Human embryonic stem cell-derived cardiomyocyte therapy in mouse permanent ischemia and ischemia-reperfusion models
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Ischemic heart diseases are still a threat to human health. Human pluripotent stem cell-based transplantation exhibits great promise in cardiovascular disease therapy, including heart ischemia. The purpose of this study was to compare the efficacy of human embryonic stem cell-derived cardiomyocyte (ESC-CM) therapy in two heart ischemia models, namely, permanent ischemia (PI) and myocardial ischemia reperfusion (IR).
Human embryonic stem cell-derived cardiomyocytes were differentiated from engineered human embryonic stem cells (ESC-Rep) carrying green fluorescent protein (GFP), herpes simplex virus-1 thymidine kinase (HSVtk), and firefly luciferase (Fluc). Two different heart ischemia models were generated by the ligation of the left anterior descending artery (LAD), and ESC-Rep-derived cardiomyocytes (ESC-Rep-CMs) were transplanted into the mouse hearts. Cardiac function was analyzed to evaluate the outcomes of ESC-Rep-CM transplantation. Bioluminescence signal analysis was performed to assess the cell engraftment. Finally, the inflammation response was analyzed by real-time PCR and ELISA.
Cardiac function was significantly improved in the PI group with ESC-Rep-CM injection compared to the PBS-injected control, as indicated by increased left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), as well as reduced fibrotic area. However, minimal improvement by ESC-Rep-CM injection was detected in the IR mouse model. We observed similar engraftment efficiency between PI and IR groups after ESC-Rep-CM injection. However, the restricted inflammation was observed after the injection of ESC-Rep-CMs in the PI group, but not in the IR group. Transplantation of ESC-Rep-CMs can partially preserve the heart function via regulating the inflammation response in the PI model, while little improvement of cardiac function in the IR model may be due to the less dynamic inflammation response by the mild heart damage.
Our findings identified the anti-inflammatory effect of ESC-CMs as a possible therapeutic mechanism to improve cardiac function in the ischemic heart.
KeywordsIschemic heart disease Embryonic stem cell Cardiomyocyte Cell therapy
Embryonic stem cell
Embryonic stem cell-derived cardiomyocyte
Green fluorescent protein
Left anterior descending artery
Left ventricular ejection fraction
Left ventricular fraction shortening
Ischemic heart disease is a leading cause of death worldwide . Cardiac ischemia, known as myocardial infarction (MI), usually triggers massive cardiomyocyte death. Due to the limited regenerative capacity of the heart , most patients who suffered from permanent ischemia (PI) will develop into heart failure. Currently, reperfusion strategies, such as thrombolytic therapy or primary percutaneous coronary intervention, are still the standard and most effective therapeutic treatment for acute myocardial infarction . Despite the potential to salvage myocardial ischemia, ischemia reperfusion (IR) usually leads to paradoxical cardiomyocyte dysfunction and worsens heart damage in what is known as ischemia-reperfusion injury [4, 5]. In contrast to IR, permanent ischemia significantly changes heart structure and function . It remains uncertain whether transplantation outcomes are correlated with the ischemic microenvironment in the heart. Thus, preclinical studies in animal models of heart ischemia are necessary to fully evaluate the cell therapeutic efficacy.
Over the past decades, extensive efforts have been made to repair heart tissue by promoting cardiac regeneration [7, 8], through inducing cardiomyocyte proliferation or reprogramming fibroblasts into cardiomyocytes [9, 10, 11]. Nevertheless, the limited cardiomyocyte proliferation rate and lower direct reprogramming efficiency restrict their application in heart repair . In recent years, pluripotent stem cell-based regenerative therapy has shown great promise in heart repair and functional improvement . Among several cell types derived from human pluripotent stem cells, cardiomyocytes hold great promise for cardiac repair, and human embryonic stem cell-derived cardiomyocytes (ESC-CMs) can improve cardiac function and attenuate myocardial remodeling after myocardial infarction [14, 15, 16, 17]. However, the efficacy of ESC-CM-mediated cardiac repair is still controversial. We speculated that different ischemia models may result in different heart repair and cell retention outcomes.
Embryonic stem cell-derived cardiomyocytes exhibited great application perspective in heart disease therapy, while the efficiency of cell transplantation is still low, and the underlying mechanisms need to be further investigated . Early reperfusion after ischemia benefits heart function in the clinic . However, whether this operation could improve cell transplantation efficiency was still unknown. The animal PI and IR models are widely used in heart repair studies . To provide evidence for optimizing cell transplantation strategy, this study investigated the efficacy of ESC-CM therapy in the two different heart ischemia models. Meanwhile, we explored the possible reasons for distinct cardiac repair outcomes of ESC-CMs in these two models.
