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. Author manuscript; available in PMC: 2018 Sep 1.
Published in final edited form as: Circ Res. 2017 Jul 25;121(6):e22–e36. doi: 10.1161/CIRCRESAHA.117.310803

Paracrine Effects of the Pluripotent Stem Cell-Derived Cardiac Myocytes Salvage the Injured Myocardium

Atsushi Tachibana 1,2, Michelle R Santoso 1, Morteza Mahmoudi 1, Praveen Shukla 1, Lei Wang 1,3, Mihoko Bennett 4, Andrew B Goldstone 5, Mouer Wang 1, Masahiro Fukushi 2, Antje D Ebert 1,6,7, Y Joseph Woo 5, Eric Rulifson 1, Phillip C Yang 1
PMCID: PMC5783162  NIHMSID: NIHMS895550  PMID: 28743804

Abstract

Rationale

Cardiac myocytes derived from pluripotent stem cells have demonstrated the potential to mitigate damage of the infarcted myocardium and improve left ventricular ejection fraction (LVEF). However, the mechanism underlying the functional benefit is unclear.

Objective

To evaluate whether the transplantation of cardiac lineage differentiated derivatives enhance myocardial viability and restore LVEF more effectively than undifferentiated pluripotent stem cells after a myocardial injury (MI). Herein, we utilize novel multimodality evaluation of human embryonic stem cells (hESCs), hESC-derived cardiac myocytes (hCMs), human induced pluripotent stem cells (iPSCs), and iPSC-derived cardiac myocytes (iCMs) in a murine MI model.

Methods and Results

Permanent ligation of the left anterior descending coronary artery was induced in immunosuppressed mice. Intra-myocardial injection was performed with (1) hESCs (n=9), (2) iPSCs (n=8), (3) hCMs (n=9), (4) iCMs (n=14) and (5) PBS control (n=10). LVEF and myocardial viability, measured by cardiac-MRI and manganese-enhanced MRI (MEMRI), respectively, was significantly improved in hCM- and iCM-treated mice compared to pluripotent stem cell- or control-treated mice. Bioluminescence imaging (BLI) revealed limited cell engraftment in all treated groups, suggesting that the cell secretions may underlie the repair mechanism. To determine the paracrine effects of the transplanted cells, cytokines from supernatants from all groups were assessed in vitro. Gene expression and immunohistochemistry analyses of the murine myocardium demonstrated significant up-regulation of the pro-migratory, pro-angiogenic, and anti-apoptotic targets in groups treated with cardiac lineage cells compared to pluripotent stem cell and control groups.

Conclusions

This study demonstrates that the cardiac phenotype of hCMs and iCMs salvages the injured myocardium more effectively than undifferentiated stem cells through their differential paracrine effects.

Keywords: Cell therapy, cell transplantation, myocardial infarction, magnetic resonance imaging (MRI), manganese-enhanced MRI (MEMRI)

Subject Term: Cell Therapy, Myocardial Biology, Magnetic Resonance Imaging (MRI), Stem Cells

INTRODUCTION

Cardiovascular disease is the leading cause of death in the US1, 2. Following ischemic injury, the myocardium is damaged, leading to pathological dilatation of the left ventricle (LV) and reduction of left ventricular ejection fraction (LVEF). Cardiac myocytes, with limited regenerative capacity, are injured, infarcted, and replaced by fibroblasts, resulting in the build-up of non-contractile collagenous scars3. This physiologic adaptation progressively decreases the heart’s pumping capacity, leading to heart failure4. Despite recent innovation in surgical, medical, and percutaneous therapeutics, the overall survival of advanced heart failure patients over a 5-year period remains a dismal 50%1. Therefore, alternative approaches are necessary to restore the myocardium post-infarct.

An important development over the past decade has been the emergence of stem cell therapy. Various adult and pluripotent stem cells, including bone marrow stem cells, mesenchymal stem cells, cardiac progenitor cells, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) have been investigated in both clinical and pre-clinical studies5-11. While adult stem cells have demonstrated some clinical benefit, the potential of hESC- and iPSC-derived cardiac myocytes are not fully explored. hESCs and iPSCs differentiate robustly into a highly purified population of functional cardiac myocytes in vitro12, 13. Functional improvement of the injured myocardium has been observed in murine, rodent, and porcine models that received either hESCs-derived cardiac myocytes (hCMs)12, 14-20 or iPSCs-derived cardiac myocytes (iCMs)21, 22. Additionally, pluripotent stem cell-derived cardiac myocytes have been shown to engraft and improve heart function more effectively than non-cardiac derivatives12, 17-19. However, the engraftment is not permanent and the mechanism of action underlying the advantages of cardiac lineage differentiation is not clearly understood. It appears that the injured cardiac myocytes are salvaged via paracrine release of cytokines and growth factors from the transplanted hCMs and iCMs, which result in functional improvement. Paracrine factors constitute the key contributors with pleiotropic effects, including anti-inflammation, anti-apoptosis, anti-fibrosis and pro-angiogenesis. Thus, transplanted stem cells may accelerate the restoration of the injured cardiac tissue through physiologic and sustained release of key cytokines22-25.

