RESUMEN
Nonsurgical bleeding occurs in a significant proportion of patients implanted with continuous-flow ventricular assist devices (CF-VADs) and is associated with nonphysiologic flow with diminished pulsatility. An in vitro vascular pulse perfusion model seeded with adult human aortic endothelial cells (HAECs) was used to identify biomarkers sensitive to changes in pulsatility. Diminished pulsatility resulted in an ~45% decrease in von Willebrand factor (vWF) levels from 9.80 to 5.32 ng/ml (n = 5, p < 0.05) and a threefold increase in angiopoietin-2 (ANGPT-2) levels from 775.29 to 2471.93 pg/ml (n = 5, p < 0.05) in cultured HAECs. These changes are in agreement with evaluation of patient blood samples obtained pre-CF-VAD implant and 30-day postimplant: a decrease in plasma vWF level by 50% from ~45.59 to ~22.49 µg/ml (n = 15, p < 0.01) and a 64% increase in plasma ANGPT-2 level from 7,073 to 11,615 pg/ml (n = 8, p < 0.05). This study identified vWF and ANGPT-2 as highly sensitive to changes in pulsatility, in addition to interleukin-6 (IL-6), IL-8, and tumor necrosis-α (TNF-α). These biomarkers may help determine the optimal level of pulsatility and help identify patients at high risk of nonsurgical bleeding.
Asunto(s)
Corazón Auxiliar , Enfermedades de von Willebrand , Adulto , Humanos , Factor de von Willebrand , Células Endoteliales , Corazón Auxiliar/efectos adversos , Angiopoyetina 2 , Hemorragia/etiología , Biomarcadores , Enfermedades de von Willebrand/etiologíaRESUMEN
OBJECTIVE AND DESIGN: Phagocytosis and clearance of apoptotic cells are essential for inflammation resolution, efficient wound healing, and tissue homeostasis. MicroRNAs are critical modulators of macrophage polarization and function. The current study aimed to investigate the role of miR-181c-5p in macrophage phagocytosis. MATERIALS AND METHODS: miR-181c-5p was identified as a potential candidate in microRNA screening of RAW264.7 macrophages fed with apoptotic cells. To investigate the role of miR-181c-5p in phagocytosis, the expression of miR-181c-5p was assessed in phagocyting bone marrow-derived macrophages. Phagocytosis efficiency was measured by fluorescence microscopy. Gain- and loss-of-function studies were performed using miR-181c-5p-specific mimic and inhibitor. The expression of the phagocytosis-associated genes and proteins of interest was evaluated by RT2 profiler PCR array and western blotting, respectively. RESULTS: miR-181c-5p expression was significantly upregulated in the phagocyting macrophages. Furthermore, mimic-induced overexpression of miR-181c-5p resulted in the increased phagocytic ability of macrophages. Moreover, overexpression of miR-181c-5p resulted in upregulation of WAVE-2 in phagocyting macrophages, suggesting that miR-181c-5p may regulate cytoskeletal arrangement during macrophage phagocytosis. CONCLUSION: Altogether, our data provide a novel function of miR-181c-5p in macrophage biology and suggest that targeting macrophage miR-181c-5p in injured tissues might improve clearance of dead cells and lead to efficient inflammation resolution.
Asunto(s)
MicroARNs , Humanos , Inflamación , Activación de Macrófagos , Macrófagos/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , FagocitosisRESUMEN
Due to the rapidly growing number of older people worldwide and the concomitant increase in cardiovascular complications, there is an urgent need for age-related cardiac disease modeling and drug screening platforms. In the present study, we developed a cardiac tissue chip model that incorporates hemodynamic loading and mimics essential aspects of the infarcted aging heart. We induced cellular senescence in H9c2 myoblasts using low-dose doxorubicin treatment. These senescent cells were then used to engineer cardiac tissue fibers, which were subjected to hemodynamic stresses associated with pressure-volume changes in the heart. Myocardial ischemia was modeled in the engineered cardiac tissue via hypoxic treatment. Our results clearly show that acute low-dose doxorubicin treatment-induced senescence, as evidenced by morphological and molecular markers, including enlarged and flattened nuclei, DNA damage response foci, and increased expression of cell cycle inhibitor p16INK4a, p53, and ROS. Under normal hemodynamic load, the engineered cardiac tissues demonstrated cell alignment and retained cardiac cell characteristics. Our senescent cardiac tissue model of hypoxia-induced myocardial infarction recapitulated the pathological disease hallmarks such as increased cell death and upregulated expression of ANP and BNP. In conclusion, the described methodology provides a novel approach to generate stress-induced aging cardiac cell phenotypes and engineer cardiac tissue chip models to study the cardiovascular disease pathologies associated with aging.
