RESUMEN
Lamin A/C (LMNA) is one of the most frequently mutated genes associated with dilated cardiomyopathy (DCM). DCM related to mutations in LMNA is a common inherited cardiomyopathy that is associated with systolic dysfunction and cardiac arrhythmias. Here we modelled the LMNA-related DCM in vitro using patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Electrophysiological studies showed that the mutant iPSC-CMs displayed aberrant calcium homeostasis that led to arrhythmias at the single-cell level. Mechanistically, we show that the platelet-derived growth factor (PDGF) signalling pathway is activated in mutant iPSC-CMs compared to isogenic control iPSC-CMs. Conversely, pharmacological and molecular inhibition of the PDGF signalling pathway ameliorated the arrhythmic phenotypes of mutant iPSC-CMs in vitro. Taken together, our findings suggest that the activation of the PDGF pathway contributes to the pathogenesis of LMNA-related DCM and point to PDGF receptor-ß (PDGFRB) as a potential therapeutic target.
Asunto(s)
Cardiomiopatía Dilatada/genética , Lamina Tipo A/genética , Mutación , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patología , Calcio/metabolismo , Células Cultivadas , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/genética , Haploinsuficiencia/genética , Homeostasis , Humanos , Técnicas In Vitro , Células Madre Pluripotentes Inducidas/patología , Modelos Biológicos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Degradación de ARNm Mediada por Codón sin Sentido , ARN Mensajero/análisis , ARN Mensajero/genética , Análisis de la Célula IndividualRESUMEN
Patient-specific pluripotent stem cells (PSCs) can be generated via nuclear reprogramming by transcription factors (i.e., induced pluripotent stem cells, iPSCs) or by somatic cell nuclear transfer (SCNT). However, abnormalities and preclinical application of differentiated cells generated by different reprogramming mechanisms have yet to be evaluated. Here we investigated the molecular and functional features, and drug response of cardiomyocytes (PSC-CMs) and endothelial cells (PSC-ECs) derived from genetically relevant sets of human iPSCs, SCNT-derived embryonic stem cells (nt-ESCs), as well as in vitro fertilization embryo-derived ESCs (IVF-ESCs). We found that differentiated cells derived from isogenic iPSCs and nt-ESCs showed comparable lineage gene expression, cellular heterogeneity, physiological properties, and metabolic functions. Genome-wide transcriptome and DNA methylome analysis indicated that iPSC derivatives (iPSC-CMs and iPSC-ECs) were more similar to isogenic nt-ESC counterparts than those derived from IVF-ESCs. Although iPSCs and nt-ESCs shared the same nuclear DNA and yet carried different sources of mitochondrial DNA, CMs derived from iPSC and nt-ESCs could both recapitulate doxorubicin-induced cardiotoxicity and exhibited insignificant differences on reactive oxygen species generation in response to stress condition. We conclude that molecular and functional characteristics of differentiated cells from human PSCs are primarily attributed to the genetic compositions rather than the reprogramming mechanisms (SCNT vs. iPSCs). Therefore, human iPSCs can replace nt-ESCs as alternatives for generating patient-specific differentiated cells for disease modeling and preclinical drug testing.
