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Children with autism spectrum disorder (ASD) are often prescribed psychotropic medications but pharmacotherapy is typically conducted and evaluated based on clinical judgement without reference to objective measurement of treatment effectiveness and combined efficacy of pharmacological-behavioral interventions. We describe an interdisciplinary review team (IRT) model at a human services organization for children with ASD that was designed to standardize a process of psychotropic medication monitoring through (1) coordinated involvement of medical, nursing, behavior analyst, and special education professionals, (2) parent-guardian participation, (3) data-driven decision making, and (4) high-level administrative support. Our description includes case illustrations of medication reduction-elimination trials with five students and social validity assessment of IRT clinicians, nurses, and parent-guardians. Key components of the IRT model are emphasized with associated practice and research recommendations.
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The Production Assistance for Cellular Therapies (PACT) Program, is funded and supported by the US Department of Health and Human Services' National Institutes of Health (NIH) National Heart Lung and Blood Institute (NHLBI) to advance development of somatic cell and genetically modified cell therapeutics in the areas of heart, lung, and blood diseases. The program began in 2003, continued under two competitive renewals, and ended June 2021. PACT has supported cell therapy product manufacturing, investigational new drug enabling preclinical studies, and translational services, and has provided regulatory assistance for candidate cell therapy products that may aid in the repair and regeneration of damaged/diseased cells, tissues, and organs. PACT currently supports the development of novel cell therapies through five cell processing facilities. These facilities offer manufacturing processes, analytical development, technology transfer, process scale-up, and preclinical development expertise necessary to produce cell therapy products that are compliant with Good Laboratory Practices, current Good Manufacturing Practices, and current Good Tissue Practices regulations. The Emmes Company, LLC, serves as the Coordinating Center and assists with the management and coordination of PACT and its application submission and review process. This paper discusses the impact and accomplishments of the PACT program on the cell therapy field and its evolution over the duration of the program. It highlights the work that has been accomplished and provides a foundation to build future programs with similar goals to advance cellular therapeutics in a coordinated and centralized programmatic manner to support unmet medical needs within NHLBI purview.
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Tratamiento Basado en Trasplante de Células y Tejidos/economía , Financiación Gubernamental , National Heart, Lung, and Blood Institute (U.S.) , Academias e Institutos , Regulación Gubernamental , Estados UnidosRESUMEN
Canavan disease (CD) is a fatal leukodystrophy caused by mutation of the aspartoacylase (ASPA) gene, which leads to deficiency in ASPA activity, accumulation of the substrate N-acetyl-L-aspartate (NAA), demyelination, and spongy degeneration of the brain. There is neither a cure nor a standard treatment for this disease. In this study, human induced pluripotent stem cell (iPSC)-based cell therapy is developed for CD. A functional ASPA gene is introduced into patient iPSC-derived neural progenitor cells (iNPCs) or oligodendrocyte progenitor cells (iOPCs) via lentiviral transduction or TALEN-mediated genetic engineering to generate ASPA iNPC or ASPA iOPC. After stereotactic transplantation into a CD (Nur7) mouse model, the engrafted cells are able to rescue major pathological features of CD, including deficient ASPA activity, elevated NAA levels, extensive vacuolation, defective myelination, and motor function deficits, in a robust and sustainable manner. Moreover, the transplanted mice exhibit much prolonged survival. These genetically engineered patient iPSC-derived cellular products are promising cell therapies for CD. This study has the potential to bring effective cell therapies, for the first time, to Canavan disease children who have no treatment options. The approach established in this study can also benefit many other children who have deadly genetic diseases that have no cure.
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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.
