Sze Yun Lim

One-step derivation of cardiomyocytes and mesenchymal stem cells from human pluripotent stem cells

Cardiomyocytes (CMs) and mesenchymal stem cells (MSCs) are important cell types for cardiac repair post myocardial infarction. Here we proved that both CMs and MSCs can be simultaneously generated from human induced pluripotent stem cells (hiPSCs) via a pro-mesoderm differentiation strategy. Two hiPSC lines, hiPSC (1) and hiPSC (2) were generated from human dermal fibroblasts using OCT-4, SOX-2, KLF-4, c-Myc via retroviral-based reprogramming. H9 human embryonic stem cells (hESCs) served as control. CMs and MSCs were co-generated from hiPSCs and hESCs via embryoid body-dependent cardiac differentiation protocol involving a serum-free and insulin-depleted medium containing a p38 MAPK inhibitor, SB 203580. Comparing to bone marrow and umbilical cord blood-derived MSCs, hiPSC-derived MSCs (iMSCs) expressed common MSC markers and were capable of adipogenesis, osteogenesis and chondrogenesis. Moreover, iMSCs continuously proliferated for more than 32 population doublings without cellular senescence and showed superior pro-angiogenic and wound healing properties. In summary, we generated a large number of homogenous MSCs in conjunction with CMs in a low-cost and efficient one step manner. Functionally competent CMs and MSCs co-generated from hiPSCs may be useful for autologous cardiac repair.

Electrophysiology of human cardiac atrial and ventricular telocytes

Telocytes (TCs) with exceptionally long cellular processes of telopodes have been described in human epicardium to act as structural supporting cells in the heart. We examined myocardial chamber‐specific TCs identified in atrial and ventricular fibroblast culture using immunocytochemistry and studied their electrophysiological property by whole‐cell patch clamp. Atrial and ventricular TCs with extended telopodes and alternating podoms and podomers that expressed CD34, c‐Kit and PDGFR‐β were identified. These cells expressed large conductance Ca2+‐activated K+ current (BKCa) and inwardly rectifying K+ current (IKir), but not transient outward K+ current (Ito) and ATP‐sensitive potassium current (KATP). The active channels were functionally competent with demonstrated modulatory response to H2S and transforming growth factor (TGF)‐β1 whereby H2S significantly inhibited the stimulatory effect of TGF‐β1 on current density of both BKCa and IKir. Furthermore, H2S attenuated TGF‐β1‐stimulated KCa1.1/Kv1.1 (encode BKCa) and Kir2.1 (encode IKir) expression in TCs. Our results show that functionally competent K+ channels are present in human atrial and ventricular TCs and their modulation may have significant implications in myocardial physiopathology.

G-CSF for stem cell therapy in acute myocardial infarction: friend or foe?

Stem cell-based therapy has emerged as a potential therapeutic option for patients with acute myocardial infarction. The ability of granulocyte colony-stimulating factor (G-CSF) to mobilize endogenous stem cells as well as to protect cardiomyocytes at risk via paracrine effects has attracted considerable attention. In the past decade, a number of clinical trials were carried out to study the efficacy of G-CSF in cardiac repair. These trials showed variable outcomes in terms of improved cardiac contractile function and suppressed left ventricular negative remodelling. Critical examinations of these results have raised doubts concerning the effectiveness of G-CSF in modulating functional recovery. However, these cumulative clinical experiences are helpful in the understanding of mechanisms and roles of signalling pathways in regulating homing and engraftment of bone marrow stem cells to the infarcted heart. In this review, we discuss some of the observations that may have influenced the clinical outcomes. Improving strategies that target the critical aspects of G-CSF-driven cardiac therapy may provide a better platform to augment clinical benefits in future trials.

