The Activation of the LIMK/Cofilin Signaling Pathway via Extracellular Matrix-Integrin Interactions Is Critical for the Generation of Mature and Vascularized Cardiac Organoids.

The generation of mature and vascularized human pluripotent stem cell-derived cardiac organoids (hPSC-COs) is necessary to ensure the validity of drug screening and disease modeling. This study investigates the effects of cellular aggregate (CA) stemness and self-organization on the generation of mature and vascularized hPSC-COs and elucidates the mechanisms underlying cardiac organoid (CO) maturation and vascularization. COs derived from 2-day-old CAs with high stemness (H-COs) and COs derived from 5-day-old CAs with low stemness (L-COs) were generated in a self-organized microenvironment via Wnt signaling induction. This study finds that H-COs exhibit ventricular, structural, metabolic, and functional cardiomyocyte maturation and vessel networks consisting of endothelial cells, smooth muscle cells, pericytes, and basement membranes compared to L-COs. Transcriptional profiling shows the upregulation of genes associated with cardiac maturation and vessel formation in H-COs compared with the genes in L-COs. Through experiments with LIMK inhibitors, the activation of ROCK-LIMK-pCofilin via ECM-integrin interactions leads to cardiomyocyte maturation and vessel formation in H-COs. Furthermore, the LIMK/Cofilin signaling pathway induces TGFß/NODAL and PDGF pathway activation for the maturation and vascularization of H-COs. The study demonstrates for the first time that LIMK/Cofilin axis activation plays an important role in the generation of mature and vascularized COs.

Detecting early-stage malignant melanoma using a calcium switch-enriched exosome subpopulation containing tumor markers as a sample.

An exosome species containing CD63 as a marker of melanoma was isolated from bulk exosome population and used as a sample for detecting malignant melanoma. A calcium binding protein (CBP) was produced and then used to raise monoclonal antibody. The antibody was sensitive to a conformational change of CBP caused by Ca2+ binding. Immuno-magnetic beads were prepared by immobilizing the conformation-sensitive binder and subsequent binding of CBP conjugated with the capture antibody specific to CD63. These immuno-beads were used to isolate CD63-positive exosome from a bulk exosome sample (normal or melanoma) based on the 'calcium switch-on/off' mechanism through magnetic separation. After recovery, the subpopulation sample was analyzed by immunoassays for cavelion1 (Cav1), CD81, and CD9 as sub-subpopulation markers. Normalized signals of Cav1 and/or CD81 over CD9 were higher in melanoma samples than in normal samples, depending on clinical stages (I, II, and IV) of patients. This was in contrast to assay results for the bulk exosome population that showed a completely mixed state of melanoma and normal samples. These results showed that an exosome subpopulation sample prepared using a 'Ca2+-dependent switch' technology might be useful for diagnosing malignant melanoma at an early stage to increase 5-year survival rates.

Modeling Hypoxic Stress In Vitro Using Human Embryonic Stem Cells Derived Cardiomyocytes Matured by FGF4 and Ascorbic Acid Treatment.

Mature cardiomyocytes (CMs) obtained from human pluripotent stem cells (hPSCs) have been required for more accurate in vitro modeling of adult-onset cardiac disease and drug discovery. Here, we found that FGF4 and ascorbic acid (AA) induce differentiation of BG01 human embryonic stem cell-cardiogenic mesoderm cells (hESC-CMCs) into mature and ventricular CMs. Co-treatment of BG01 hESC-CMCs with FGF4+AA synergistically induced differentiation into mature and ventricular CMs. FGF4+AA-treated BG01 hESC-CMs robustly released acute myocardial infarction (AMI) biomarkers (cTnI, CK-MB, and myoglobin) into culture medium in response to hypoxic injury. Hypoxia-responsive genes and potential cardiac biomarkers proved in the diagnosis and prognosis of coronary artery diseases were induced in FGF4+AA-treated BG01 hESC-CMs in response to hypoxia based on transcriptome analyses. This study demonstrates that it is feasible to model hypoxic stress in vitro using hESC-CMs matured by soluble factors.

LEFTY-PITX2 signaling pathway is critical for generation of mature and ventricular cardiac organoids in human pluripotent stem cell-derived cardiac mesoderm cells.