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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
BACKGROUND: Advancing structural and functional maturation of stem cell-derived cardiomyocytes remains a key challenge for applications in disease modeling, drug screening, and heart repair. Here, we sought to advance cardiomyocyte maturation in engineered human myocardium (EHM) toward an adult phenotype under defined conditions. METHODS: We systematically investigated cell composition, matrix, and media conditions to generate EHM from embryonic and induced pluripotent stem cell-derived cardiomyocytes and fibroblasts with organotypic functionality under serum-free conditions. We used morphological, functional, and transcriptome analyses to benchmark maturation of EHM. RESULTS: EHM demonstrated important structural and functional properties of postnatal myocardium, including: (1) rod-shaped cardiomyocytes with M bands assembled as a functional syncytium; (2) systolic twitch forces at a similar level as observed in bona fide postnatal myocardium; (3) a positive force-frequency response; (4) inotropic responses to ß-adrenergic stimulation mediated via canonical ß1- and ß2-adrenoceptor signaling pathways; and (5) evidence for advanced molecular maturation by transcriptome profiling. EHM responded to chronic catecholamine toxicity with contractile dysfunction, cardiomyocyte hypertrophy, cardiomyocyte death, and N-terminal pro B-type natriuretic peptide release; all are classical hallmarks of heart failure. In addition, we demonstrate the scalability of EHM according to anticipated clinical demands for cardiac repair. CONCLUSIONS: We provide proof-of-concept for a universally applicable technology for the engineering of macroscale human myocardium for disease modeling and heart repair from embryonic and induced pluripotent stem cell-derived cardiomyocytes under defined, serum-free conditions.
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Células Madre Embrionarias/trasplante , Insuficiencia Cardíaca/terapia , Células Madre Pluripotentes Inducidas/trasplante , Miocitos Cardíacos/trasplante , Ingeniería de Tejidos/métodos , Remodelación Ventricular/fisiología , Animales , Diferenciación Celular/fisiología , Células Madre Embrionarias/fisiología , Insuficiencia Cardíaca/patología , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Miocardio/citología , Miocardio/patología , Miocitos Cardíacos/fisiología , Impresión Tridimensional , Ratas , Ratas DesnudasRESUMEN
Adolescent non-suicidal self-injury (NSSI) and suicidality are serious health concerns; however, factors that contribute to the transition from NSSI to suicide ideation and suicide attempts are unclear. To address this gap, we investigated whether demographic characteristics, child maltreatment, and psychiatric factors are associated with the level suicidality among adolescents with a history of self-injury. Participants were three groups of adolescent inpatient self-injurers (n = 397, 317 female), aged 13-18 years (M = 15.44, SD = 1.36): (a) non-ideators (n = 96; no current suicide ideation and no lifetime suicide attempts), (b) suicide ideators (n = 149; current ideation and no lifetime attempts), and (c) suicide attempters (n = 152; current ideation and at least one lifetime attempt). Participants completed interviews assessing psychiatric diagnoses, suicidality, and NSSI characteristics, as well as questionnaires on childhood trauma, psychiatric symptoms, and risky behavior engagement. Depression severity was associated with greater odds being a suicide ideator (p < 0.001, OR = 1.04) and an attempter (p < 0.001, OR = 1.05) compared to a non-ideator. Suicide attempters used more NSSI methods and reported greater risky behavior engagement than non-ideators (p = 0.03, OR = 1.29 and p = 0.03, OR = 1.06, respectively) and ideators (p = 0.015, OR = 1.25 and p = 0.04, OR = 1.05, respectively); attempters used more severe NSSI methods (e.g., burning). Our results identify a wide range of risk markers for increasing lethality in a sample at high risk for suicide mortality; future research is needed to refine risk assessments for adolescent self-injurers and determine the clinical utility of using risk markers for screening and intervention.