Asunto(s)
Enfermedades Cardiovasculares , Infarto del Miocardio , Anciano , Envejecimiento , Enfermedades Cardiovasculares/complicaciones , Senescencia Celular/genética , Doxorrubicina , Corazón , Humanos , Infarto del Miocardio/patologíaRESUMEN
Doxorubicin (DOX, an anthracycline) is a widely used chemotherapy agent against various forms of cancer; however, it is also known to induce dose-dependent cardiotoxicity leading to adverse complications. Investigating the underlying molecular mechanisms and strategies to limit DOX-induced cardiotoxicity might have potential clinical implications. Our previous study has shown that expression of microRNA-377 (miR-377) increases in cardiomyocytes (CMs) after cardiac ischemia-reperfusion injury in mice, but its specific role in DOX-induced cardiotoxicity has not been elucidated. In the present study, we investigated the effect of anti-miR-377 on DOX-induced cardiac cell death, remodeling, and dysfunction. We evaluated the role of miR-377 in CM apoptosis, its target analysis by RNA sequencing, and we tested the effect of AAV9-anti-miR-377 on DOX-induced cardiotoxicity and mortality. DOX administration in mice increases miR-377 expression in the myocardium. miR-377 inhibition in cardiomyocyte cell line protects against DOX-induced cell death and oxidative stress. Furthermore, RNA sequencing and Gene Ontology (GO) analysis revealed alterations in a number of cell death/survival genes. Intriguingly, we observed accelerated mortality and enhanced myocardial remodeling in the mice pretreated with AAV9-anti-miR-377 followed by DOX administration as compared to the AAV9-scrambled-control-pretreated mice. Taken together, our data suggest that in vitro miR-377 inhibition protects against DOX-induced cardiomyocyte cell death. On the contrary, in vivo administration of AAV9-anti-miR-377 increases mortality in DOX-treated mice.
RESUMEN
Cardiac tissue surrogates show promise for restoring mechanical and electrical function in infarcted left ventricular (LV) myocardium. For these cardiac surrogates to be usefulin vivo, they are required to support synchronous and forceful contraction over the infarcted region. These design requirements necessitate a thickness sufficient to produce a useful contractile force, an area large enough to cover an infarcted region, and prevascularization to overcome diffusion limitations. Attempts to meet these requirements have been hampered by diffusion limits of oxygen and nutrients (100-200 µm) leading to necrotic regions. This study demonstrates a novel layer-by-layer (LbL) fabrication method used to produce tissue surrogates that meet these requirements and mimic normal myocardium in form and function. Thick (1.5-2 mm) LbL cardiac tissues created from human induced pluripotent stem cell-derived cardiomyocytes and endothelial cells were assessed,in vitro, over a 4-week period for viability (<5.6 ± 1.4% nectrotic cells), cell morphology, viscoelastic properties and functionality. Viscoelastic properties of the cardiac surrogates were determined via stress relaxation response modeling and compared to native murine LV tissue. Viscoelastic characterization showed that the generalized Maxwell model of order 4 described the samples well (0.7 Asunto(s)
Células Endoteliales
, Células Madre Pluripotentes Inducidas
, Animales
, Humanos
, Ratones
, Miocardio
, Miocitos Cardíacos
, Ingeniería de Tejidos/métodos
RESUMEN
Preeclampsia (PE) and vascular dysfunction are major causes of maternal and neonatal morbidity and mortality. Although extensively studied, the complete understanding of the pathophysiology behind PE remains unclear. Current reports indicate that exosomes are essential mediators in PE-related cardiovascular disease (CVDs). Exosomes are synthesized from multivesicular bodies (MVB) and contain functionally active microRNAs miRNAs). These miRNAs have been shown to mediate physiological and pathological functions through autocrine, paracrine, and endocrine signaling mechanisms. The role of miRNAs in pregnant women with PE has been studied extensively. However, little is known about the effect of exosomal miRNAs (exomiR) in PE. This paper will review and discuss the existing evidence for exomiR function in PE and highlight the need for future studies to explore the role that exomiR signatures have in cardiovascular dysfunction associated with PE.