The generation of mature ventricular cardiomyocytes (CMs) resembling adult CMs from human pluripotent stem cells (hPSCs) is necessary for disease modeling and drug discovery. To investigate the effect of self-organizing capacity on the generation of mature cardiac organoids (COs), we generated cardiac mesoderm cell-derived COs (CMC-COs) and CM-derived COs (CM-COs) and evaluated COs. CMC-COs exhibited more organized sarcomere structures and mitochondria, well-arranged t-tubule structures, and evenly distributed intercalated discs. Increased expressions of ventricular CM, cardiac metabolic, t-tubule formation, K+ ion channel, and junctional markers were confirmed in CMC-COs. Mature ventricular-like function such as faster motion vector speed, decreased beats per min, increased peak-to-peak duration, and prolonged APD50 and APD90 were observed in CMC-COs. Transcriptional profiling revealed that extracellular matrix-integrin, focal adhesion, and LEFTY-PITX2 signaling pathways are upregulated in CMC-COs. LEFTY knockdown affected ECM-integrin-FA signaling pathways in CMC-COs. Here, we found that high self-organizing capacity of CMCs is critical for the generation of mature and ventricular COs. We also demonstrated that LEFTY-PITX2 signaling plays key roles for CM maturation and specification into ventricular-like CM subtype in CMC-COs. CMC-COs are an attractive resource for disease modeling and drug discovery.

Transplantation of 3D bio-printed cardiac mesh improves cardiac function and vessel formation via ANGPT1/Tie2 pathway in rats with acute myocardial infarction.

A novel tissue engineering strategy using 3D bio-print technology has become a promising therapeutic method for acute myocardial infarction (AMI) in an animal model. However, the application of 3D bio-printed tissue remains limited due to poor graft survival. Therefore, it is a scientific priority to enhance graft survival by precisely adjusting the 3D environment of encapsulated cells. In this study, novel transplantable 3D cardiac mesh (cMesh) tissue with a porous mesh structure was presented using human cardiomyocytes, human cardiac fibroblasts, and gelatin-methacryloyl-collagen hydrogel. Cardiomyocytes and cardiac fibroblasts were well spreaded. The cardiomyocytes were connected with a gap junction channel in bio-printed cMesh and a 3D cardiac patch with an aggregated structure. Porous cMesh demonstrated structural advantages by increased phosphorylation of mTOR, AKT, and ERK signals associated with cell survival. Transplanted cMesh in rats with AMI improved long-term graft survival, vessel formation, and stabilization, reduced fibrosis, increased left ventricle thickness, and enhanced cardiac function. Our results suggest that porous cMesh provides structural advantages and a positive therapeutic effect in an AMI animal model.

Thymosin ß4-Enhancing Therapeutic Efficacy of Human Adipose-Derived Stem Cells in Mouse Ischemic Hindlimb Model.

Thymosin ß4 (Tß4) is a G-actin sequestering protein that contributes to diverse cellular activities, such as migration and angiogenesis. In this study, the beneficial effects of combined cell therapy with Tß4 and human adipose-derived stem cells (hASCs) in a mouse ischemic hindlimb model were investigated. We observed that exogenous treatment with Tß4 enhanced endogenous TMSB4X mRNA expression and promoted morphological changes (increased cell length) in hASCs. Interestingly, Tß4 induced the active state of hASCs by up-regulating intracellular signaling pathways including the PI3K/AKT/mTOR and MAPK/ERK pathways. Treatment with Tß4 significantly increased cell migration and sprouting from microbeads. Moreover, additional treatment with Tß4 promoted the endothelial differentiation potential of hASCs by up-regulating various angiogenic genes. To evaluate the in vivo effects of the Tß4-hASCs combination on vessel recruitment, dorsal window chambers were transplanted, and the co-treated mice were found to have a significantly increased number of microvessel branches. Transplantation of hASCs in combination with Tß4 was found to improve blood flow and attenuate limb or foot loss post-ischemia compared to transplantation with hASCs alone. Taken together, the therapeutic application of hASCs combined with Tß4 could be effective in enhancing endothelial differentiation and vascularization for treating hindlimb ischemia.

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).

Correction: Multidimensional assembly using layer-by-layer deposition for synchronized cardiac macro tissues.

[This corrects the article DOI: 10.1039/D0RA01577F.].

Multidimensional assembly using layer-by-layer deposition for synchronized cardiac macro tissues.