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Conducta Autodestructiva , Ideación Suicida , Intento de Suicidio , Adolescente , Análisis de Varianza , Depresión/complicaciones , Depresión/epidemiología , Depresión/terapia , Femenino , Humanos , Pacientes Internos , Entrevista Psicológica , Masculino , Análisis Multivariante , Escalas de Valoración Psiquiátrica , Asunción de Riesgos , Conducta Autodestructiva/complicaciones , Conducta Autodestructiva/epidemiología , Conducta Autodestructiva/psicología , Conducta Autodestructiva/terapia , Intento de Suicidio/psicología , Encuestas y CuestionariosRESUMEN
OBJECTIVE: Presently, little is known about what factors predict adolescent psychiatric rehospitalization. Thus, the present study tested whether a battery of demographic and clinical characteristics predicted readmission within 6 months of discharge. METHODS: Participants were 165 adolescents (112 females) aged 13-19 years (mean = 15.61, SD = 1.48) admitted to an acute residential treatment program between November 25, 2013, and November 18, 2014. Patients met diagnostic criteria (DSM-IV-TR) for current major depressive disorder or dysthymia. At admission, participants completed a battery of clinical interviews and questionnaires assessing demographics, early life stress, comorbid diagnoses, psychiatric symptoms, suicidality, self-injury, and risky behavior engagement. At discharge, psychiatric symptoms were reassessed. Readmission to the same residential service was monitored over a 6-month period following discharge. RESULTS: Overall, 12.1% of adolescents were rehospitalized. We conducted a series of Cox regression survival analyses to test demographic and clinical predictors of patients' time to readmission. More frequent self-injurious behaviors in the month prior to hospitalization was significantly associated with a more rapid time to rehospitalization (ß = 0.05, SE = .02, Wald1 = 4.35, P = .037, OR = 1.05, 95% CI = 1.003-1.10). CONCLUSIONS: It is critical to more effectively manage self-injury during the treatment of depressed adolescents, as this is the strongest predictor of later rehospitalization.
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Trastorno Depresivo/epidemiología , Trastorno Depresivo/terapia , Readmisión del Paciente/estadística & datos numéricos , Tratamiento Domiciliario , Adolescente , Manejo de Caso , Terapia Combinada , Trastorno Depresivo/diagnóstico , Trastorno Depresivo/psicología , Femenino , Humanos , Entrevista Psicológica , Funciones de Verosimilitud , Masculino , Massachusetts , Psicometría/estadística & datos numéricos , Medición de Riesgo/estadística & datos numéricos , Conducta Autodestructiva/diagnóstico , Conducta Autodestructiva/epidemiología , Conducta Autodestructiva/psicología , Conducta Autodestructiva/terapia , Encuestas y CuestionariosRESUMEN
In familial pulmonary arterial hypertension (FPAH), the autosomal dominant disease-causing BMPR2 mutation is only 20% penetrant, suggesting that genetic variation provides modifiers that alleviate the disease. Here, we used comparison of induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from three families with unaffected mutation carriers (UMCs), FPAH patients, and gender-matched controls to investigate this variation. Our analysis identified features of UMC iPSC-ECs related to modifiers of BMPR2 signaling or to differentially expressed genes. FPAH-iPSC-ECs showed reduced adhesion, survival, migration, and angiogenesis compared to UMC-iPSC-ECs and control cells. The "rescued" phenotype of UMC cells was related to an increase in specific BMPR2 activators and/or a reduction in inhibitors, and the improved cell adhesion could be attributed to preservation of related signaling. The improved survival was related to increased BIRC3 and was independent of BMPR2. Our findings therefore highlight protective modifiers for FPAH that could help inform development of future treatment strategies.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Células Endoteliales/citología , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/prevención & control , Células Madre Pluripotentes Inducidas/citología , Mutación/genética , Secuencia de Bases , Proteína Morfogenética Ósea 4/farmacología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Edición Génica , Regulación de la Expresión Génica/efectos de los fármacos , Heterocigoto , Humanos , Hipertensión Pulmonar/patología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/genética , Fosforilación/efectos de los fármacos , Análisis de Secuencia de ARN , Transducción de Señal/efectos de los fármacos , Proteínas Smad/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Many reprogramming methods can generate human induced pluripotent stem cells (hiPSCs) that closely resemble human embryonic stem cells (hESCs). This has led to assessments of how similar hiPSCs are to hESCs, by evaluating differences in gene expression, epigenetic marks and differentiation potential. However, all previous studies were performed using hiPSCs acquired from different laboratories, passage numbers, culturing conditions, genetic backgrounds and reprogramming methods, all of which may contribute to the reported differences. Here, by using high-throughput sequencing under standardized cell culturing conditions and passage number, we compare the epigenetic signatures (H3K4me3, H3K27me3 and HDAC2 ChIP-seq profiles) and transcriptome differences (by RNA-seq) of hiPSCs generated from the same primary fibroblast population by using six different reprogramming methods. We found that the reprogramming method impacts the resulting transcriptome and that all hiPSC lines could terminally differentiate, regardless of the reprogramming method. Moreover, by comparing the differences between the hiPSC and hESC lines, we observed a significant proportion of differentially expressed genes that could be attributed to polycomb repressive complex targets.