The aim of this investigation is to evaluate whether cardiac differentiation of pluripotent stem cells may repair the ischemic myocardium. Systematic investigation of undifferentiated pluripotent stem cells (hESCs, iPSCs) and their cardiac-differentiated derivatives (hCMs, iCMs) is performed, employing novel multi-modality evaluations of cell engraftment, myocardial viability, and cardiac function. These in vivo findings are then validated by ex vivo cellular and molecular assays to confirm paracrine mechanism of action.

METHODS

Detailed experimental methods are available in the Online Supplement

Cell preparations

hESCs line H7 were purchased from WiCell Research Institute26. Monoclonal iPSCs lines were generated by infection of blood mononuclear cells with non-integrating Sendai virus delivering OCT3/4, SOX2, KLF4, L-MYC, LIN-28, short-hairpin RNA for P53 and EBNA127, 28. Zinc finger nuclease technology was used to integrate a reporter gene containing firefly luciferase29.

Electrophysiology of hCMs and iCMs

Whole cell action potentials were recorded by standard patch-clamp technique, as previously described13, 30, 31.

Acute myocardial injury (MI) model and intra-myocardial delivery of cells

All experimental protocols were approved by Administrative Panel on Laboratory Animal Care (APLAC) at Stanford School of Medicine. MIs were induced in severe combined immunodeficient (SCID) beige male mice (90-120 days; Charles River Laboratories) by ligation of the mid left anterior descending coronary artery (LAD) through a left thoracotomy. A blinded surgeon injected 60 μl of following cells (500,000) in a 50:50 mixture of PBS and Matrigel (Corning) into the anterolateral wall: (1) hESCs (n=9), (2) iPSCs (n=8), (3) hCMs (n=9), (4) iCMs (n=14) and (5) PBS control (n=10).

Magnetic resonance imaging (MRI)

Mice were imaged at week 2 and 4 after LAD ligation to evaluate LVEF and myocardial viability. Mice were injected with 0.7 cc/kg of SeeMore (Eagle Vision Pharmaceutical) intraperitoneally prior to manganese-enhanced MRI (MEMRI) acquisition32, 33. MRI scanning was randomized and the reader for the study interpretation was blinded.

Bioluminescence imaging (BLI)

Optical BLI was performed at week 2 and 4 after LAD ligation.

Ex vivo immunohistochemistry of the myocardium

Immunohistochemistry assays of mouse myocardium were performed at week 4 after LAD ligation. Sectioning and H&E staining were performed by a blinded pathologist. For other immunochemical analyses, the specimen demonstrated experimental conditions and required specific techniques to assess the reagents. These conditions may have unblinded the researcher.

Evaluation of the in vitro release of the cytokines from cultured cells

hESCs, iPSCs, hCMs, and iCMs (500,000 cells) were cultured in normoxic conditions for 24 hours. Their supernatants were harvested and analyzed by Luminex Multiplex Assay (Thermo Fisher Scientific).

Ex vivo real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of the myocardium tissues

Total RNA was isolated from cell-treated, control, and wild-type myocardium at the end of the study, reverse transcribed into cDNA, and analyzed for gene expression by real-time RT-PCR.

RESULTS

Electrophysiologic characterization of the in vitro hCMs and iCMs

To validate the cardiac phenotype and compare the contractility of hESC- and iPSC-derived cardiac myocytes, we performed action potential patch-clamp analyses. At days 22-28 of differentiation, sample populations of hCMs and iCMs displayed action potentials (AP) of three major cardiac subtypes, ventricular- (V-), atrial- (A-), and nodal-like (N-like) cardiac myocytes. By comparisons of multiple key action potentials, we determined that hCMs and iCMs were electrophysiologically and functionally similar (Online Table I and Online Figure I).

Sustained engraftment of hCMs and iCMs correlated with improved LVEF

To test whether cardiac differentiation improves cardiac function, we measured LVEF by cardiac MRI (Figure 1A-B). At week 4, mice treated with cardiac myocytes exhibited significantly improved LVEF compared to the control group (hCMs 36.1%*, iCMs 36.6%* vs. control 19.8%, week 4, *p<0.05). The hCM and iCM groups also showed a significantly improved LVEF compared to their undifferentiated pluripotent stem cells (hESC, iPSC groups) at week 4. Notably, the hCM and iCM groups demonstrated significantly improved LVEF between week 2 and 4. In contrast, mice treated with hESCs, iPSCs, or the control had decreased LVEF between week 2 and 4 (Figure 1A-B). These findings suggest that treatment with differentiated cardiac myocytes not only improve LVEF significantly compared to pluripotent stem cells or control, but prevent the progressive deterioration of LV function.