Asunto(s)
Enfermedades Cardiovasculares/genética , Exosomas/genética , MicroARNs/fisiología , Preeclampsia/genética , Enfermedades Cardiovasculares/diagnóstico , Enfermedades Cardiovasculares/fisiopatología , Femenino , Humanos , MicroARNs/análisis , Preeclampsia/diagnóstico , Preeclampsia/fisiopatología , Embarazo , Complicaciones Cardiovasculares del Embarazo/diagnóstico , Complicaciones Cardiovasculares del Embarazo/genética , Complicaciones Cardiovasculares del Embarazo/fisiopatología , Transcriptoma/fisiologíaRESUMEN
DNA damage accrued in induced pluripotent stem cell (iPSC)-derived cardiomyocytes during in vitro culture practices lessens their therapeutic potential. We determined whether DNA-damage-free iPSCs (DdF-iPSCs) can be selected using stabilization of p53, a transcription factor that promotes apoptosis in DNA-damaged cells, and differentiated them into functionally competent DdF cardiomyocytes (DdF-CMs). p53 was activated using Nutlin-3a in iPSCs to selectively kill the DNA-damaged cells, and the stable DdF cells were cultured further and differentiated into CMs. Both DdF-iPSCs and DdF-CMs were then characterized. We observed a significant decrease in the expression of reactive oxygen species and DNA damage in DdF-iPSCs compared with control (Ctrl) iPSCs. Next-generation RNA sequencing and Ingenuity Pathway Analysis revealed improved molecular, cellular, and physiological functions in DdF-iPSCs. The differentiated DdF-CMs had a compact beating frequency between 40 and 60 beats/min accompanied by increased cell surface area. Additionally, DdF-CMs were able to retain the improved molecular, cellular, and physiological functions after differentiation from iPSCs, and, interestingly, cardiac development network was prominent compared with Ctrl-CMs. Enhanced expression of various ion channel transcripts in DdF-CMs implies DdF-CMs are of ventricular CMs and mature compared with their counterparts. Our results indicated that DdF-iPSCs could be selected through p53 stabilization using a small-molecule inhibitor and differentiated into ventricular DdF-CMs with fine-tuned molecular signatures. These iPSC-derived DdF-CMs show immense clinical potential in repairing injured myocardium.NEW & NOTEWORTHY Culture-stress-induced DNA damage in stem cells lessens their performance. A robust small-molecule-based approach, by stabilizing/activating p53, to select functionally competent DNA-damage-free cells from a heterogeneous population of cells is demonstrated. This protocol can be adopted by clinics to select DNA-damage-free cells before transplanting them to the host myocardium. The intact DNA-damage-free cells exhibited with fine-tuned molecular signatures and improved cellular functions. DNA-damage-free cardiomyocytes compared with control expressed superior cardiomyocyte functional properties, including, but not limited to, enhanced ion channel signatures. These DNA-intact cells would better engraft, survive, and, importantly, improve the cardiac function of the injured myocardium.
Asunto(s)
Diferenciación Celular , Daño del ADN , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología , Células Cultivadas , Técnicas de Reprogramación Celular/métodos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Canales Iónicos/genética , Canales Iónicos/metabolismo , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/fisiología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0219442.].
RESUMEN
Free from the limitations posed by exogenous scaffolds or extracellular matrix-based materials, scaffold-free engineered tissues have immense clinical potential. Biomaterials may produce adverse responses, interfere with cell-cell interaction, or affect the extracellular matrix integrity of cells. The scaffold-free Kenzan method can generate complex tissues using spheroids on an array of needles but could be inefficient in terms of time, as it moves and places only a single spheroid at a time. We aimed to design and construct a novel scaffold-free bioprinter that can print an entire layer of spheroids at once, effectively reducing the printing time. The bioprinter was designed using computer-aided design software and constructed from machined, 3D printed, and commercially available parts. The printing efficiency and the operating precision were examined using Zirconia and alginate beads, which mimic spheroids. In less than a minute, the printer could efficiently pick and transfer the beads to the printing surface and assemble them onto the 4 × 4 needles. The average overlap coefficient between layers was measured and found to be 0.997. As a proof of concept using human induced pluripotent stem cell-derived spheroids, we confirmed the ability of the bioprinter to place cellular spheroids onto the needles efficiently to print an entire layer of tissue. This novel layer-by-layer, scaffold-free bioprinter is efficient and precise in operation and can be easily scaled to print large tissues.