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Understanding individual susceptibility to drug-induced cardiotoxicity is key to improving patient safety and preventing drug attrition. Human induced pluripotent stem cells (hiPSCs) enable the study of pharmacological and toxicological responses in patient-specific cardiomyocytes (CMs) and may serve as preclinical platforms for precision medicine. Transcriptome profiling in hiPSC-CMs from seven individuals lacking known cardiovascular disease-associated mutations and in three isogenic human heart tissue and hiPSC-CM pairs showed greater inter-patient variation than intra-patient variation, verifying that reprogramming and differentiation preserve patient-specific gene expression, particularly in metabolic and stress-response genes. Transcriptome-based toxicology analysis predicted and risk-stratified patient-specific susceptibility to cardiotoxicity, and functional assays in hiPSC-CMs using tacrolimus and rosiglitazone, drugs targeting pathways predicted to produce cardiotoxicity, validated inter-patient differential responses. CRISPR/Cas9-mediated pathway correction prevented drug-induced cardiotoxicity. Our data suggest that hiPSC-CMs can be used in vitro to predict and validate patient-specific drug safety and efficacy, potentially enabling future clinical approaches to precision medicine.
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Perfilación de la Expresión Génica , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/citología , Tacrolimus/efectos adversos , Tiazolidinedionas/efectos adversos , Sistemas CRISPR-Cas/genética , Muerte Celular/efectos de los fármacos , Edición Génica , Genoma Humano , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Humanos , Secuencias Invertidas Repetidas/genética , Miocitos Cardíacos/metabolismo , Rosiglitazona , Resultado del TratamientoRESUMEN
The use of cells derived from pluripotent stem cells (PSCs) for regenerative therapies confers a considerable risk for neoplastic growth and teratoma formation. Preclinical and clinical assessment of such therapies will require suitable monitoring strategies to understand and mitigate these risks. Here we generated human-induced pluripotent stem cells (iPSCs), selected clones that continued to express reprogramming factors after differentiation into cardiomyocytes, and transplanted these cardiomyocytes into immunocompromised rat hearts post-myocardial infarction. We compared magnetic resonance imaging (MRI), cardiac ultrasound, and serum biomarkers for their ability to delineate teratoma formation and growth. MRI enabled the detection of teratomas with a volume >8 mm(3). A combination of three plasma biomarkers (CEA, AFP, and HCG) was able to detect teratomas with a volume >17 mm(3) and with a sensitivity of more than 87%. Based on our findings, a combination of serum biomarkers with MRI screening may offer the highest sensitivity for teratoma detection and tracking.