The fabrication of biomimetic structures for tissues and organs is emerging in the fields of biomedical engineering and precision medicine. While current progress in biomedical research provides a number of biofabrication methods, the construction of multi-dimensional cardiac tissue is highly challenging due to difficulties in the maturation and synchronization of cardiomyocytes (CMs) in conjunction with other types of cells, such as myofibroblasts and endothelial cells. Here, we show a simple fabrication methodology to construct multi-dimensional cardiac macro tissue (mCMT) by layer-by-layer (LBL) deposition of cells on micro patterned PDMS. mCMTs formed by LBL deposition of pluripotent stem cell (PSC)-derived cardiomyocytes and cardiac fibroblasts formed 3D patterned structures with synchronized beating characteristics. We also demonstrate that cardiac maturation factors such as the gene expression of MLC2v and cTNI and formation of sarcomeres in mCMTs were significantly enhanced by LBL deposition and growth factors during the maturation process. Fabrication of matured mCMTs with synchronized beating enables providing an efficient platform for evaluating the efficacy and toxicity of drug candidates. These results have important implications because mCMTs are applicable to diverse in vitro studies and drug screening methods that require tissue-like structures and functions in a physiological environment.

Modulating cardiomyocyte and fibroblast interaction using layer-by-layer deposition facilitates synchronisation of cardiac macro tissues.

Maturation and synchronisation of heart cells, including cardiomyocytes and fibroblasts, are essential to develop functional biomimetic cardiac tissues for regenerative medicine and drug discovery. Synchronisation of cells in the biomimetic cardiac tissue requires the structural integrity and functional maturation of cardiomyocytes with other cell types. However, it is challenging to synchronise the beating of macroscale cardiac tissues and induce maturation of cardiomyocytes derived from stem cells. Here, we developed a simple assembly technology to modulate cell-cell interactions by combining layer-by-layer (LBL) deposition and centrifugation of cells with collagen type I to control cell-cell interactions for the preparation of cardiac macro tissues (CMTs). We found that maturation of cardiomyocytes in CMTs was largely enhanced by growth factors FGF-4 and ascorbic acid, but synchronisation of cardiac beating required LBL deposition of cardiomyocytes and cardiac fibroblasts in addition to the growth factors during the maturation process. Our findings have important implications because incorporation of cardiac fibroblasts into the cardiomyocyte layer is a prerequisite for synchronised beating of macroscale cardiac tissues in addition to growth factors to facilitate maturation of stem cell-derived cardiomyocytes.

Fimasartan reduces neointimal formation and inflammation after carotid arterial injury in apolipoprotein E knockout mice.

BACKGROUND: The beneficial effects of angiotensin II type 1 receptor blockers (ARBs) on atherosclerosis have been demonstrated in numerous studies. We investigated the effects of fimasartan on reducing neointimal formation and systemic inflammation after carotid artery (CA) injury in Apolipoprotein E knockout (ApoE KO) mice. METHODS: ApoE KO mice were randomly allocated to Group I (without CA injury), Group II (without CA injury + Fimasartan), Group III (CA injury), and Group IV (CA injury + Fimasartan). Fimasartan was orally administered everyday starting 3 days before iatrogenic left CA injury. RESULTS: At 28 days, neointimal hyperplasia and the inflammatory cytokines including TNFα, IL-6, ICAM, and MMP-9 in the peripheral blood were significantly reduced in Groups II and IV compared to Groups I and III, respectively. All fimasartan-administered groups revealed significant increases of CD4+CD25+Foxp3+ regulatory T (Treg) cells with increased plasma levels of IL-10 and TGFß. In addition, increased CD8+ T cells by fimasartan were correlated with reduced smooth muscle cell (SMC) proliferation in the neointima in Groups II and IV. Furthermore, the populations of Treg and CD8+ T cells in total splenocytes were increased in Groups II and IV compared to Groups I and III, respectively. The enlargement of spleens due to CA injury in the Group III was attenuated by fimasartan, as shown in the Group IV. These data indicate that fimasartan significantly reduced SMC proliferation in neointima and increased Treg cells in ApoE KO CA injury mice. CONCLUSIONS: This study suggests fimasartan could be an efficient strategy for reduction of atherosclerotic progression, with a decrease in immune response and systemic inflammation.

Regulating response and leukocyte adhesion of human endothelial cell by gradient nanohole substrate.