Figure 1. Effects of hESCs, iPSCs, hCMs and iCMs on LVEF and myocardial viability.

Figure 1

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(A) LVEF of hESCs, hCMs, and control at week 2 and 4 after cell transplantation. LVEF is significantly greater in hCMs at week 4 vs. control or hESCs at week 4 or hCMs at week 2. (B) LVEF of iPSCs, iCMs, and control at week 2 and 4 after cell transplantation. LVEF is significantly greater in iCMs at week 4 vs. control or iPSCs at week 4 or iCMs at week 2. (C) Short axis acquisitions during diastole and systole at mid-LV. The hCM and iCM treated mice exhibited higher contractility compared to hESC, iPSC, and control groups. (D) Myocardial viability of hESCs, hCMs, and control at week 2 and 4 after cell transplantation. Myocardial viability is significantly greater in hCMs at week 4 vs. control and hESCs at week 4 or hCMs at week 2. (E) Myocardial viability of iPSCs, iCMs, and control at week 2 and 4 after cell transplantation. Myocardial viability is significantly greater in hCMs at week 4 vs. control and hESCs at week 4 or hCMs at week 2. Conversely, the control group had significantly reduced myocardial viability between week 2 and 4. (F) There is a significant positive correlation between LVEF and myocardial viability (R=0.76, p<0.001). (G) Myocardial viability in the PIR as visualized by MEMRI, highlighted with red arrows. Data are presented as mean ± S.E.M., *p<0.05.

Though LVEF remains the gold standard in evaluating cardiac function, we sought to further characterize the extent of injury and repair in the peri-infarct region (PIR). We directly quantified the volume of the viable myocardium by MEMRI at week 2 and 4 (Figure 1D-G). MEMRI demonstrated significantly greater myocardial viability in the hCM and iCM groups compared to the control (hCMs 73.4%*, iCMs 65.6%* vs. control 46.0%, week 4, *p<0.05). Consistent with the LVEF results, mice treated with cardiac lineage cells (hCMs or iCMs) showed significantly improved myocardial viability compared to the hESC and iPSC groups, respectively. Furthermore, MRI measurements, including LV end-diastolic volume, myocardial viability volume, and infarct volume, were also performed (Online Table II, Online Figure II), demonstrating reduced infarct volume in hCM- and iCM-treated animals. Finally, a significant correlation was found between LVEF and myocardial viability (R=0.76, p<0.001, Figure 1F).

To determine if these functional improvements correlated with sustained engraftment, we performed BLI to track the luciferase-tagged stem cells or cardiac myocytes (Figure 2A-E, Online Table II). Functional improvement paralleled the viability of the transplanted cells. As expected, BLI signal in cardiac myocyte-treated mice was greater than that of the control group at week 2, as expected (hCMs 3813.8p/s/cm2/sr* and iCMs 3709.7p/s/cm2/sr* vs. control 1474.8p/s/cm2/sr, *p<0.05, week 2). However, these BLI signals were reduced by week 4, indicating decreasing engraftment. While hCMs and iCMs appeared to engraft in the myocardium between week 2 and 4, little to no signal was found in the hESC or iPSC groups by week 2 and was virtually absent by week 4. These findings suggest that cardiac differentiation reduces death of implanted cells in the myocardium. BLI measurements of all groups can be found in Online Table II. To derive a relationship between BLI signal and absolute cell number, in vitro BLI analyses were performed using iCMs as a representative cell line. Results demonstrate a significant linear correlation between cell number and BLI signal (R=0.99, p<0.0001, Online Figure III). BLI was thus validated as a tool to monitor viable cell number. Using this linear correlation, we estimated absolute transplanted cell numbers in each treatment group (Figure 2D-E).

Figure 2. Stem cell engraftment measured by BLI.

Figure 2

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(A) BLI signals of hESCs, hCMs, and control at week 2 and 4. The hCM group demonstrated sustained engraftment throughout the 4-week period while the hESC group showed low levels of engraftment or cell death by week 2. (B) Similarly, the iCM group indicated greater cell engraftment at week 2 and 4 vs. control. The iPSC group had measureable engraftment signals at week 2 but showed no evidence of sustained engraftment at week 4. (C) Robust engraftment in hCMs and iCMs at week 2 decreased by week 4. (D) Absolute cell numbers were estimated in hESCs and hCMs, and (E) in iPSCs and iCMs. Data are presented as mean ± S.E.M., *p<0.05.