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Biomarcadores de Tumor/sangre , Imagen por Resonancia Magnética/métodos , Células Madre Pluripotentes/patología , Teratoma/sangre , Teratoma/diagnóstico , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Gadolinio , Corazón/fisiopatología , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Inflamación/patología , Lentivirus/metabolismo , Masculino , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Miocitos Cardíacos/trasplante , Fenotipo , Células Madre Pluripotentes/efectos de los fármacos , Ratas Desnudas , Teratoma/irrigación sanguínea , Carga Tumoral/efectos de los fármacosRESUMEN
BACKGROUND: Suicide is the second leading cause of death among adolescents, and depressed youth are six times more likely to make suicide attempts as compared to non-depressed adolescents. The present study examined the unique and interactive effects of two well-established correlates of suicidality - childhood sexual abuse (CSA) and disinhibition - in predicting suicide attempts among depressed adolescents. METHOD: Participants were 163 adolescents (125 females) aged 13-18 (M=15.60, SD=1.27) diagnosed with Major Depressive Disorder (n=95, 58.3%) and/or Dysthymia (n=69, 42.3%) recruited from an acute residential treatment service. Participants completed interviews assessing psychopathology and suicidality, self-report measures of depressive symptoms and CSA, and a computerized disinhibition task. RESULTS: Consistent with hypotheses, CSA moderated the association between disinhibition and adolescents' report of their past year and lifetime suicide attempts. Specifically, higher disinhibition was associated with a greater likelihood of having made a suicide attempt among adolescents with a history of CSA, but not among those without. The same pattern of results held in analyses of suicide attempt frequency. LIMITATIONS: Primary findings were based on observational, cross-sectional data, and therefore, causal relationships cannot be inferred. The gender imbalance in the sample precluded stratifying our analyses by gender. CSA was ascertained by self-report; replication of the results with more objective measures is warranted. CONCLUSIONS: Our findings indicate that CSA and disinhibition may work together to predict elevated suicide risk, and these results have implications for early identification efforts in youth at high risk for suicide.
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Conducta del Adolescente/psicología , Abuso Sexual Infantil/psicología , Trastorno Depresivo Mayor/psicología , Inhibición Psicológica , Intento de Suicidio/prevención & control , Intento de Suicidio/psicología , Adolescente , Estudios Transversales , Trastorno Depresivo Mayor/complicaciones , Femenino , Humanos , MasculinoRESUMEN
To meet the need of a large quantity of hPSC-derived cardiomyocytes (CM) for pre-clinical and clinical studies, a robust and scalable differentiation system for CM production is essential. With a human pluripotent stem cells (hPSC) aggregate suspension culture system we established previously, we developed a matrix-free, scalable, and GMP-compliant process for directing hPSC differentiation to CM in suspension culture by modulating Wnt pathways with small molecules. By optimizing critical process parameters including: cell aggregate size, small molecule concentrations, induction timing, and agitation rate, we were able to consistently differentiate hPSCs to >90% CM purity with an average yield of 1.5 to 2×10(9) CM/L at scales up to 1L spinner flasks. CM generated from the suspension culture displayed typical genetic, morphological, and electrophysiological cardiac cell characteristics. This suspension culture system allows seamless transition from hPSC expansion to CM differentiation in a continuous suspension culture. It not only provides a cost and labor effective scalable process for large scale CM production, but also provides a bioreactor prototype for automation of cell manufacturing, which will accelerate the advance of hPSC research towards therapeutic applications.
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Diferenciación Celular , Células Madre Pluripotentes/citología , Actinina/metabolismo , Técnicas de Cultivo de Célula , Línea Celular , Regulación de la Expresión Génica , Humanos , Microscopía Fluorescente , Miocitos Cardíacos/citología , Células Madre Pluripotentes/metabolismo , ARN/química , ARN/aislamiento & purificación , Análisis de Secuencia de ARN , Troponina I/metabolismo , Troponina T/metabolismo , Vía de Señalización WntRESUMEN
RATIONALE: Tissue engineering approaches may improve survival and functional benefits from human embryonic stem cell-derived cardiomyocyte transplantation, thereby potentially preventing dilative remodeling and progression to heart failure. OBJECTIVE: Assessment of transport stability, long-term survival, structural organization, functional benefits, and teratoma risk of engineered heart muscle (EHM) in a chronic myocardial infarction model. METHODS AND RESULTS: We constructed EHMs from human embryonic stem cell-derived cardiomyocytes and released them for transatlantic shipping following predefined quality control criteria. Two days of shipment did not lead to adverse effects on cell viability or contractile performance of EHMs (n=3, P=0.83, P=0.87). One month after ischemia/reperfusion injury, EHMs were implanted onto immunocompromised rat hearts to simulate chronic ischemia. Bioluminescence imaging showed stable engraftment with no significant cell loss between week 2 and 12 (n=6, P=0.67), preserving ≤25% of the transplanted cells. Despite high engraftment rates and attenuated disease progression (change in ejection fraction for EHMs, -6.7±1.4% versus control, -10.9±1.5%; n>12; P=0.05), we observed no difference between EHMs containing viable and nonviable human cardiomyocytes in this chronic xenotransplantation model (n>12; P=0.41). Grafted cardiomyocytes showed enhanced sarcomere alignment and increased connexin 43 expression at 220 days after transplantation. No teratomas or tumors were found in any of the animals (n=14) used for long-term monitoring. CONCLUSIONS: EHM transplantation led to high engraftment rates, long-term survival, and progressive maturation of human cardiomyocytes. However, cell engraftment was not correlated with functional improvements in this chronic myocardial infarction model. Most importantly, the safety of this approach was demonstrated by the lack of tumor or teratoma formation.