Asunto(s)
Daño del ADN , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/trasplante , Animales , Apoptosis , Diferenciación Celular , Separación Celular , Estenosis Coronaria/terapia , Vasos Coronarios , Modelos Animales de Enfermedad , Genes p53 , Supervivencia de Injerto , Imidazoles/farmacología , Células Madre Pluripotentes Inducidas/química , Ligadura , Ratones , Ratones SCID , Miocitos Cardíacos/citología , Piperazinas/farmacologíaRESUMEN
Functional myocardium derived from human induced pluripotent stem cells (hiPSCs) can be impactful for cardiac disease modeling, drug testing, and the repair of injured myocardium. However, when hiPSCs are differentiated into cardiomyocytes, they do not possess characteristics of mature myocytes which limits their application in these endeavors. We hypothesized that mechanical and electrical stimuli would enhance the maturation of hiPSC-derived cardiomyocyte (hiPSC-CM) spheroids on both a structural and functional level, potentially leading to a better model for drug testing as well as cell therapy. Spheroids were generated with hiPSC-CM. For inducing mechanical stimulation, they were placed in a custom-made device with PDMS channels and exposed to cyclic, uniaxial stretch. Spheroids were electrically stimulated in the C-Pace EP from IONOptix for 7 days. Following the stimulations, the spheroids were then analyzed for cardiomyocyte maturation. Both stimulated groups of spheroids possessed enhanced transcript and protein expressions for key maturation markers, such as cTnI, MLC2v, and MLC2a, along with improved ultrastructure of the hiPSC-CMs in both groups with enhanced Z-band/Z-body formation, fibril alignment, and fiber number. Optical mapping showed that spheroids exposed to electrical stimulation were able to capture signals at increasing rates of pacing up to 4 Hz, which failed in unstimulated spheroids. Our results clearly indicate that a significantly improved myocyte maturation can be achieved by culturing iPSC-CMs as spheroids and exposing them to cyclic, uniaxial stretch and electrical stimulation.
RESUMEN
OBJECTIVE: Cardiovascular research and regenerative strategies have been significantly limited by the lack of relevant cell culture models that can recreate complex hemodynamic stresses associated with pressure-volume changes in the heart. METHODS: To address this issue, we designed a biomimetic cardiac tissue chip (CTC) model where encapsulated cardiac cells can be cultured in three-dimensional (3-D) fibres and subjected to hemodynamic loading to mimic pressure-volume changes seen in the left ventricle. These 3-D fibres are suspended within a microfluidic chamber between two posts and integrated within a flow loop. Various parameters associated with heart function, like heart rate, peak-systolic pressure, end-diastolic pressure and volume, end-systolic pressure and volume, and duration ratio between systolic and diastolic, can all be precisely manipulated, allowing culture of cardiac cells under developmental, normal, and disease states. RESULTS: We describe two examples of how the CTC can significantly impact cardiovascular research by reproducing the pathophysiological mechanical stresses associated with pressure overload and volume overload. Our results using H9c2 cells, a cardiomyogenic cell line, clearly show that culture within the CTC under pathological hemodynamic loads accurately induces morphological and gene expression changes, similar to those seen in both hypertrophic and dilated cardiomyopathy. Under pressure overload, the cells within the CTC see increased hypertrophic remodeling and fibrosis, whereas cells subject to prolonged volume overload experience significant changes to cellular aspect ratio through thinning and elongation of the engineered tissue. CONCLUSIONS: These results demonstrate that the CTC can be used to create highly relevant models where hemodynamic loading and unloading are accurately reproduced for cardiovascular disease modeling.