The mortality of the mice in each treatment group was the following: hESCs 22%, iPSCs 25%, hCMs 11%, iCMs 43%, and control 20%. There were no significant differences between the groups.

Histological evidence of hCM and iCM engraftment

To validate our BLI results, myocardial sections were stained for human cTnT and human nuclear antibody (Figure 3). We found a small volume of viable transplanted cells (hCMs 0.20 × 10−3 mm3, iCMs 1.2 × 10−3 mm3). Despite this limited engraftment, viable myocardium in the PIR greatly increased from week 2 to 4 in hCM-treated mice (+1.09 mm3) and iCM-treated mice (+2.37 mm3), demonstrating a volume of more than 1000-fold greater than the xenograft. This significant discrepancy suggests that the microscopic population of engrafted cardiac cells could not account for the entire volume of increased viable myocardium by replacing scar tissue; instead, surviving hCMs and iCMs may have salvaged the hibernating and injured myocardium by the widespread effects of their cytokines. No teratomas were detected in all groups (Online Figure IV).

Figure 3. Immunofluorescence staining at the site of hCMs and iCMs engraftment.

Figure 3

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(A) Sections of hCM-treated myocardia were stained with human nuclear antibody and human cardiac troponin T at the site of injection. Hoechst 33342 was used to visualize nuclei (blue). Co-localization identified transplanted hCMs at week 4. (B) The iCM group also demonstrated cell engraftment at week 4. However, engraftment volume is significantly lower than the volume of increased myocardial viability in both hCM and iCM groups.

Cytokine production of the transplanted hCMs and iCMs

We studied the paracrine mechanism of action of the hCMs and iCMs to explain the significant discrepancy between the volume of increased myocardial viability and of engrafted cardiac stem cells. To assess the paracrine effects of the transplanted cells, we analyzed the supernatant of each cell type by 63-plex Luminex immunoassay of human cytokines. There were significant differences in the cytokines of cardiac derivatives and pluripotent stem cells (Online Figure V). Analysis demonstrated significant up-regulation of the following families of cytokines in hCMs vs. hESCs: anti-apoptosis (tumor necrosis factor α [TNF-α]†); pro-angiogenesis (Interleukin 8 [IL8]*, granulocyte colony stimulating factor [GCSF]*, and vascular endothelial growth factor [VEGF]*); and pro-cell migration (TNF-α†, vascular cell adhesion molecule 1 [VCAM1]*, and plasminogen activator inhibitor 1 [PAI1]*; *p<0.05 and †p<0.08). Similarly, the iCMs showed significantly increased cytokine production when compared to iPSCs: anti-apoptosis (TNF-α*), pro-angiogenesis (GCSF*, VEGF* and placenta growth factor 1 [PIGF1]*), and pro-cell migration (TNF-α*, VCAM1*, PAI1* and stromal cell derived factor 1α [SDF1α]*; *p<0.05). We hypothesized that these factors enhanced cell engraftment, promoted angiogenesis, increased cell proliferation, and inhibited apoptosis, which may be the basis for the repair of the injured cardiac myocytes in the PIR.

Cardiac myocyte transplantation reduces apoptosis in the PIR of the recipient myocardium

To evaluate the effect of cytokines on the host myocardium, we examined gene expression in the PIR at week 4. RT-PCR in the hCM group demonstrated an upward trend of anti-apoptosis (TNF-α‡) compared to the hESC and control groups (‡p<0.3, Figure 4A-B). Similarly, the iCM group showed up-regulation of anti-apoptotic genes compared to control group (Akt1* and TNF-α*, *p<0.05, Figure 4E-F).

Figure 4. Anti-apoptotic effects of stem cell therapy in the PIR of the murine myocardium.

Figure 4

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(A) Relative gene expression of Akt1 and (B) TNF-α in the hESC, hCM and control groups. There was no significant difference in Akt1 expression; there was, however, a trend towards TNF-α upregulation in the hCM group compared to the control group. (C) Analysis of TUNEL-stained slides localized at the PIR indicated significantly less DNA fragmentation, and thus less apoptosis, in the hCM group compared to the control group. (D) Representative IHC images. (E) Akt1 and (F) TNF-α gene expression is significantly upregulated in iCM group compared to the control and the iPSC groups. (G) IHC confirmed a significant fewer TUNEL-positive cells in the PIR of iCM-treated mice compared to iPSC and control groups. (H) Representative IHC images. Data are presented as mean ± S.E.M., ‡p<0.3, *p<0.05.