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Células Madre Embrionarias/trasplante , Supervivencia de Injerto , Trasplante de Corazón/métodos , Infarto del Miocardio/cirugía , Miocitos Cardíacos/trasplante , Músculos Papilares/trasplante , Ingeniería de Tejidos/métodos , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Línea Celular , Supervivencia Celular , Conexina 43/metabolismo , Modelos Animales de Enfermedad , Células Madre Embrionarias/inmunología , Células Madre Embrionarias/metabolismo , Trasplante de Corazón/efectos adversos , Xenoinjertos , Humanos , Inmunosupresores/farmacología , Masculino , Contracción Miocárdica , Infarto del Miocardio/inmunología , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/inmunología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Músculos Papilares/inmunología , Músculos Papilares/metabolismo , Músculos Papilares/patología , Músculos Papilares/fisiopatología , Ratas Desnudas , Ratas Sprague-Dawley , Volumen Sistólico , Factores de Tiempo , TransfecciónRESUMEN
ß-adrenergic signaling pathways mediate key aspects of cardiac function. Its dysregulation is associated with a range of cardiac diseases, including dilated cardiomyopathy (DCM). Previously, we established an iPSC model of familial DCM from patients with a mutation in TNNT2, a sarcomeric protein. Here, we found that the ß-adrenergic agonist isoproterenol induced mature ß-adrenergic signaling in iPSC-derived cardiomyocytes (iPSC-CMs) but that this pathway was blunted in DCM iPSC-CMs. Although expression levels of several ß-adrenergic signaling components were unaltered between control and DCM iPSC-CMs, we found that phosphodiesterases (PDEs) 2A and PDE3A were upregulated in DCM iPSC-CMs and that PDE2A was also upregulated in DCM patient tissue. We further discovered increased nuclear localization of mutant TNNT2 and epigenetic modifications of PDE genes in both DCM iPSC-CMs and patient tissue. Notably, pharmacologic inhibition of PDE2A and PDE3A restored cAMP levels and ameliorated the impaired ß-adrenergic signaling of DCM iPSC-CMs, suggesting therapeutic potential.
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Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Células Madre Pluripotentes Inducidas/fisiología , Receptores Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/farmacología , Cardiomiopatía Dilatada/patología , Diferenciación Celular , Células Cultivadas , Epigénesis Genética , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/patología , Isoproterenol/farmacología , Modelos Cardiovasculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Miocitos Cardíacos/fisiología , Transducción de Señal , Troponina T/genética , Troponina T/metabolismo , Regulación hacia ArribaAsunto(s)
Células Madre Adultas/trasplante , Exosomas , Terapia Genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/uso terapéutico , MicroARNs/uso terapéutico , Células Madre Multipotentes/trasplante , Infarto del Miocardio/terapia , Isquemia Miocárdica/terapia , Animales , Femenino , MasculinoRESUMEN
There is a need for physical standards (reference materials) to ensure both reproducibility and consistency in the production of somatic cell types from human pluripotent stem cell (hPSC) sources. We have outlined the need for reference materials (RMs) in relation to the unique properties and concerns surrounding hPSC-derived products and suggest in-house approaches to RM generation relevant to basic research, drug screening, and therapeutic applications. hPSCs have an unparalleled potential as a source of somatic cells for drug screening, disease modeling, and therapeutic application. Undefined variation and product variability after differentiation to the lineage or cell type of interest impede efficient translation and can obscure the evaluation of clinical safety and efficacy. Moreover, in the absence of a consistent population, data generated from in vitro studies could be unreliable and irreproducible. Efforts to devise approaches and tools that facilitate improved consistency of hPSC-derived products, both as development tools and therapeutic products, will aid translation. Standards exist in both written and physical form; however, because many unknown factors persist in the field, premature written standards could inhibit rather than promote innovation and translation. We focused on the derivation of physical standard RMs. We outline the need for RMs and assess the approaches to in-house RM generation for hPSC-derived products, a critical tool for the analysis and control of product variation that can be applied by researchers and developers. We then explore potential routes for the generation of RMs, including both cellular and noncellular materials and novel methods that might provide valuable tools to measure and account for variation. Multiparametric techniques to identify "signatures" for therapeutically relevant cell types, such as neurons and cardiomyocytes that can be derived from hPSCs, would be of significant utility, although physical RMs will be required for clinical purposes.