Understanding signals in the microenvironment that regulate endothelial cell behavior are important in tissue engineering. Although many studies have examined the cellular effects of nanotopography, no study has investigated the functional regulation of human endothelial cells grown on nano-sized gradient hole substrate. We examined the cellular response of human umbilical vein endothelial cells (HUVECs) by using a gradient nanohole substrate (GHS) with three different types of nanohole patterns (HP): which diameters were described in HP1, 120-200 nm; HP2, 200-280 nm; HP3, 280-360 nm. In results, HP2 GHS increased the attachment and proliferation of HUVECs. Also, gene expression of focal adhesion markers in HUVECs was significantly increased on HP2 GHS. In vitro tube formation assay showed the enhancement of tubular network formation of HUVECs after priming on GHS compared to Flat. Furthermore, leukocyte adhesion was also reduced in the HUVECs in a hole-diameter dependent manner. To summarize, optimal proliferations with reduced leukocyte adhesion of HUVECs were achieved by gradient nanohole substrate with 200-280 nm-sized holes.

Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells.

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular reactions. However, it is difficult to determine the relation between the size of a nanostructure and the responses of cells owing to the lack of proper screening tools. Here, we show the development of reproducible and cost-effective gradient nanopattern plates for the manipulation of cellular responses. Using anodic aluminum oxide (AAO) as a master mold, gradient nanopattern plates with nanopillars of increasing diameter ranges [120-200 nm (GP 120/200), 200-280 nm (GP 200/280), and 280-360 nm (GP 280/360)] were fabricated by a thermal imprinting technique. These gradient nanopattern plates were designed to mimic the various sizes of nanotopography in the ECM and were used to screen the responses of human endothelial colony-forming cells (hECFCs). In this protocol, we describe the step-by-step procedure of fabricating gradient nanopattern plates for cell engineering, techniques of cultivating hECFCs from human peripheral blood, and culturing hECFCs on nanopattern plates.

Manipulation of the response of human endothelial colony-forming cells by focal adhesion assembly using gradient nanopattern plates.

Nanotopography plays a pivotal role in the regulation of cellular responses. Nonetheless, little is known about how the gradient size of nanostructural stimuli alters the responses of endothelial progenitor cells without chemical factors. Herein, the fabrication of gradient nanopattern plates intended to mimic microenvironment nanotopography is described. The gradient nanopattern plates consist of nanopillars of increasing diameter ranges [120-200 nm (GP 120/200), 200-280 nm (GP 200/280), and 280-360 nm (GP 280/360)] that were used to screen the responses of human endothelial colony-forming cells (hECFCs). Nanopillars with a smaller nanopillar diameter caused the cell area and perimeter of hECFCs to decrease and their filopodial outgrowth to increase. The structure of vinculin (a focal adhesion marker in hECFCs) was also modulated by nanostructural stimuli of the gradient nanopattern plates. Moreover, Rho-associated protein kinase (ROCK) gene expression was significantly higher in hECFCs cultured on GP 120/200 than in those on flat plates (no nanopillars), and ROCK suppression impaired the nanostructural-stimuli-induced vinculin assembly. These results suggest that the gradient nanopattern plates generate size-specific nanostructural stimuli suitable for manipulation of the response of hECFCs, in a process dependent on ROCK signaling. This is the first evidence of size-specific nanostructure-sensing behavior of hECFCs. SIGNIFICANCE: Nano feature surfaces are of growing interest as materials for a controlled response of various cells. In this study, we successfully fabricated gradient nanopattern plates to manipulate the response of blood-derived hECFCs without any chemical stimulation. Interestingly, we find that the sensitive nanopillar size for manipulation of hECFCs is range between 120 nm and 200 nm, which decreased the area and increased the filopodial outgrowth of hECFCs. Furthermore, we only modulate the nanopillar size to increase ROCK expression can be an attractive method for modulating the cytoskeletal integrity and focal adhesion of hECFCs.

Nanopillar Surface Topology Promotes Cardiomyocyte Differentiation through Cofilin-Mediated Cytoskeleton Rearrangement.