Asunto(s)
Diferenciación Celular , Metilación de ADN , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Células Madre Embrionarias Humanas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Técnicas de Transferencia Nuclear , Células Endoteliales/citología , Estudio de Asociación del Genoma Completo , Células Madre Embrionarias Humanas/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citologíaRESUMEN
The ability to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes (CMs) makes them an attractive source for repairing injured myocardium, disease modeling, and drug testing. Although current differentiation protocols yield hPSC-CMs to >90% efficiency, hPSC-CMs exhibit immature characteristics. With the goal of overcoming this limitation, we tested the effects of varying passive stretch on engineered heart muscle (EHM) structural and functional maturation, guided by computational modeling. Human embryonic stem cells (hESCs, H7 line) or human induced pluripotent stem cells (IMR-90 line) were differentiated to hPSC-derived cardiomyocytes (hPSC-CMs) in vitro using a small molecule based protocol. hPSC-CMs were characterized by troponin+ flow cytometry as well as electrophysiological measurements. Afterwards, 1.2 × 106 hPSC-CMs were mixed with 0.4 × 106 human fibroblasts (IMR-90 line) (3:1 ratio) and type-I collagen. The blend was cast into custom-made 12-mm long polydimethylsiloxane reservoirs to vary nominal passive stretch of EHMs to 5, 7, or 9 mm. EHM characteristics were monitored for up to 50 days, with EHMs having a passive stretch of 7 mm giving the most consistent formation. Based on our initial macroscopic observations of EHM formation, we created a computational model that predicts the stress distribution throughout EHMs, which is a function of cellular composition, cellular ratio, and geometry. Based on this predictive modeling, we show cell alignment by immunohistochemistry and coordinated calcium waves by calcium imaging. Furthermore, coordinated calcium waves and mechanical contractions were apparent throughout entire EHMs. The stiffness and active forces of hPSC-derived EHMs are comparable with rat neonatal cardiomyocyte-derived EHMs. Three-dimensional EHMs display increased expression of mature cardiomyocyte genes including sarcomeric protein troponin-T, calcium and potassium ion channels, ß-adrenergic receptors, and t-tubule protein caveolin-3. Passive stretch affects the structural and functional maturation of EHMs. Based on our predictive computational modeling, we show how to optimize cell alignment and calcium dynamics within EHMs. These findings provide a basis for the rational design of EHMs, which enables future scale-up productions for clinical use in cardiovascular tissue engineering. Stem Cells 2018;36:265-277.
Asunto(s)
Biología Computacional/métodos , Miocardio/citología , Línea Celular , Citometría de Flujo , Humanos , Miocardio/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Ingeniería de Tejidos/métodosRESUMEN
The last decade has seen impressive progress in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) that makes them ideal tools to repair injured hearts. To achieve an optimal outcome, advanced molecular imaging methods are essential to accurately track these transplanted cells in the heart. Herein, we demonstrate for the first time that a class of photoacoustic nanoparticles (PANPs) incorporating semiconducting polymers (SPs) as contrast agents can be used in the photoacoustic imaging (PAI) of transplanted hESC-CMs in living mouse hearts. This is achieved by virtue of two benefits of PANPs. First, strong PA signals and specific spectral features of SPs allow PAI to sensitively detect and distinguish a small number of PANP-labeled cells (2,000) from background tissues in vivo. Second, the PANPs show a high efficiency for hESC-CM labeling without adverse effects on cell structure, function, and gene expression. Assisted by ultrasound imaging, the delivery and engraftment of hESC-CMs in living mouse hearts can be assessed by PANP-based PAI with high spatial resolution (~100 µm). In summary, this study explores and validates a novel application of SPs as a PA contrast agent to track labeled cells with high sensitivity and accuracy in vivo, highlighting the advantages of integrating PAI and PANPs to advance cardiac regenerative therapies.
Asunto(s)
Válvula Aórtica/diagnóstico por imagen , Endocarditis Bacteriana/diagnóstico por imagen , Radioisótopos de Flúor , Imagen Molecular , Tomografía Computarizada por Tomografía de Emisión de Positrones , Radiofármacos , Trisacáridos , Animales , Válvula Aórtica/microbiología , Proteínas Bacterianas/metabolismo , Modelos Animales de Enfermedad , Endocarditis Bacteriana/microbiología , Humanos , Proteínas de Transporte de Membrana , Ratones , Polisacáridos/metabolismo , Valor Predictivo de las Pruebas , Staphylococcus aureus/metabolismoRESUMEN
Diffuse intrinsic pontine glioma (DIPG) remains a fatal brainstem tumor demanding innovative therapies. As B7-H3 (CD276) is expressed on central nervous system (CNS) tumors, we designed B7-H3-specific chimeric antigen receptor (CAR) T cells, confirmed their preclinical efficacy, and opened BrainChild-03 (NCT04185038), a first-in-human phase I trial administering repeated locoregional B7-H3 CAR T cells to children with recurrent/refractory CNS tumors and DIPG. Here, we report the results of the first three evaluable patients with DIPG (including two who enrolled after progression), who received 40 infusions with no dose-limiting toxicities. One patient had sustained clinical and radiographic improvement through 12 months on study. Patients exhibited correlative evidence of local immune activation and persistent cerebrospinal fluid (CSF) B7-H3 CAR T cells. Targeted mass spectrometry of CSF biospecimens revealed modulation of B7-H3 and critical immune analytes (CD14, CD163, CSF-1, CXCL13, and VCAM-1). Our data suggest the feasibility of repeated intracranial B7-H3 CAR T-cell dosing and that intracranial delivery may induce local immune activation. SIGNIFICANCE: This is the first report of repeatedly dosed intracranial B7-H3 CAR T cells for patients with DIPG and includes preliminary tolerability, the detection of CAR T cells in the CSF, CSF cytokine elevations supporting locoregional immune activation, and the feasibility of serial mass spectrometry from both serum and CSF. This article is highlighted in the In This Issue feature, p. 1.