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Investigación Biomédica , Evaluación Preclínica de Medicamentos , Células Madre Pluripotentes , Investigación Biomédica/instrumentación , Investigación Biomédica/métodos , Investigación Biomédica/normas , Investigación Biomédica/tendencias , Evaluación Preclínica de Medicamentos/economía , Evaluación Preclínica de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/normas , Evaluación Preclínica de Medicamentos/tendencias , Humanos , Estándares de ReferenciaRESUMEN
AIMS: High-fat diet-induced obesity (DIO) is a major contributor to type II diabetes and micro- and macro-vascular complications leading to peripheral vascular disease (PVD). Metabolic abnormalities of induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from obese individuals could potentially limit their therapeutic efficacy for PVD. The aim of this study was to compare the function of iPSC-ECs from normal and DIO mice using comprehensive in vitro and in vivo assays. METHODS AND RESULTS: Six-week-old C57Bl/6 mice were fed with a normal or high-fat diet. At 24 weeks, iPSCs were generated from tail tip fibroblasts and differentiated into iPSC-ECs using a directed monolayer approach. In vitro functional analysis revealed that iPSC-ECs from DIO mice had significantly decreased capacity to form capillary-like networks, diminished migration, and lower proliferation. Microarray and ELISA confirmed elevated apoptotic, inflammatory, and oxidative stress pathways in DIO iPSC-ECs. Following hindlimb ischaemia, mice receiving intramuscular injections of DIO iPSC-ECs had significantly decreased reperfusion compared with mice injected with control healthy iPSC-ECs. Hindlimb sections revealed increased muscle atrophy and presence of inflammatory cells in mice receiving DIO iPSC-ECs. When pravastatin was co-administered to mice receiving DIO iPSC-ECs, a significant increase in reperfusion was observed; however, this beneficial effect was blunted by co-administration of the nitric oxide synthase inhibitor, N(ω)-nitro-l-arginine methyl ester. CONCLUSION: This is the first study to provide evidence that iPSC-ECs from DIO mice exhibit signs of endothelial dysfunction and have suboptimal efficacy following transplantation in a hindlimb ischaemia model. These findings may have important implications for future treatment of PVD using iPSC-ECs in the obese population.