We performed correlation analysis to determine if there was any relationship between the in vitro cytokine production and the resultant gene expression in vivo in the recipient mouse myocardium. The hCMs demonstrated a significant correlation between the in vitro cytokine production vs. in vivo gene expression of anti-apoptotic cytokine (TNF-α and GCSF) vs. gene (TNF-α; R=0.85 and p=0.04). Similarly, the iCM and iPSC groups showed a significant correlation in anti-apoptotic cytokines (TNF-α and GCSF) vs. genes (Akt1 and TNF-α; R=0.70 and p=0.012). This relationship suggests that the increased production of anti-apoptotic cytokines by the hCMs and iCMs contributed to the reduction of apoptosis in the murine myocardium.

To validate the up-regulation of anti-apoptotic genes in the recipient myocardium, tissues were probed with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) complex and co-stained with mouse cardiac troponin 3 (TNNI3) to detect DNA fragmentation. Fluorescence emitted by the TUNEL complex was normalized to Hoechst 33342 nuclear staining in TNNI3-positive cells. Consistent with our cytokine production and gene expression findings, TUNEL (apoptosis) signal in the PIR of hCM or iCM groups was significantly downregulated compared to control, and trended downwards compared to hESC or iPSC groups, respectively (Figure 4C-D, G-H). Apoptosis in remote region cardiac myocytes was similar across all groups (Online Figure VI).

Increased cell migration in the hCM/iCM-treated murine myocardium

Our Luminex data indicated production of pleiotropic cytokines with anti-apoptotic and pro-migratory effects. TNF-α, PAI1, VCAM1, and SDF1α were secreted at particularly high levels in hCMs and iCMs supernatant. TNF-α, which mediates both apoptosis and migration, was upregulated in the hCM‡- and iCM*-treated myocardium (‡p<0.3, *p<0.05) (Figure 4B, F). The relationship between in vivo TNF-α gene expression and in vitro pro-migratory cytokine production was particularly significant (genes: TNF-α vs. cytokines: TNF-α, PAI1 and VCAM1; R=0.80 and p=0.019). Additionally, connexin-43, a pro-migratory gene, was up-regulated in the hCM and iCM groups (Figure 5A, D).

Figure 5. Pro-migratory effects of stem cell therapy in the PIR of the murine myocardium.

Figure 5

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(A) Relative gene expression of connexin-43 in the hESC, hCM and control groups. There is an upward trend in hCMs vs. control. (B) IHC indicates signficant overexpression of CXCR4, a downstream effector of pro-migration cytokines and mRNA in the hCM group compared to the hESC group. (C) Representative IHC images. (D) Connexin-43 gene trends upwards in the iCM group. (E) CXCR4 is significantly overexpressed in the iCM group. (F) Representative IHC images. Data are presented as mean ± S.E.M., ‡p<0.3, *p<0.05.

We verified these cytokine and gene expression findings by IHC analysis of pro-migration marker, CXCR434, 35. Consistent with PCR results, CXCR4 expression was significantly up-regulated in hCM- or iCM-treated myocardium compared to pluripotent stem cell groups (Figure 5B-C, E-F). These findings indicate that the increased secretion of pro-migratory cytokines by hCMs and iCMs correlate with the up-regulated migratory genes and markers in the PIR, which may have ultimately contributed to improved cardiac function post-MI.

Angiogenesis is promoted in the hCM/iCM-treated murine myocardium

Angiogenic cytokines (IL8, VEGF, PIGF1, and GCSF) were significantly increased in hCM and iCM groups (compared to hESC and iPSC groups, respectively). Ex vivo RT-PCR analysis revealed that the iCM-treated myocardium demonstrated up-regulated angiogenic genes (Akt1*, VEGF‡, *p<0.05, ‡p<0.3 vs. control group, Figure 6A, D). Secretion of angiogenic cytokines in iCMs in vitro and expression of VEGF in iCM group ex vivo were significantly correlated (cytokines: VEGF, PIGF1, and GCSF vs. genes: Akt1 and VEGF; R=0.74 and p=0.00049). To verify activation of the pro-angiogenic pathway in the recipient myocardium, we measured expression of CD31 by IHC. CD31, a marker for angiogenesis36, 37, was significantly overexpressed in the hCM- and iCM-treated myocardium (Figure 6B-C, E-F, p<0.05 vs. hESC/iPSC groups), confirming the effect of pro-angiogenic cytokines from hCM and iCM supernatant.

Figure 6. Pro-angiogenic effects of stem cell therapy in the PIR of the murine myocardium.