Nanoscaled surface patterning is an emerging potential method of directing the fate of stem cells. We adopted nanoscaled pillar gradient patterned cell culture plates with three diameter gradients [280-360 (GP 280/360), 200-280 (GP 200/280), and 120-200 nm (GP 120/200)] and investigated their cell fate-modifying effect on multipotent fetal liver kinase 1-positive mesodermal precursor cells (Flk1+ MPCs) derived from embryonic stem cells. We observed increased cell proliferation and colony formation of the Flk1+ MPCs on the nanopattern plates. Interestingly, the 200-280 nm-sized (GP 200/280) pillar surface dramatically increased cardiomyocyte differentiation and expression of the early cardiac marker gene Mesp1. The gradient nanopattern surface-induced cardiomyocytes had cardiac sarcomeres with mature cardiac gene expression. We observed Vinculin and p-Cofilin-mediated cytoskeleton reorganization during this process. In summary, the gradient nanopattern surface with 200-280 nm-sized pillars enhanced cardiomyocyte differentiation in Flk1+ MPCs.

Talin Modulation by a Synthetic N-Acylurea Derivative Reduces Angiogenesis in Human Endothelial Cells.

Talin is a focal adhesion protein that activates integrins and recruits other focal adhesion proteins. Talin regulates the interactions between integrins and the extracellular matrix, which are critical for endothelial cells during angiogenesis. In this study, we successfully synthesized a novel talin modulator, N-((2-(1H-indol-3-yl)ethyl)carbamoyl)-2-(benzo[d][1,3]dioxol-5-yloxy)acetamide, referred to as KCH-1521. KCH-1521 was determined to bind talin and modulate downstream signaling molecules of talin. After 24 h of treatment, KCH-1521 changed the cell morphology of human umbilical vein endothelial cells (HUVECs) and reduced focal adhesion protein expression including vinculin and paxillin. Talin downstream signaling is regulated via focal adhesion kinase (FAK), kinase B (AKT), and extracellular signal-regulated kinase (ERK) pathways, however, treatment with KCH-1521 decreased phosphorylation of FAK, AKT, and ERK, leading to reduction of cell proliferation, survival, and angiogenesis. Interestingly, the expression of various angiogenic genes was significantly decreased after treatment with KCH-1521. Also, in vitro tube forming assay revealed that KCH-1521 reduced angiogenic networks in a time-dependent manner. To investigate the reversibility of its effects, KCH-1521 was removed after treatment. HUVECs recovered their morphology through rearrangement of the cytoskeleton and the expression of angiogenic genes was also recovered. By further optimization and in vivo studies of KCH-1521, a novel drug of talin modulation could be used to achieve therapeutic anti-angiogenesis for vascular diseases and cancers.

Transplantation of Adipose-Derived Stem Cell Sheet Attenuates Adverse Cardiac Remodeling in Acute Myocardial Infarction.

Adipose-derived stem cell (ADSC) transplantation has been proposed to improve cardiac function and acute myocardial infarction (AMI). Recently, cell sheet technology has been investigated for its potential applicability in cardiac injury. However, a detailed comparison of the functional recovery in the injured myocardium between cell sheets and conventional cell injection has not been adequately examined. ADSCs were isolated from the inguinal fat tissue of ICR mice. Three groups of AMI induction only (sham), intramyocardial injection of ADSCs (imADSC), and ADSC sheet transplantation (shADSC) were compared by using rat AMI models. Engraftment of ADSCs was better sustained through 28 days in the shADSC group compared with the imADSC group. Ejection fraction was improved in both imADSC and shADSC groups compared with the sham group. Ventricular wall thickness in the infarct zone was higher in the shADSC group compared with both imADSC and sham groups. Growth factor and cytokine expression in the implanted heart tissue were higher in the shADSC group compared with both imADSC and sham groups. Furthermore, only the shADSC group showed donor-derived vessels at the peri-infarct zone. Taken together, these results indicate that, although shADSC resulted in a similar improvement in left ventricular systolic function, it significantly promoted cellular engraftment and upregulated growth factor and cytokine expression, and, ultimately, attenuated adverse cardiac remodeling in rat AMI models compared with imADSC.

Intrinsic FGF2 and FGF5 promotes angiogenesis of human aortic endothelial cells in 3D microfluidic angiogenesis system.