Asunto(s)
Enfermedades Cardiovasculares/fisiopatología , Modelos Cardiovasculares , Miocardio/citología , Análisis de Matrices Tisulares/instrumentación , Técnicas de Cultivo de Tejidos/instrumentación , Animales , Células Cultivadas , Diseño de Equipo , Fibroblastos/citología , Corazón/fisiología , Ratas , Análisis de Matrices Tisulares/métodos , Técnicas de Cultivo de Tejidos/métodosRESUMEN
The role of p53 transactivation domain (p53-TAD), a multifunctional and dynamic domain, on DNA repair and retaining DNA integrity in human induced pluripotent stem cells (hiPSCs) has never been studied. p53-TAD was knocked out in iPSCs using CRISPR/Cas9 and was confirmed by DNA sequencing. p53-TAD knockout (KO) cells were characterized by accelerated proliferation, decreased population doubling time, and unaltered Bcl-2, Bcl-2-binding component 3, insulin-like growth factor 1 receptor, and Bax and altered Mdm2, p21, and p53-induced death domain transcript expression. In p53-TAD KO cells, the p53-regulated DNA repair proteins xeroderma pigmentosum group A, DNA polymerase H, and DNA-binding protein 2 expression were found to be reduced compared with p53 wild-type cells. Exposure to a low dose of doxorubicin (Doxo) induced similar DNA damage and DNA damage response (DDR) as measured by RAD50 and MRE11 expression, checkpoint kinase 2 activation, and γH2A.X recruitment at DNA strand breaks in both cell groups, indicating that silence of p53-TAD does not affect the DDR mechanism upstream of p53. After removal of Doxo, p53 wild-type hiPSCs underwent DNA repair, corrected their damaged DNA, and restored DNA integrity. Conversely, p53-TAD KO hiPSCs did not undergo complete DNA repair and failed to restore DNA integrity. More importantly, continuous culture of p53-TAD KO hiPSCs underwent G2/M cell cycle arrest and expressed the cellular senescent marker p16INK4a. Our data clearly show that silence of the TAD of p53 did not affect DDR but affected the DNA repair process, implying the crucial role of p53-TAD in maintaining DNA integrity. Therefore, activating p53-TAD domain using small molecules may promote DNA repair and integrity of cells and prevent cellular senescence.
Asunto(s)
Reparación del ADN , Células Madre Pluripotentes Inducidas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ácido Anhídrido Hidrolasas/metabolismo , Puntos de Control del Ciclo Celular , Células Cultivadas , Quinasa de Punto de Control 2/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Proteínas de Unión al ADN/metabolismo , Doxorrubicina/toxicidad , Inestabilidad Genómica , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Proteína Homóloga de MRE11/metabolismo , Dominios Proteicos , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The microenvironment of native heart tissue may be better replicated when cardiomyocytes are cultured in three-dimensional clusters (i.e., spheroids) than in monolayers or as individual cells. Thus, we differentiated human cardiac lineage-induced pluripotent stem cells in cardiomyocytes (hiPSC-CMs) and allowed them to form spheroids and spheroid fusions that were characterized in vitro and evaluated in mice after experimentally induced myocardial infarction (MI). Synchronized contractions were observed within 24 h of spheroid formation, and optical mapping experiments confirmed the presence of both Ca2+ transients and propagating action potentials. In spheroid fusions, the intraspheroid conduction velocity was 7.0 ± 3.8 cm/s on days 1- 2 after formation, whereas the conduction velocity between spheroids increased significantly ( P = 0.003) from 0.8 ± 1.1 cm/s on days 1- 2 to 3.3 ± 1.4 cm/s on day 7. For the murine MI model, five-spheroid fusions (200,000 hiPSC-CMs/spheroid) were embedded in a fibrin patch and the patch was transplanted over the site of infarction. Later (4 wk), echocardiographic measurements of left ventricular ejection fraction and fractional shortening were significantly greater in patch-treated animals than in animals that recovered without the patch, and the engraftment rate was 25.6% or 30% when evaluated histologically or via bioluminescence imaging, respectively. The exosomes released from the spheroid patch seemed to increase cardiac function. In conclusion, our results established the feasibility of using hiPSC-CM spheroids and spheroid fusions for cardiac tissue engineering, and, when fibrin patches containing hiPSC-CM spheroid fusions were evaluated in a murine MI model, the engraftment rate