Cell delivery and tracking in post-myocardial infarction cardiac stem cell therapy: an introduction for clinical researchers

Stem cell-based therapy for patients with post-infarct heart failure is a relatively new and revolutionary concept in cardiology. Despite the encouraging results from pre-clinical studies, outcomes from most clinical trials remain moderately positive while the clinical benefits are largely attributed to transplanted cell-associated paracrine effects in stimulating angiogenesis and protecting endogenous cardiomyocytes. This scenario indicates that there may be a considerably protracted iterative process of conceptual and procedural refinement before true clinical benefits can be fully materialized. At present, many pressing questions regarding cell therapy remain unanswered. In addition to the primary interest in determining the ideal type of stem cells with best cardiogenic potential in vitro and in vivo, there are growing concerns on the impact of the host cardiac milieu on the transplanted cells, including their survival, migration, engraftment, and trans-differentiation as well as contribution to left ventricular function. Effective cell delivery and tracking methods are central to the unraveling of these questions. To date, cell-delivery modalities are yet to be optimized and strategies for safe and effective assessment of cells transplanted in the recipients are to be established. In this review, we discuss cell delivery and tracking modalities that are adopted in the current pre-clinical and clinical studies. We further discussed emerging technologies that are poised to impact the success of cell therapy.

Differential effect of myocardial matrix and integrins on cardiac differentiation of human mesenchymal stem cells

Dysregulation of matrix synthesis during myocardial fibrosis in post-infarct ventricular remodeling contributes to ventricular dysfunction. Bone marrow stem cell transplantation prevents functional deterioration following myocardial infarction. However, effect of myocardial extracellular matrix (ECM) on stem cell differentiation is poorly understood. We investigate the role of collagen matrices and integrin system in cardiac differentiation and engraftment of stem cells in infarcted myocardium. Sternum-derived bone marrow mesenchymal stem cells (MSCs) were differentiated into cardiomyocyte-like cells (CLCs). They were characterized using RT-PCR, immunofluorescence, flow cytometry and functional integrin neutralization assays. CLCs were injected into peri-infarct borders of injured myocardium of Wistar rats one week following left anterior descending (LAD) artery ligation. Cardiac function was analyzed via pressure-volume relationships. Cardiac differentiated CLCs displayed collagen V specificity, which was absent in undifferentiated MSCs. Collagen V, but not collagen I matrix, promoted attachment, proliferation and cardiac differentiation of CLCs. In contrast to beta(1), alpha(v) integrin contributed minimally in the attachment of CLCs on collagen matrices. However, inhibition of alpha(v)beta(3,) but not alpha(2)beta(1) integrin, selectively attenuated troponin T, sarcomeric alpha-actin and ryanodine 2 receptor gene expression in CLCs. Both MSC and CLC transplantation prevented chamber dilatation and improved contractile function. However, systolic activity in MSC transplanted animals was accompanied by heightened wall stress as demonstrated by elevated myocardial end-diastolic pressure and prolonged tissue relaxation time. Localization of CLCs in the vicinity of collagen V-expressing myofibers promoted their integration into cardiac syncytium. CLCs may facilitate hemodynamic recovery by preserving tissue elasticity in the peri-infarct borders that sustains contractile efficiency for functional recovery in an actively remodeling infarcted myocardium.

iPSC-derived human mesenchymal stem cells improve myocardial strain of infarcted myocardium

We investigated global and regional effects of myocardial transplantation of human induced pluripotent stem cell (iPSC)‐derived mesenchymal stem cells (iMSCs) in infarcted myocardium. Acute myocardial infarction (MI) was induced by ligation of left coronary artery of severe combined immunodeficient mice before 2 × 105 iMSCs or cell‐free saline were injected into peri‐infarcted anterior free wall. Sham‐operated animals received no injection. Global and regional myocardial function was assessed serially at 1‐week and 8‐week by segmental strain analysis by using two dimensional (2D) speckle tracking echocardiography. Early myocardial remodelling was observed at 1‐week and persisted to 8‐week with global contractility of ejection fraction and fractional area change in saline‐ (32.96 ± 14.23%; 21.50 ± 10.07%) and iMSC‐injected (32.95 ± 10.31%; 21.00 ± 7.11%) groups significantly depressed as compared to sham control (51.17 ± 11.69%, P < 0.05; 34.86 ± 9.82%, P < 0.05). However, myocardial dilatation was observed in saline‐injected animals (4.40 ± 0.62 mm, P < 0.05), but not iMSCs (4.29 ± 0.57 mm), when compared to sham control (3.74 ± 0.32 mm). Furthermore, strain analysis showed significant improved basal anterior wall strain (28.86 ± 8.16%, P < 0.05) in the iMSC group, but not saline‐injected (15.81 ± 13.92%), when compared to sham control (22.18 ± 4.13%). This was corroborated by multi‐segments deterioration of radial strain only in saline‐injected (21.50 ± 5.31%, P < 0.05), but not iMSC (25.67 ± 12.53%), when compared to sham control (34.88 ± 5.77%). Improvements of the myocardial strain coincided with the presence of interconnecting telocytes in interstitial space of the infarcted anterior segment of the heart. Our results show that localized injection of iMSCs alleviates ventricular remodelling, sustains global and regional myocardial strain by paracrine‐driven effect on neoangiogenesis and myocardial deformation/compliance via parenchymal and interstitial cell interactions in the infarcted myocardium.