Asunto(s)
Neoplasias del Tronco Encefálico , Glioma Pontino Intrínseco Difuso , Humanos , Antígenos B7 , Neoplasias del Tronco Encefálico/terapia , Linfocitos TRESUMEN
Human pluripotent stem cells (PSCs), which are composed of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide an opportunity to advance cardiac cell therapy-based clinical trials. However, an important hurdle that must be overcome is the risk of teratoma formation after cell transplantation due to the proliferative capacity of residual undifferentiated PSCs in differentiation batches. To tackle this problem, we propose the use of a minimal noncardiotoxic doxorubicin dose as a purifying agent to selectively target rapidly proliferating stem cells for cell death, which will provide a purer population of terminally differentiated cardiomyocytes before cell transplantation. In this study, we determined an appropriate in vitro doxorubicin dose that (a) eliminates residual undifferentiated stem cells before cell injection to prevent teratoma formation after cell transplantation and (b) does not cause cardiotoxicity in ESC-derived cardiomyocytes (CMs) as demonstrated through contractility analysis, electrophysiology, topoisomerase activity assay, and quantification of reactive oxygen species generation. This study establishes a potentially novel method for tumorigenic-free cell therapy studies aimed at clinical applications of cardiac cell transplantation.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Doxorrubicina/administración & dosificación , Células Madre Embrionarias/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Células Madre Pluripotentes/citología , Animales , Apoptosis/efectos de los fármacos , Cardiotoxicidad/etiología , Cardiotoxicidad/prevención & control , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Tratamiento Basado en Trasplante de Células y Tejidos/efectos adversos , Relación Dosis-Respuesta a Droga , Doxorrubicina/farmacología , Células Madre Embrionarias/trasplante , Regulación de la Expresión Génica/efectos de los fármacos , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/efectos de los fármacos , Humanos , Ratones SCID , Especies Reactivas de Oxígeno/metabolismo , Teratoma/prevención & controlRESUMEN
AIMS: Stem cell therapy has shown promise for treating myocardial infarction via re-muscularization and paracrine signalling in both small and large animals. Non-human primates (NHPs), such as rhesus macaques (Macaca mulatta), are primarily utilized in preclinical trials due to their similarity to humans, both genetically and physiologically. Currently, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are delivered into the infarcted myocardium by either direct cell injection or an engineered tissue patch. Although both approaches have advantages in terms of sample preparation, cell-host interaction, and engraftment, how the iPSC-CMs respond to ischaemic conditions in the infarcted heart under these two different delivery approaches remains unclear. Here, we aim to gain a better understanding of the effects of hypoxia on iPSC-CMs at the transcriptome level. METHODS AND RESULTS: NHP iPSC-CMs in both monolayer culture (2D) and engineered heart tissue (EHT) (3D) format were exposed to hypoxic conditions to serve as surrogates of direct cell injection and tissue implantation in vivo, respectively. Outcomes were compared at the transcriptome level. We found the 3D EHT model was more sensitive to ischaemic conditions and similar to the native in vivo myocardium in terms of cell-extracellular matrix/cell-cell interactions, energy metabolism, and paracrine signalling. CONCLUSION: By exposing NHP iPSC-CMs to different culture conditions, transcriptome profiling improves our understanding of the mechanism of ischaemic injury.