was much higher than the rates we have achieved via the direct intramyocardial injection. NEW & NOTEWORTHY Spheroids fuse in culture to produce structures with uniformly distributed cells. Furthermore, human cardiac lineage-induced pluripotent stem cells in cardiomyocytes in adjacent fused spheroids became electromechanically coupled as the fusions matured in vitro, and when the spheroids were combined with a biological matrix and administered as a patch over the infarcted region of mouse hearts, the engraftment rate exceeded 25%, and the treatment was associated with significant improvements in cardiac function via a paracrine mechanism, where exosomes released from the spheroid patch.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Infarto del Miocardio/terapia , Miocitos Cardíacos/trasplante , Esferoides Celulares/trasplante , Animales , Señalización del Calcio , Células Cultivadas , Células HEK293 , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Contracción Miocárdica , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Esferoides Celulares/metabolismo , Trasplante de Células Madre/métodosRESUMEN
Synthetic oxytocin (sOT) is widely used during labor, yet little is known about its effects on fetal brain development despite evidence that it reaches the fetal circulation. Here, we tested the hypothesis that sOT would affect early neurodevelopment by investigating its effects on neural progenitor cells (NPC) from embryonic day 14 rat pups. NPCs expressed the oxytocin receptor (OXTR), which was downregulated by 45% upon prolonged treatment with sOT. Next, we examined the effects of sOT on NPC death, apoptosis, proliferation, and differentiation using antibodies to NeuN (neurons), Olig2 (oligodendrocytes), and GFAP (astrocytes). Treated NPCs were analysed with unbiased high-throughput immunocytochemistry. Neither 6 nor 24 h exposure to 100 pM or 100 nM sOT had an effect on viability as assessed by PI or CC-3 immunocytochemistry. Similarly, sOT had negligible effect on NPC proliferation, except that the overall rate of NPC proliferation was higher in the 24 h compared to the 6 h group regardless of sOT exposure. The most significant finding was that sOT exposure caused NPCs to select a predominantly neuronal lineage, along with a concomitant decrease in glial cells. Collectively, our data suggest that perinatal exposure to sOT can have neurodevelopmental consequences for the fetus, and support the need for in vivo anatomical and behavioral studies in offspring exposed to sOT in utero.
Asunto(s)
Células-Madre Neurales/efectos de los fármacos , Oxitocina/toxicidad , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Femenino , Humanos , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neurogénesis/efectos de los fármacos , Neuroglía/citología , Neuroglía/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Oligodendroglía/citología , Oligodendroglía/efectos de los fármacos , Oxitocina/administración & dosificación , Oxitocina/metabolismo , Placenta/metabolismo , Embarazo , Efectos Tardíos de la Exposición Prenatal , Ratas , Ratas Sprague-Dawley , Receptores de Oxitocina/metabolismoRESUMEN
Human induced pluripotent stem cell (hiPSC)-derived cardio-myocytes (hiPSC-CMs) hold great promise for cardiovascular disease modeling and regenerative medicine. However, these cells are both structurally and functionally -immature, primarily due to their differentiation into cardiomyocytes occurring under static culture which only reproduces biomolecular cues and ignores the dynamic hemo-dynamic cues that shape early and late heart development during cardiogenesis. To evaluate the effects of hemodynamic stimuli on hiPSC-CM maturation, we used the biomimetic cardiac tissue model to reproduce the hemodynamics and pressure/volume changes associated with heart development. Following 7 days of gradually increasing stimulation, we show that hemodynamic loading results in (a) enhanced alignment of the cells and extracellular matrix, (b) significant increases in genes associated with physiological hypertrophy, (c) noticeable changes in sarcomeric organization and potential changes to cellular metabolism, and (d) a significant increase in fractional shortening, suggestive of a positive force frequency response. These findings suggest that culture of hiPSC-CMs under conditions that accurately reproduce hemodynamic cues results in structural orga-nization and molecular signaling consistent with organ growth and functional maturation.