Asunto(s)
Células Endoteliales/fisiología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Células Madre Pluripotentes Inducidas/fisiología , Óxido Nítrico/fisiología , Obesidad/fisiopatología , Pravastatina/farmacología , Análisis de Varianza , Animales , Apoptosis/fisiología , Diferenciación Celular , Dieta Alta en Grasa , Inhibidores Enzimáticos/farmacología , Fibroblastos/fisiología , Miembro Posterior/irrigación sanguínea , Inyecciones Intramusculares , Isquemia/fisiopatología , Isquemia/prevención & control , Ratones Endogámicos C57BL , Músculo Esquelético , Enfermedades Musculares/prevención & control , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Estrés Oxidativo/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Distribución Aleatoria , Daño por Reperfusión/fisiopatología , Transducción de SeñalRESUMEN
Nearly 8% of the human population carries an inactivating point mutation in the gene that encodes the cardioprotective enzyme aldehyde dehydrogenase 2 (ALDH2). This genetic polymorphism (ALDH2*2) is linked to more severe outcomes from ischemic heart damage and an increased risk of coronary artery disease (CAD), but the underlying molecular bases are unknown. We investigated the ALDH2*2 mechanisms in a human model system of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from individuals carrying the most common heterozygous form of the ALDH2*2 genotype. We showed that the ALDH2*2 mutation gave rise to elevated amounts of reactive oxygen species and toxic aldehydes, thereby inducing cell cycle arrest and activation of apoptotic signaling pathways, especially during ischemic injury. We established that ALDH2 controls cell survival decisions by modulating oxidative stress levels and that this regulatory circuitry was dysfunctional in the loss-of-function ALDH2*2 genotype, causing up-regulation of apoptosis in cardiomyocytes after ischemic insult. These results reveal a new function for the metabolic enzyme ALDH2 in modulation of cell survival decisions. Insight into the molecular mechanisms that mediate ALDH2*2-related increased ischemic damage is important for the development of specific diagnostic methods and improved risk management of CAD and may lead to patient-specific cardiac therapies.
Asunto(s)
Aldehído Deshidrogenasa/genética , Diferenciación Celular , Células Madre Pluripotentes Inducidas/enzimología , Isquemia Miocárdica/enzimología , Isquemia Miocárdica/genética , Miocitos Cardíacos/enzimología , Polimorfismo Genético , Aldehído Deshidrogenasa/antagonistas & inhibidores , Aldehído Deshidrogenasa/metabolismo , Aldehído Deshidrogenasa Mitocondrial , Aldehídos/metabolismo , Apoptosis , Puntos de Control del Ciclo Celular , Línea Celular , Inhibidores Enzimáticos/farmacología , Predisposición Genética a la Enfermedad , Heterocigoto , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/patología , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Isquemia Miocárdica/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Estrés Oxidativo , Fenotipo , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Factores de Tiempo , Transfección , Adulto JovenRESUMEN
BACKGROUND: Despite the promise shown by stem cells for restoration of cardiac function after myocardial infarction, the poor survival of transplanted cells has been a major issue. Hypoxia-inducible factor-1 (HIF1) is a transcription factor that mediates adaptive responses to ischemia. Here, we hypothesize that codelivery of cardiac progenitor cells (CPCs) with a nonviral minicircle plasmid carrying HIF1 (MC-HIF1) into the ischemic myocardium can improve the survival of transplanted CPCs. METHODS AND RESULTS: After myocardial infarction, CPCs were codelivered intramyocardially into adult NOD/SCID mice with saline, MC-green fluorescent protein, or MC-HIF1 versus MC-HIF1 alone (n=10 per group). Bioluminescence imaging demonstrated better survival when CPCs were codelivered with MC-HIF1. Importantly, echocardiography showed mice injected with CPCs+MC-HIF1 had the highest ejection fraction 6 weeks after myocardial infarction (57.1±2.6%; P=0.002) followed by MC-HIF1 alone (48.5±2.6%; P=0.04), with no significant protection for CPCs+MC-green fluorescent protein (44.8±3.3%; P=NS) when compared with saline control (38.7±3.2%). In vitro mechanistic studies confirmed that cardiac endothelial cells produced exosomes that were actively internalized by recipient CPCs. Exosomes purified from endothelial cells overexpressing HIF1 had higher contents of miR-126 and miR-210. These microRNAs activated prosurvival kinases and induced a glycolytic switch in recipient CPCs, giving them increased tolerance when subjected to in vitro hypoxic stress. Inhibiting both of these miRs blocked the protective effects of the exosomes. CONCLUSIONS: In summary, HIF1 can be used to modulate the host microenvironment for improving survival of transplanted cells. The exosomal transfer of miRs from host cells to transplanted cells represents a unique mechanism that can be potentially targeted for improving survival of transplanted cells.