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(A) RT-PCR of the PIR showed no significant difference in VEGF expression in the hCM group compared to the hESC or control groups. (B) However, IHC of CD31 showed significant overexpression of CD31 in the hCM-treated vs. hESC-treated PIR. (C) Representative IHC images. (D) There was upregulation of VEGF in the iCM group compared to the iPSC and control groups, suggesting increased angiogenesis, which was confirmed by (E) IHC of CD31. (F) Representative IHC images. Data are presented as mean ± S.E.M., ‡p<0.3, *p<0.05.

Salvage vs. regeneration of the cell-treated myocardium

We investigated whether the restoration of the injured murine myocardium may be due to salvage of the injured cardiac myocytes or proliferation of new cells. To make this distinction, we studied expression of MLC2v, a marker for myocyte contractility in the PIR, and monitored host cell proliferation by injecting EdU (5-ethynyl-2′-deoxyuridine), a nucleoside analog of thymidine that is actively incorporated into DNA during DNA synthesis. IHC analysis of the PIR showed little EdU signal in the PIR with no statistically significant difference between cardiac and pluripotent stem cell treatment groups (data not shown). There was, however, an upward trend of MLC2v expression in the hCM- or iCM-treated myocardium compared to control group (Figure 7A, D). Similarly, IHC detected significant overexpression of MLC2v in the hCM and iCM groups compared to the hESC and iPSC groups, respectively (Figure 7B-C, E-F). These findings suggest that functional improvements in the hCM- or iCM-treated myocardium are not caused predominantly by recruitment of proliferating cells to regenerate de novo myocardium. Rather, cardiac stem cell therapy more likely restores the injured cardiac myocytes in the PIR.

Figure 7. Salvage vs. regeneration of the stem cell therapy in the PIR.

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(A) MLC2v gene expression was greater in the hCM group compared to the control and had an upward trend compared to the hESC group. (B) The PIR was identified using H&E staining and analyzed for MLC2v expression. (C) Representative H&E and IHC images for MLC2v and EdU. (D) MLC2v was overexpressed in the iCM group compared to the control group. (E) There was significant MLC2v expression in the iCM group compared to the iPSC group. (F) Representative H&E and IHC images for MLC2v and EdU. Data are presented as mean ± S.E.M., ‡p<0.3, *p<0.05.

We further explored the size of surviving cardiac myocytes in the PIR and remote region. The cross-sectional area of cardiac myocytes in the PIR of hCM and iCM groups were significantly greater than that of myocytes in the PIR of pluripotent stem cell-treated or control groups (Online Figure VIIA, C). Myocytes in the remote regions were similar across all groups (Online Figure VIIB, D). Myocyte nuclear density measurements revealed no significant difference between groups in the PIR or in the remote region (Online Figure VIIE-H). These findings suggest that the recovery of the contractile elements may be linked to compensatory hypertrophy of the injured cardiac myocytes, which underlies the mechanism of action of salvage in the PIR. The improved survival, enhanced contractility, and hypertrophy of the injured cardiac myocytes by the paracrine effects of cells improve LVEF and myocardial viability post-MI.

DISCUSSION

Acute MI results in cardiac myocyte death and progressive reduction in heart function, which eventually deteriorates to heart failure4. Although recent pre-clinical and clinical reports have shown that stem cell transplantation leads to improvement in LV function and viability, the underlying mechanism remains unclear25, 38. While other investigations have described the paracrine mechanism, this study is, to the best of our knowledge, the first to systematically quantify pluripotent stem cell or pluripotent stem cell-derived cardiac myocyte cytokines and their in vivo target mechanism. Additionally, we compare two variants of pluripotent stem cells (hESCs and iPSCs) and their cardiac lineage differentiated cells (hCMs and iCMs) with multiple assessments, including a direct in vivo evaluation of myocardial viability and function and in vitro measurement of myocyte cross-sectional area and immunohistology of myocardial apoptosis, fibrosis, angiogenesis, and cell migration. This rigorous and novel approach allowed real-time tracking of fundamental stem cell biology in vivo to enhance our understanding of the mechanism underlying myocardial restoration.

Results from this investigation indicate that pluripotent stem cell-derived cardiac myocytes are therapeutically more advantageous than their undifferentiated counterparts or the control in an ischemic MI model. Benefits of cardiac cell therapy, such as a significant improvement in LVEF and myocardial viability are attributed to enhanced engraftment as detected by BLI and confirmed by immunostaining of the hCM- and iCM-treated heart tissues at week 4. However, a significant volumetric discrepancy between the engrafted cells and the enhanced myocardial viability demonstrates clearly that the engrafted cells did not regenerate the myocardium but instead may have salvaged the existing injured cardiac myocytes in the PIR.