Asunto(s)
Técnicas de Cultivo de Célula/instrumentación , Diferenciación Celular , Hemodinámica , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología , Biomimética/instrumentación , Biomimética/métodos , Técnicas de Cultivo de Célula/métodos , Línea Celular , Diseño de Equipo , Humanos , Miocitos Cardíacos/ultraestructura , Sarcómeros/ultraestructuraRESUMEN
BACKGROUND: Here, we generated human cardiac muscle patches (hCMPs) of clinically relevant dimensions (4 cm × 2 cm × 1.25 mm) by suspending cardiomyocytes, smooth muscle cells, and endothelial cells that had been differentiated from human induced-pluripotent stem cells in a fibrin scaffold and then culturing the construct on a dynamic (rocking) platform. METHODS: In vitro assessments of hCMPs suggest maturation in response to dynamic culture stimulation. In vivo assessments were conducted in a porcine model of myocardial infarction (MI). Animal groups included: MI hearts treated with 2 hCMPs (MI+hCMP, n=13), MI hearts treated with 2 cell-free open fibrin patches (n=14), or MI hearts with neither experimental patch (n=15); a fourth group of animals underwent sham surgery (Sham, n=8). Cardiac function and infarct size were evaluated by MRI, arrhythmia incidence by implanted loop recorders, and the engraftment rate by calculation of quantitative polymerase chain reaction measurements of expression of the human Y chromosome. Additional studies examined the myocardial protein expression profile changes and potential mechanisms of action that related to exosomes from the cell patch. RESULTS: The hCMPs began to beat synchronously within 1 day of fabrication, and after 7 days of dynamic culture stimulation, in vitro assessments indicated the mechanisms related to the improvements in electronic mechanical coupling, calcium-handling, and force generation, suggesting a maturation process during the dynamic culture. The engraftment rate was 10.9±1.8% at 4 weeks after the transplantation. The hCMP transplantation was associated with significant improvements in left ventricular function, infarct size, myocardial wall stress, myocardial hypertrophy, and reduced apoptosis in the periscar boarder zone myocardium. hCMP transplantation also reversed some MI-associated changes in sarcomeric regulatory protein phosphorylation. The exosomes released from the hCMP appeared to have cytoprotective properties that improved cardiomyocyte survival. CONCLUSIONS: We have fabricated a clinically relevant size of hCMP with trilineage cardiac cells derived from human induced-pluripotent stem cells. The hCMP matures in vitro during 7 days of dynamic culture. Transplantation of this type of hCMP results in significantly reduced infarct size and improvements in cardiac function that are associated with reduction in left ventricular wall stress. The hCMP treatment is not associated with significant changes in arrhythmogenicity.
Asunto(s)
Células Endoteliales/trasplante , Células Madre Pluripotentes Inducidas/trasplante , Infarto del Miocardio/cirugía , Miocardio/patología , Miocitos Cardíacos/trasplante , Miocitos del Músculo Liso/trasplante , Regeneración , Trasplante de Células Madre/métodos , Ingeniería de Tejidos/métodos , Animales , Diferenciación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/patología , Miocitos del Músculo Liso/patología , Recuperación de la Función , Regeneración/genética , Sus scrofa , Factores de Tiempo , Andamios del Tejido , Trasplante Heterólogo , Función Ventricular Izquierda , Remodelación VentricularRESUMEN
Vascular endothelial growth factor (VEGF) is a well-characterized proangiogenic cytokine that has been shown to promote neovascularization in hearts of patients with ischemic heart disease but can also lead to adverse effects depending on the dose and mode of delivery. We investigated whether prolonged exposure to a low dose of VEGF could be achieved by encapsulating VEGF in polylactic coglycolic acid nanoparticles and whether treatment with VEGF-containing nanoparticles improved cardiac function and protected against left ventricular remodeling in the hearts of mice with experimentally induced myocardial infarction. Polylactic coglycolic acid nanoparticles with a mean diameter of ~113 nm were generated via double emulsion and loaded with VEGF; the encapsulation efficiency was 53.5 ± 1.7% (107.1 ± 3.3 ng VEGF/mg nanoparticles). In culture, VEGF nanoparticles released VEGF continuously for at least 31 days, and in a murine myocardial infarction model, VEGF nanoparticle administration was associated with significantly greater vascular density in the peri-infarct region, reductions in infarct size, and improvements in left ventricular contractile function 4 wk after treatment. Thus, our study provides proof of principle that nanoparticle-mediated delivery increases the angiogenic and therapeutic potency of VEGF for the treatment of ischemic heart disease. NEW & NOTEWORTHY Vascular endothelial growth factor (VEGF) is a well-characterized proangiogenic cytokine but has a short half-life and a rapid clearance rate. When encapsulated in nanoparticles, VEGF was released for 31 days and improved left ventricular function in infarcted mouse hearts. These observations indicate that our new platform increases the therapeutic potency of VEGF.