Hydrogen sulphide suppresses human atrial fibroblast proliferation and transformation to myofibroblasts

Cardiac fibroblasts are crucial in pathophysiology of the myocardium whereby their aberrant proliferation has significant impact on cardiac function. Hydrogen sulphide (H2S) is a gaseous modulator of potassium channels on cardiomyocytes and has been reported to attenuate cardiac fibrosis. Yet, the mechanism of H2S in modulating proliferation of cardiac fibroblasts remains poorly understood. We hypothesized that H2S inhibits proliferative response of atrial fibroblasts through modulation of potassium channels. Biophysical property of potassium channels in human atrial fibroblasts was examined by whole‐cell patch clamp technique and their cellular proliferation in response to H2S was assessed by BrdU assay. Large conductance Ca2+‐activated K+ current (BKCa), transient outward K+ current (Ito) and inwardly rectifying K+ current (IKir) were found in human atrial fibroblasts. Current density of BKCa (IC50 = 69.4 μM; n = 6), Ito (IC50 = 55.1 μM; n = 6) and IKir (IC50 = 78.9 μM; n = 6) was significantly decreased (P < 0.05) by acute exposure to NaHS (a H2S donor) in atrial fibroblasts. Furthermore, NaHS (100–500 μM) inhibited fibroblast proliferation induced by transforming growth factor‐β1 (TGF‐β1; 1 ng/ml), Ang II (100 nM) or 20% FBS. Pre‐conditioning of fibroblasts with NaHS decreased basal expression of Kv4.3 (encode Ito), but not KCa1.1 (encode BKCa) and Kir2.1 (encode IKir). Furthermore, H2S significantly attenuated TGF‐β1–stimulated Kv4.3 and α‐smooth muscle actin expression, which coincided with its inhibition of TGF‐β–induced myofibroblast transformation. Our results show that H2S attenuates atrial fibroblast proliferation via suppression of K+ channel activity and moderates their differentiation towards myofibroblasts.

iPSC‐derived human cardiac progenitor cells improve ventricular remodelling via angiogenesis and interstitial networking of infarcted myocardium

We investigate the effects of myocardial transplantation of human induced pluripotent stem cell (iPSC)‐derived progenitors and cardiomyocytes into acutely infarcted myocardium in severe combined immune deficiency mice. A total of 2 × 105 progenitors, cardiomyocytes or cell‐free saline were injected into peri‐infarcted anterior free wall. Sham‐operated animals received no injection. Myocardial function was assessed at 2‐week and 4‐week post‐infarction by using echocardiography and pressure‐volume catheterization. Early myocardial remodelling was observed at 2‐week with echocardiography derived stroke volume (SV) in saline (20.45 ± 7.36 μl, P < 0.05) and cardiomyocyte (19.52 ± 3.97 μl, P < 0.05) groups, but not in progenitor group (25.65 ± 3.61 μl), significantly deteriorated as compared to sham control group (28.41 ± 4.41 μl). Consistently, pressure–volume haemodynamic measurements showed worsening chamber dilation in saline (EDV: 23.24 ± 5.01 μl, P < 0.05; ESV: 17.08 ± 5.82 μl, P < 0.05) and cardiomyocyte (EDV: 26.45 ± 5.69 μl, P < 0.05; ESV: 18.03 ± 6.58 μl, P < 0.05) groups by 4‐week post‐infarction as compared to control (EDV: 15.26 ± 2.96 μl; ESV: 8.41 ± 2.94 μl). In contrast, cardiac progenitors (EDV: 20.09 ± 7.76 μl; ESV: 13.98 ± 6.74 μl) persistently protected chamber geometry against negative cardiac remodelling. Similarly, as compared to sham control (54.64 ± 11.37%), LV ejection fraction was preserved in progenitor group from 2‐(38.68 ± 7.34%) to 4‐week (39.56 ± 13.26%) while cardiomyocyte (36.52 ± 11.39%, P < 0.05) and saline (35.34 ± 11.86%, P < 0.05) groups deteriorated early at 2‐week. Improvements of myocardial function in the progenitor group corresponded to increased vascularization (16.12 ± 1.49/mm2 to 25.48 ± 2.08/mm2 myocardial tissue, P < 0.05) and coincided with augmented networking of cardiac telocytes in the interstitial space of infarcted zone.

