (Gnecchi et al. 2008).
There were some limitations in this study. Because this study confirmed significant cardioprotective effects via sphere formation using CSCs, further experiments using other stem cell types will be necessary. In addition, although our study investigated the downstream pathways of CXCR4 and SDF-1α, further investigations should be conducted to confirm the exact mechanisms of antiapoptotic effects. Because functional improvements were assessed by echocardiography, the results may be subjective. Finally, xenograft transplantation was performed by mCSC implantation into rat models with AMI in our study. However, mesenchymal stem cells, including CSCs, have been reported to have low immunogenicity and antigen presentation capabilities. MSCs could moderate T-cell mediated immunological responses and assist cell homing to the ischemic site, thereby inducing immune tolerance to suppress rejection response to xenograft transplantation (Yagi et al. 2010;Potian et al. 2003). Future study should be investigated regarding recipient immune responses after CSC transplantation into AMI models.

Conclusions
In this study, poly-HEMA was capable of producing unattached and floating S-GECS. S-GECS showed significantly reduced antiapoptotic marker levels and increased levels of hypoxic and growth factors compared with A-GECS. These cardioprotective effects were confirmed to be related with the CXCR4 and SDF-1α pathways. S-GECS transplantation into infarcted hearts could reduce the infarct size and improve cardiac function. Our results suggest that the transplanted S-GECS may possess cardioprotective roles in the infarcted myocardium due to paracrine effects, thereby contributing to the improvement of cardiac functions after AMI.
Additional file 1: Table S1. Primers used for real-time PCR in this study. Figure S1. Characterization of GECS. (A) Representative immunofluorescence images and flow cytometry of CSCs positive for CD29, CD44, CD71, CD106, and Sca-1. All results are representative; scale bars represent 100 μm. (B) Quantitative RT-PCR analysis of apoptotic, hypoxic, and growth factors in S-GECS for 24 and 48 h, each in quadruplicate. *p < 0.05 vs. 24 h. Figure S2. Mouse cytokines/chemokines antibody array panels of A-GECS and S-GECS lysates.