Asunto(s)
Inductores de la Angiogénesis/administración & dosificación , Infarto del Miocardio/tratamiento farmacológico , Nanopartículas , Neovascularización Fisiológica/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/administración & dosificación , Función Ventricular Izquierda/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacos , Inductores de la Angiogénesis/química , Animales , Células Cultivadas , Preparaciones de Acción Retardada , Modelos Animales de Enfermedad , Portadores de Fármacos , Composición de Medicamentos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Ratones Endogámicos NOD , Ratones SCID , Contracción Miocárdica/efectos de los fármacos , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Recuperación de la Función , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/químicaRESUMEN
p53 is an important modulator of stem cell fate, but its role in cardiac progenitor cells (CPCs) is unknown. Here, we tested the effects of a single extra-copy of p53 on the function of CPCs in the presence of oxidative stress mediated by doxorubicin in vitro and type-1 diabetes in vivo. CPCs were obtained from super-p53 transgenic mice (p53-tg), in which the additional allele is regulated in a manner similar to the endogenous protein. Old CPCs with increased p53 dosage showed a superior ability to sustain oxidative stress, repair DNA damage and restore cell division. With doxorubicin, a larger fraction of CPCs carrying an extra-copy of the p53 allele recruited γH2A.X reestablishing DNA integrity. Enhanced p53 expression resulted in a superior tolerance to oxidative stress in vivo by providing CPCs with defense mechanisms necessary to survive in the milieu of the diabetic heart; they engrafted in regions of tissue injury and in three days acquired the cardiomyocyte phenotype. The biological advantage provided by the increased dosage of p53 in CPCs suggests that this genetic strategy may be translated to humans to increase cellular engraftment and growth, critical determinants of successful cell therapy for the failing heart.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Células Madre/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Western Blotting , Diferenciación Celular/genética , Proliferación Celular/genética , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Expresión Génica , Corazón/fisiopatología , Histonas/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Miocitos Cardíacos/citología , Miocitos Cardíacos/trasplante , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante de Células Madre/métodos , Células Madre/citología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) in tissues different from their organ of origin remains unclear. We tested the hypothesis that c-kit-BMCs are functionally heterogeneous and only a subgroup of these cells possesses cardiomyogenic potential. Population-based assays fall short of identifying the properties of individual stem cells, imposing on us the introduction of single cell-based approaches to track the fate of c-kit-BMCs in the injured heart; they included viral gene-tagging, multicolor clonal-marking and transcriptional profiling. Based on these strategies, we report that single mouse c-kit-BMCs expand clonally within the infarcted myocardium and differentiate into specialized cardiac cells. Newly-formed cardiomyocytes, endothelial cells, fibroblasts and c-kit-BMCs showed in their genome common sites of viral integration, providing strong evidence in favor of the plasticity of a subset of BMCs expressing the c-kit receptor. Similarly, individual c-kit-BMCs, which were infected with multicolor reporters and injected in infarcted hearts, formed cardiomyocytes and vascular cells organized in clusters of similarly colored cells. The uniform distribution of fluorescent proteins in groups of specialized cells documented the polyclonal nature of myocardial regeneration. The transcriptional profile of myogenic c-kit-BMCs and whole c-kit-BMCs was defined by RNA sequencing. Genes relevant for engraftment, survival, migration, and differentiation were enriched in myogenic c-kit-BMCs, a cell subtype which could not be assigned to a specific hematopoietic lineage. Collectively, our findings demonstrate that the bone marrow comprises a category of cardiomyogenic, vasculogenic and/or fibrogenic c-kit-positive cells and a category of c-kit-positive cells that retains an undifferentiated state within the damaged heart.