Human embryonic stem cell culture
For routine maintenance, undifferentiated ESCs were cultured on growth factor-reduced Matrigel (Corning, USA)-precoated dishes in complete mTeSR™1 medium (STEMCELL Technologies, Canada) and passaged every ~ 4 days using 0.5 mM EDTA (Sigma, USA). Rho-associated protein kinase (ROCK) inhibitor thiazovivin (Selleck Chemicals, USA) was added during cell passaging to prevent dissociation-induced ESC apoptosis.
Generation of reporter-engineered human embryonic stem cells (ESC-Rep)
The DNA fragment containing green fluorescent protein (GFP), Herpes Simplex Virus-1 Thymidine Kinase (HSVtk), and firefly luciferase (Fluc) was inserted to AAVS1 locus of PPP1R12C gene through CRISPR/Cas9-mediated homologous recombination. The donor plasmid contains CAG promoter-derived three reporter genes using 2A peptide fusion method for co-expression. A splice acceptor element and a 2A linker were placed in front of the puromycin-polyA cassette, which expressed the puromycin resistance gene for positive clone selection. After transfection, puromycin-resistant and GFP+ cells were enriched by puromycin (1 μg/mL), and single GFP+ cell clone was picked for expansion. Finally, the expression of three reporter genes and pluripotency markers were confirmed. The edited human embryonic stem cell line was named as ESC-Rep.
ESCs were split and cultured as described above. When cells reached ~ 90% confluence, cardiomyocyte differentiation was initiated by changing the culture medium to differentiation medium CDM3 . During cardiomyocyte differentiation, cells were treated with 5 μM of the glycogen synthase kinase 3-β inhibitor CHIR99021 (Sigma, USA) on days 0–2 and 2 μM of the Wnt pathway inhibitor Wnt-C59 (Selleck Chemicals, USA) on days 4–6. The medium was changed daily, and spontaneous beating was noted from day 9. For cardiomyocyte purification, the cells were replated and cultured in CDM3L medium, which consisted of glucose-free RPMI 1640 (Thermo Fisher, USA) and 5 mM sodium dl-lactate (Sigma, USA). Cardiomyocytes were then split at 1:4 with 0.25% trypsin (Sigma, USA) containing 0.1 mM EDTA and seeded on 0.1% gelatin-coated dishes in CDM3.
Because female mice have less pronounced maladaptive remodeling and higher survival rate after heart injury , female severe combined immune deficiency (SCID/beige) mice (8 weeks old) were used in this study to exclude the gender influence on the heart function. Mice were randomly grouped into 5 groups: Sham control (n = 15), PI groups that received permanent coronary occlusion followed by PBS (n = 15) or ESC-Rep-CM injection (n = 18), and IR groups subjected to transient ischemia for 30 min followed by reperfusion and injection of PBS (n = 15) or ESC-Rep-CMs (n = 18). After left thoracotomy, the left anterior descending artery (LAD) was ligated, either permanently (PI groups) or temporarily (IR groups), with a 7–0 prolene suture 1 mm caudally from the tip of the left auricle. In the IR group, the LAD was transiently occluded for 30 min before removing the suture. After the PI or IR operation, a total of 1 × 106 ESC-Rep-CMs in 9 μL PBS were injected in equal portions into three sites beneath the ligation position. In the PBS group, an equal volume of PBS was injected at the same positions. To evaluate the influence of inflammatory microenvironment in the functional improvement of IR hearts, the mice were randomized into 5 groups (n = 5 for each group) including Sham control, PBS, ESC-Rep-CMs, IL-10, and ESC-Rep-CMs+IL-10. The mouse recombinant IL-10 (50 μg/kg) was subcutaneously injected into the mice in IL-10 and ESC-Rep-CMs+IL-10 groups at days 0, 1, and 3 post-surgery.
The Visual Sonics Vevo 2100 system equipped with a medium-frequency (30 MHz) MS-400 transducer was used for evaluating the cardiac function as previously reported . Generally, two-dimensional long-axis and short-axis left ventricle imaging were collected for analysis. M-mode tracings were recorded through the septum and posterior LV walls to measure LV dimension and wall thickness. Left ventricular end-diastolic diameter and end-systolic diameter were measured and used to calculate left ventricular ejection fraction (LVEF) and fraction shorting (LVFS). Pulse-wave Doppler was recorded from the apical 4-chamber view. E (the peak early transmitral flow velocity), A (the peak late transmitral flow velocity), and E/A (the ratio of the peak early transmitral flow velocity to the peak late transmitral flow velocity) were measured and analyzed as previously described . In addition to the cardiac functional evaluation by echocardiography, the heart fibrosis area and fibrotic marker expression were also detected to determine the injury degree of heart.