To understand the role of cytokines from transplanted cells, we performed in vitro analyses of the cell supernatants. Six paracrine factors (IL8, TNF-α, GCSF, VCAM1, VEGF, and PAI1) were found to be significantly more abundant in hCMs compared to hESCs. iCMs also produced seven paracrine factors (SDF1α, PIGF1, TNF-α, GCSF, VCAM1, VEGF and PAI1) at significantly higher levels than iPSCs. These paracrine factors lie upstream of important remodeling and healing pathways in the infarcted myocardium, which may have contributed to the anti-apoptosis (TNF-α)39, pro-angiogenesis (IL8, PIGF1, GCSF and VEGF)40-44, and pro-cell migration (SDF1α, TNF-α, VCAM1 and PAI1)45-48 of injured cardiac myocytes in the PIR. To correlate the observed cytokine secretions with their subsequent genetic regulation in vivo, we probed the explanted hearts for evidence of transcriptional and/or translational effects of these cytokines, specifically in their known targets.

RT-PCR and immunohistochemistry analyses at week 4 showed significant correlation with the observed Luminex data. Up-regulation of Akt1 has been reported to play a critical role in cell survival and angiogenesis41, 49. Akt1 is activated by GCSF secretion and has been noted in iCMs through the PI3K/Akt pathway42. GCSF has also been reported to activate the intracellular signaling cascade of Jak/STAT pathway, which induces the expression of angiogenesis factors50. In this study, we found significant increases in both VEGF and GCSF cytokine production, and associated VEGF and Akt1 gene expression in the myocardium of the iCM-treatment group. This finding is consistent with other studies, which report that PIGF1 cytokine secretion enhances the VEGF gene expression from the autocrine pathway and contributes to the angiogenesis in myocardial infarction44, 51. Our data are also in agreement with up-regulation of PIGF1 cytokine secretion and VEGF gene expression in the iCM group. Ultimately, angiogenesis, as quantified by CD31 IHC, was up-regulated in both hCM and iCM groups.

Moreover, TNF-α and connexin-43, which promote anti-apoptosis39 and cell migration45, 52, was significantly up-regulated in the supernatant of cardiac stem cells and correlated with the significantly increased gene expression of TNF-α in hCM- and iCM-treated heart tissue. SDF1α was also significantly up-regulated in the iCM supernatant compared to the iPSC. Apoptosis, as measured by TUNEL IHC, was significantly downregulated in cardiac stem cell-treated myocardium.

While apoptosis in the post-infarct myocardium is extensively studied, the role of migration is less understood. Our data associate transplantation of cardiac stem cells in the PIR with enhanced cell migration. SDF1α, also known as CXC12, is a chemokine ligand of CXCR4 and is known to be upregulated following myocardial ischemia53. Injection of CXC12 was reported to reduce infarct size and increase cardiac function after MI54, 55. It thus follows that SDF1α secreted by hCMs and iCMs may activate CXCR4 to induce the same cardioprotective benefits. While the role of CXCR4 in the ischemic heart is largely studied as it pertains to progenitor cell recruitment, other studies report that it activates Akt1 within resident endothelial cells and cardiac myocytes55-57.

We explored whether enhanced cardiac function is attributed to regeneration or salvage of the myocardium. EdU injections and comparisons of nuclear density showed no substantial evidence of proliferating cells in any groups; however, hCMs and iCMs up-regulated gene and protein expression of MLC2v in the PIR to enhance cardiac contractility58. These findings suggest that salvage of the injured cardiac myocytes, rather than recruitment of proliferative cells, underlie the significantly greater myocardial viability in the PIR and improved LVEF associated with cardiac stem cell therapy. Closer inspection of PIR cardiac myocytes revealed significantly larger cells in the hCM and iCM groups compared to the hESC and iPSC groups, respectively, or in comparison to the remote region of either group. These data indicate that the increase in cardiac myocyte size and recovery of the contractile element may accompany salvage of injured cardiac myocytes. Canonically, post-infarct hypertrophy is strongly associated with poor outcomes59; some studies show, however, that hypertrophy in the absence of fibrogenesis may instead be beneficial and compensatory60. Our data, which show that the hypertrophied PIR of cardiac myocyte-treated myocardium had reduced apoptosis and fibrosis, suggesting that such hypertrophy may be physiologic and repair heart function.