Asunto(s)
Diferenciación Celular , Perfilación de la Expresión Génica , Células Madre Pluripotentes Inducidas/metabolismo , Isquemia Miocárdica/genética , Miocitos Cardíacos/metabolismo , Ingeniería de Tejidos , Transcriptoma , Animales , Hipoxia de la Célula , Uniones Célula-Matriz , Células Cultivadas , Metabolismo Energético , Redes Reguladoras de Genes , Frecuencia Cardíaca , Células Madre Pluripotentes Inducidas/patología , Macaca mulatta , Masculino , Ratones SCID , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patología , Miocitos Cardíacos/patología , Comunicación Paracrina , FenotipoRESUMEN
The growing number of victims of "stem cell tourism," the unregulated transplantation of stem cells worldwide, has raised concerns about the safety of stem cell transplantation. Although the transplantation of differentiated rather than undifferentiated cells is common practice, teratomas can still arise from the presence of residual undifferentiated stem cells at the time of transplant or from spontaneous mutations in differentiated cells. Because stem cell therapies are often delivered into anatomically sensitive sites, even small tumors can be clinically devastating, resulting in blindness, paralysis, cognitive abnormalities, and cardiovascular dysfunction. Surgical access to these sites may also be limited, leaving patients with few therapeutic options. Controlling stem cell misbehavior is, therefore, critical for the clinical translation of stem cell therapy. External beam radiation offers an effective means of delivering targeted therapy to decrease the teratoma burden while minimizing injury to surrounding organs. Additionally, this method avoids genetic manipulation or viral transduction of stem cells-which are associated with additional clinical safety and efficacy concerns. Here, we describe a protocol to create pluripotent stem cell-derived teratomas in mice and to apply external beam radiation therapy to selectively ablate these tumors in vivo.
Asunto(s)
Células Madre Pluripotentes/patología , Radioterapia/métodos , Teratoma/radioterapia , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Teratoma/patología , Resultado del TratamientoRESUMEN
Non-human primates (NHPs) can serve as a human-like model to study cell therapy using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). However, whether the efficacy of NHP and human iPSC-CMs is mechanistically similar remains unknown. To examine this, RNU rats received intramyocardial injection of 1 × 107 NHP or human iPSC-CMs or the same number of respective fibroblasts or PBS control (n = 9-14/group) at 4 days after 60-min coronary artery occlusion-reperfusion. Cardiac function and left ventricular remodeling were similarly improved in both iPSC-CM-treated groups. To mimic the ischemic environment in the infarcted heart, both cultured NHP and human iPSC-CMs underwent 24-hr hypoxia in vitro. Both cells and media were collected, and similarities in transcriptomic as well as metabolomic profiles were noted between both groups. In conclusion, both NHP and human iPSC-CMs confer similar cardioprotection in a rodent myocardial infarction model through relatively similar mechanisms via promotion of cell survival, angiogenesis, and inhibition of hypertrophy and fibrosis.
Asunto(s)
Células Madre Pluripotentes Inducidas/trasplante , Infarto del Miocardio/terapia , Miocitos Cardíacos/trasplante , Trasplante de Células Madre , Animales , Diferenciación Celular , Hipoxia de la Célula/fisiología , Supervivencia Celular/fisiología , Modelos Animales de Enfermedad , Humanos , Células Madre Pluripotentes Inducidas/citología , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/citología , Primates , RatasRESUMEN
BACKGROUND: The use of tissue engineering approaches in combination with exogenously produced cardiomyocytes offers the potential to restore contractile function after myocardial injury. However, current techniques assessing changes in global cardiac performance after such treatments are plagued by relatively low detection ability. Since the treatment is locally performed, this detection could be improved by myocardial strain imaging that measures regional contractility. METHODS AND RESULTS: Tissue engineered heart muscles (EHMs) were generated by casting human embryonic stem cell-derived cardiomyocytes with collagen in preformed molds. EHMs were transplanted (n=12) to cover infarct and border zones of recipient rat hearts 1 month after ischemia reperfusion injury. A control group (n=10) received only sham placement of sutures without EHMs. To assess the efficacy of EHMs, magnetic resonance imaging and ultrasound-based strain imaging were performed before and 4 weeks after transplantation. In addition to strain imaging, global cardiac performance was estimated from cardiac magnetic resonance imaging. Although no significant differences were found for global changes in left ventricular ejection fraction (control -9.6±1.3% versus EHM -6.2±1.9%; P=0.17), regional myocardial strain from tagged magnetic resonance imaging was able to detect preserved systolic function in EHM-treated animals compared with control (control 4.4±1.0% versus EHM 1.0±0.6%; P=0.04). However, ultrasound-based strain failed to detect any significant change (control 2.1±3.0% versus EHM 6.3±2.9%; P=0.46). CONCLUSIONS: This study highlights the feasibility of using cardiac strain from tagged magnetic resonance imaging to assess functional changes in rat models following localized regenerative therapies, which may not be detected by conventional measures of global systolic performance.