Quantitative real-time PCR
To analyze mRNA expression, cells were dissociated, and pellets of cells were snap-frozen in liquid nitrogen and stored at − 80 °C. Total RNA was isolated using Trizol Reagent (Sigma, USA), and cDNA was produced using a PrimeScript™ 1st Strand cDNA Synthesis Kit (Clontech, USA). Real-time PCR was performed using PrimeScript™ RT Master Mix (Clontech, USA) in a StepOnePlus™ Real-Time PCR System (Thermo Fisher, USA) as previously reported . Gene expression levels were normalized to the endogenous reference gene GAPDH and assessed using the comparative threshold cycle (2−ΔΔCt) method. All primer sequences are listed in Additional file 1: Table S1.
Cells and heart sections were fixed in 4% paraformaldehyde (PFA) for 10 min at room temperature (RT). After permeabilized with 0.2% Triton X-100 in PBS, samples were blocked in 5% BSA (Sigma, USA) at RT for 1 h, then incubated with indicated primary antibodies at 4 °C overnight. After washing with PBS-T (0.1% Tween 20 in PBS), cells or sections were incubated with corresponding fluorescent secondary antibodies at RT for 1 h, and then counterstained with Hoechst 33342 (Sigma, USA). Images were captured with a confocal microscope (ZEISS, Germany). Antibodies used in this study are listed in Additional file 1: Table S2.
Bioluminescence imaging in vitro and in vivo
For in vitro imaging, cells were seeded as indicated. Before analysis, cells were washed with D-PBS carefully and incubated in medium containing 150 μg/mL d-luciferin (Gold Biotechnology, USA) at 37 °C for 5 min. Signals were detected by an in vivo imaging system (PerkinElmer, USA). For in vivo imaging, mice were anesthetized and simultaneously received an intraperitoneal injection of d-luciferin at 150 mg/kg (luciferin/body weight). Ten minutes post-injection, images were recorded for 10 min with 1-min acquisition intervals. As previously described, the bioluminescence from a fixed region of interest was processed with IVIS imaging systems, Living Image 4.5 (PerkinElmer, USA), and the data for cell retention and survival were quantified in units of photons per second per centimeter squared per steradian (p/s/cm2/sr) .
Hearts from PI and IR groups at 4 weeks after surgery were harvested, fixed, dehydrated, and immersed in O.C.T. compound. Serial 5-μm-thick frozen sections were collected following a standard protocol. To determine the fibrotic degree of the heart, sections were stained with Masson’s trichrome (MT) using the Trichrome Stain kit (Sigma, USA) according to the manufacturer’s instructions. Collagen deposition was visualized as blue staining. Immunofluorescence staining for human nuclear antigen (HNA) was used to locate ESC-Rep-CM engraftment.
On days 1, 3, and 7 after surgery, blood was obtained via retroorbital bleeding and allowed to clot at RT. The levels of inflammation factors in sera, namely, TNF-α, IL-6, and IL-10, were measured using the Mouse TNF alpha ELISA kit (Abcam, USA), Mouse IL-6 ELISA kit (Abcam, USA), and Mouse IL-10 ELISA kit (Abcam, USA) as per the manufacturer’s instructions.
Determination of MPO activity
MPO activity was measured according to the instructions of a MPO kit (Jiancheng Bio, China). Briefly, the homogenized tissue samples were sonicated to release the MPO from the tissue into the supernatant. After the addition of o-dianisidine hydrochloride and hydrogen peroxide, MPO activity was detected at 460 nm according to the spectrophotometer method.
Comparisons between two groups were analyzed using Student’s t test. Comparisons in multiple groups were analyzed with one-way analysis of variance (ANOVA) or two-way repeated-measures analysis of variance with the Bonferroni post hoc test. Statistical significance was denoted by a p value of less than 0.05. Data are presented as the mean ± SEM.
Generation and identification of ESC-derived cardiomyocytes
Video S1. The representative movie of spontaneous beating cardiomyocytes derived from reporter cells at day 20 after purification. (MP4 848 kb)
Cardiac performance after ESC-Rep-CM injection in two heart ischemia models
Retention of the transplanted ESC-Rep-CMs in mouse heart
Inflammation response in mouse heart after ESC-Rep-CM transplantation
Myocardial infarction animal models are typically induced by occlusion of the left anterior descending artery. According to the ligating time, there are two different ischemia models, which are termed PI and IR respectively in this study. Ligation for 30–45 min in an ischemia-reperfusion model is well accepted , but longer ischemia (> 50 min) results in unacceptably high mortality (nearly 100%) either during the ischemic process or in the first week after the surgery . Thus, a conservative ligating time (30 min) was used in the ischemia-reperfusion model (IR) in our study. Our data on infarct area differences between the PI and IR groups were consistent with other studies, which also observed a significantly larger infarct area after permanent ischemia . The cardiac function of IR mice was also much better than that of PI mice. In this regard, our study revealed that ESC-Rep-CM transplantation could partially preserve the cardiac function of PI mice, but showed a mild benefit in the cardiac performance of IR mouse hearts, indicating the degree of injury affected the outcome of cell therapy. Considering the similar cell retention in two different models, we speculated the degree of heart injury and the different heart remodeling might affect the outcomes of cell therapy. Meanwhile, we noticed the gradual loss of transplanted cells might attenuate the efficiency of cell therapy. Thus, the strategy of cell transplantation needs to be further optimized. Previous studies have shown the combination of biomaterial and cytokines can promote cell survival and retention in the infarct area [32, 33], and these methods should further benefit the heart function in both PI and IR models. Due to the comparable characteristics between ESCs and induced pluripotent stem cells (iPSCs) [34, 35, 36], the same therapeutic effects of ESC- and iPSC-derived cardiomyocytes in these two heart ischemia models would be expected.