The human body contains different endothelial cell types and differences in their angiogenic potential are poorly understood. We compared the functional angiogenic ability of human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) using a three-dimensional (3D) microfluidic cell culture system. HAECs and HUVECs exhibited similar cellular characteristics in a 2D culture system; however, in the 3D microfluidic angiogenesis system, HAECs exhibited stronger angiogenic potential than HUVECs. Interestingly, the expression level of fibroblast growth factor (FGF)2 and FGF5 under vascular endothelial growth factor (VEGF)-A stimulation was significantly higher in HAECs than in HUVECs. Moreover, small interfering RNA-mediated knockdown of FGF2 and FGF5 more significantly attenuated vascular sprouting induced from HAECs than HUVECs. Our results suggest that HAECs have greater angiogenic potential through FGF2 and FGF5 upregulation and could be a compatible endothelial cell type to achieve robust angiogenesis.

Intramyocardial Adipose-Derived Stem Cell Transplantation Increases Pericardial Fat with Recovery of Myocardial Function after Acute Myocardial Infarction.

Intramyocardial injection of adipose-derived stem cells (ASC) with other cell types in acute myocardial infarction (AMI) animal models has consistently shown promising clinical regenerative capacities. We investigated the effects of intramyocardial injections of mouse ASC (mASC) with mouse endothelial cells (mEC) on left ventricular function and generation of pericardial fat in AMI rats. AMI rat models were created by ligating left anterior descending coronary artery and were randomly assigned into four groups: control (n = 10), mASC (n = 10), mEC (n = 10) and mASC+mEC (n = 10) via direct intramyocardial injections, and each rat received 1x106 cells around three peri-infarct areas. Echocardiography and cardiac positron emission tomography (PET) were compared at baseline and on 28 days after AMI. Changes in left ventricular ejection fraction measured by PET, increased significantly in mASC and mASC+mEC groups compared to mEC and control groups. Furthermore, significant decreases in fibrosis were confirmed after sacrifice on 28 days in mASC and mASC+mEC groups. Successful cell engraftment was confirmed by positive Y-Chromosome staining in the transplantation region. Pericardial fat increased significantly in mASC and mASC+mEC groups compared to control group, and pericardial fat was shown to originate from the AMI rat. mASC group expressed higher adiponectin and lower leptin levels in plasma than control group. In addition, pericardial fat from AMI rats demonstrated increased phospho-AMPK levels and reduced phospho-ACC levels. Intramyocardial mASC transplantation after AMI in rats increased pericardial fat, which might play a protective role in the recovery of myocardial function after ischemic myocardial damage.

Cardiac Stem Cell Secretome Protects Cardiomyocytes from Hypoxic Injury Partly via Monocyte Chemotactic Protein-1-Dependent Mechanism.

Cardiac stem cells (CSCs) were known to secrete diverse paracrine factors leading to functional improvement and beneficial left ventricular remodeling via activation of the endogenous pro-survival signaling pathway. However, little is known about the paracrine factors secreted by CSCs and their roles in cardiomyocyte survival during hypoxic condition mimicking the post-myocardial infarction environment. We established Sca-1+/CD31- human telomerase reverse transcriptase-immortalized CSCs (Sca-1+/CD31- CSCs(hTERT)), evaluated their stem cell properties, and paracrine potential in cardiomyocyte survival during hypoxia-induced injury. Sca-1+/CD31- CSCs(hTERT) sustained proliferation ability even after long-term culture exceeding 100 population doublings, and represented multi-differentiation potential into cardiomyogenic, endothelial, adipogenic, and osteogenic lineages. Dominant factors secreted from Sca-1+/CD31- CSCs(hTERT) were EGF, TGF-ß1, IGF-1, IGF-2, MCP-1, HGF R, and IL-6. Among these, MCP-1 was the most predominant factor in Sca-1+/CD31- CSCs(hTERT) conditioned medium (CM). Sca-1+/CD31- CSCs(hTERT) CM increased survival and reduced apoptosis of HL-1 cardiomyocytes during hypoxic injury. MCP-1 silencing in Sca-1+/CD31- CSCs(hTERT) CM resulted in a significant reduction in cardiomyocyte apoptosis. We demonstrated that Sca-1+/CD31- CSCs(hTERT) exhibited long-term proliferation capacity and multi-differentiation potential. Sca-1+/CD31- CSCs(hTERT) CM protected cardiomyocytes from hypoxic injury partly via MCP-1-dependent mechanism. Thus, they are valuable sources for in vitro and in vivo studies in the cardiovascular field.