Herein, we present evidence that robust cardiac-lineage differentiated stem cell13 is essential for effective stem cell therapy of ischemic MI. The clear discrepancy between their limited cellular engraftment and disproportionate increase in the myocardial viability in PIR provides compelling evidence of cardio-protective paracrine secretion12, 17, 19 to modulate gene and protein expression in the injured cardiac myocytes to improved myocardial function (Figure 8). On the other hand, pluripotent stem cell-treated groups demonstrated decreased engraftment in vivo and Luminex assays demonstrated diminished cytokine secretion in vitro. This finding supports the critical role of paracrine factors found in the stem cells-derived cardiac myocytes. Our histological analyses support the notion that the injured myocardium was salvaged rather than regenerated de novo from transplanted cardiac stem cells. We observed reduced apoptosis and infarct size with enhanced angiogenesis, cell migration, and myocyte contractility in cardiac stem cell-treated PIR. We also observed no difference in the population of proliferating endogenous cells between all groups. Thus, the regenerative mechanism, which requires cell engraftment and direct electromechanical contribution to the myocardial function, did not represent the dominant underlying mechanism in the improved heart function. This supports other work that report stem cell-mediated myocardium regeneration to be a rare occurrence61.

Figure 8. Schematic of the Paracrine Mechanism of hCMs and iCMs Salvage of the Injured Myocardium.

Figure 8

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Summary of the correlation among immunohistology, significantly up-regulated gene expression, and the associated cytokine productions in the myocardium treated with (A) hCMs and (B) iCMs.

Limitations

This study has some limitations. First, we measured cytokine secretions from the supernatant of cultured cells. This experimental method does not provide any direct insight into the in vivo cellular secretomes and immunological response of engrafted cells after their delivery into infarcted regions. Engrafted cells in the infarcted myocardium, exposed to the hypoxic and inflammatory in vivo niche, may respond differently from the cultured conditions. Second, all surgical operations were conducted by an experienced and blinded microsurgeon. The murine MI model is consistent but the efficacy and reliability of cell injections into the infarcted mouse myocardium may vary, as the delivery efficacy is difficult to confirm. Third, the PIR of the myocardium may have included the remote area for RT-PCR due to the minute size of the murine heart. Fourth, cardiac lineage cells (hCMs and iCMs) in this acute chronic ischemia model may have a different effect in patients with advanced heart failure.

In summary, this study provides a mechanistic correlation between the fundamental biological parameters of cardiac lineage stem cells and their physiologic effects on myocardium salvage. Cardiac differentiation of pluripotent stem cells resulted in improved engraftment and subsequent restoration of LV function and myocardial viability compared to the undifferentiated pluripotent stem cells (hESCs and iPSCs) and control. This difference delineated the important effects of the underlying paracrine mechanism of action of the cardiac lineage derivatives of pluripotent stem cells. This investigation found considerable discrepancy between the microscopic volume of the engrafted cells and the volume of myocardial viability. The data indicated that hCMs and iCMs salvaged the injured myocardium effectively by their differential paracrine effects.

Supplementary Material

310803 Online Supplement

NOVELTY AND SIGNIFICANCE.

What Is Known?

  • Human embryonic stem cell (hESC)-derived cardiac myocytes (hCMs) and induced pluripotent stem cell (iPSC)-derived cardiac myocytes (iCMs) mitigate damage of the injured myocardium.

  • Cardiac function improves despite sub-optimal engraftment and eventual death of the transplanted stem cells.

What New Information Does This Article Contribute?

  • iCMs and hCMs secrete cytokines that target the restorative genes and pathways of the injured myocardium.

  • Cell therapy does not stimulate cardiac myocyte proliferation or regeneration but hypertrophy and salvage of the injured cardiac myocytes in the peri-infarct region (PIR).

Pre-clinical studies have reported functional benefit by iCMs and hCMs despite their sub-optimal engraftment in the myocardium. Little is known about the underlying mechanism. We investigated the efficacy of undifferentiated pluripotent stem cells versus their cardiac lineage derivatives in a murine myocardial injury model. Cardiac function significantly improved in the iCM- and hCM-treatment groups. Our data confirm the production of reparative cytokines that modulate anti-apoptotic, pro-angiogenic, and pro-migratory activity, resulting in cardiac myocyte hypertrophy and improved contractility in the PIR. This study demonstrates that cardiac differentiation of pluripotent stem cells improves the therapeutic efficacy through paracrine mechanism to mitigate damage of the injured myocardium.

Acknowledgments

We are grateful to Bryan B. Edwards, Masashi Kawamura, and Jay Patel for their help with mice operations.

SOURCES OF FUNDING

This work was supported by US Public Health Service grants NIH/NHLBI 5UM1 HL113456-02 (PY).

Nonstandard Abbreviations and Acronyms

LV

left ventricle

LVEF

left ventricular ejection fraction

hESCs

human embryonic stem cells

iPSCs

human induced pluripotent stem cells

hCMs

hESCs-derived cardiac myocytes

iCMs

iPSCs-derived cardiac myocytes

MI

myocardial injury

MEMRI

manganese-enhanced MRI

BLI

bioluminescence imaging

PIR

peri-infarct region

Footnotes

DISCLOSURES

None.

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