Transplanted cardiomyocytes can improve the heart function in several animal MI models through functional integration, paracrine-mediated inhibition of cardiomyocyte apoptosis, and enhancement of the vasculogenic response [15, 37, 38, 39]. To our knowledge, the extremely high rate of post-transplantation cell death is a major issue that limits the functional efficacy of cell therapy for ischemic heart disease. We therefore first evaluated the engraft efficiency of ESC-Rep-CMs in the two different cardiac ischemia models, but we did not find an obvious difference in cell retention. Thus, the distinct cardiac improvement of ESC-Rep-CM transplantation in the PI and IR is not due to grafting efficiency and might be related to the distinct changes to the microenvironment in the two models.
Cardiac inflammation response is an important event within the first week after ischemia. After injury, the inflammatory response localizes into the infarcted zone, and it leads to the migration of monocytes, neutrophils, and macrophages . Among these, neutrophils have traditionally been regarded as pro-inflammatory effectors and are undoubtedly major effectors of acute inflammation . In a mouse model, when TGF-β was specifically blocked in cardiomyocytes, cardiac function was improved due to decreased neutrophil recruitment to the heart . These results indicated that the inflammatory response was very important for heart remodeling and function. Thus, if cardiac inflammation could be repressed at the right time after injury, it may be beneficial for cardiac performance. A recent study revealed a benefit of transplanted myoblasts in inhibiting the inflammatory response in cardiac tissue after MI . We therefore asked whether inflammation repression was positively correlated with improved cardiac function. Based on our data, inflammation was alleviated after cardiomyocyte injection in the PI group with the decreasing levels of TNF-α and IL-6 and the increasing level of IL-10. MPO activity was also decreased. However, in the IR group, inflammatory response and MPO activity were not changed. This may have been caused by the less dynamic change in inflammation profile of the IR group. Because temporary ischemia time in the IR model was 30 min in our study, the cardiac function was still preserved, and the inflammation reaction was less severe than permanent ischemia. IL-10 is a potent anti-inflammatory cytokine as well as a cardio-protective factor in the ischemic heart . Consistent with previous reports on the PI model, our data showed that, even in a lower inflammatory microenvironment, injection of the anti-inflammatory factor IL-10 could mildly benefit the cardiac performance. Taken together, when cell therapy is purposed to treat ischemic heart, the microenvironment in the heart such as inflammation response should also be taken into consideration.
In conclusion, cardiomyocytes derived from ESCs could reside in mouse ischemia models, but the retention and survival of these cells were model-independent. Cardiac function was preserved in the PI group and this protection may be related to the decreased inflammation.
YY, NQ, and X-AL contributed equally to this work. WL, Z-AZ, and SH were responsible for the concept and design of this study. WL, Z-AZ, and SH were responsible for financial and administrative support. YY, X-AL, XN, and LY carried out all cellular and molecular experiments. NQ and JL carried out all animal studies. XH helped to collect mouse samples. YY, NQ, and SH were responsible for manuscript writing. ZS, WC, Z-AZ, and WL revised the manuscript. All authors read and approved the final manuscript.
This study was supported by the National Key R&D Program of China (2017YFA0103700), National Natural Science Foundation of China (81770257, 81600218), Natural Science Foundation of Jiangsu Province (BK20170002), Natural Science Foundation for Colleges and Universities in Jiangsu Province (17KJA310006), Suzhou Municipal Science and Technology Foundation (SYS201675), National Clinical Key Specialty of cardiovascular surgery, Jiangsu Clinical Research Center for Cardiovascular Surgery, Jiangsu Province’s Key Discipline/Laboratory of Medicine (XK201118), and National Center for International Research (2017B01012).
Ethics approval and consent to participate
All surgical procedures and animal care protocols in this study were approved by the Laboratory Animal Research Committee of Soochow University.
Consent for publication
The authors declare that they have no competing interests.
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