Dose-dependent systolic contribution of differentiated stem cells in post-infarct ventricular function

BACKGROUND Differentiation of bone marrow stem cells toward cardiomyocytes has been widely reported in vitro. However, optimum cell types and mechanisms leading to functional improvement in cardiac cell therapy remain unresolved. There is limited evidence showing a dose-dependent effect of transplanted cells in contributing to functional recovery. This study showed that cell transplantation of differentiated cardiomyocyte-like cells (CLCs) and undifferentiated mesenchymal stem cells (MSCs) dose-dependently improved left ventricular function in a rat myocardial infarction model. METHODS At 1 week after infarction in Wistar rats, 1 × 10(6) MSCs (n = 9) or CLCs (n = 9) and 5 × 10(6) MSCs (n = 18) or CLCs (n = 15) were injected into peri-infarcted myocardium to study their effect after 6 weeks. RESULTS High-dose CLCs exhibited a dose-response that was significantly more effective than MSCs in recovering cardiac contractility. Superiority of CLCs over MSCs was demonstrated in load-independent measurement of the end-systolic pressure-volume relationship and pre-load recruitable stroke work, but not in the end-diastolic pressure-volume relationship. These findings showed a unique systolic role of CLCs in contractility recovery. Functional improvement mediated by MSCs was mainly derived from preservation of endogenous myocyte function and restriction of chamber dilatation by enhancing intramyocardial angiogenesis during post-infarct ventricular remodeling. Engrafted CLCs showed better survival, were strategically integrated into myofiber-associated collagen V matrix, and exhibited mature sarcomeric cross-striations. Vascular differentiation, but not cardiac, was observed with MSCs. CONCLUSION These cell type-specific effects suggest that committing stem cells to a cardiac phenotype ex vivo promoted mechanical and functional integration of CLCs into the myofibrillar syncytium of infarcted myocardium.

Structural stability of neoangiogenic intramyocardial microvessels supports functional recovery in chronic ischemic myocardium

We hypothesize that combining angiopoietin-1 (ANG-1) or ANG-2 with vascular endothelial growth factor (VEGF) improves myocardial perfusion and contractile function by modulating vascular adaptation of neoangiogenic microvessels in a chronic ischemic swine model. Four weeks after occlusion of the left circumflex coronary artery (LCx), animals were injected with AdVEGF(165) (n=6), AdVEGF(165)+AdANG-1 (n=6), AdVEGF(165)+AdANG-2 (n=6) or control vector (n=5) into the left ventricular posterolateral wall. Regional perfusion by fluorescent microspheres and segmental myocardial tissue velocity by tissue Doppler imaging (TDI) were assessed at baseline, 4 weeks post occlusion and 4 weeks post therapy. Despite similar vascular growth following VEGF+ANG-1 and VEGF+ANG-2 treatments, transmural myocardial contractility improved only when VEGF was paired with ANG-1. In contrast, regional systolic function deteriorated uniformly across subepicardial, mid-myocardial and subendocardial segments in VEGF and VEGF+ANG-2 treated groups. Contractile improvement was associated with enhanced vascular stability through augmented arteriole formation, tight structural integration between VE-cadherin and beta-catenin at endothelial junctions and improved cross-talk between endothelium and myocardium. Structural stability of developing intramyocardial microvessels contributes to systolic function during ischemic neovascularization. Coordinated regulation of angiogenic revascularization that supports vascular stability is a key aspect in improving therapeutic outcomes in ischemic myocardium.