RESUMEN
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disorder with minimally effective treatment options. An important hurdle in ALS drug development is the non-invasive therapeutic access to the motor cortex currently limited by the presence of the blood-brain barrier (BBB). Focused ultrasound and microbubble (FUS+ MB) treatment is an emerging technology that was successfully used in ALS patients to temporarily open the cortical BBB. However, FUS+ MB-mediated drug delivery across ALS patients' BBB has not yet been reported. Similarly, the effects of FUS+ MB on human ALS BBB cells remain unexplored. METHODS: Here we established the first FUS+ MB-compatible, fully-human ALS patient-cell-derived BBB model based on induced brain endothelial-like cells (iBECs) to study anti-TDP-43 antibody delivery and FUS+ MB bioeffects in vitro. RESULTS: Generated ALS iBECs recapitulated disease-specific hallmarks of BBB pathology, including reduced BBB integrity and permeability, and TDP-43 proteinopathy. The results also identified differences between sporadic ALS and familial (C9orf72 expansion carrying) ALS iBECs reflecting patient heterogeneity associated with disease subgroups. Studies in these models revealed successful ALS iBEC monolayer opening in vitro with no adverse cellular effects of FUS+ MB as reflected by lactate dehydrogenase (LDH) release viability assay and the lack of visible monolayer damage or morphology change in FUS+ MB treated cells. This was accompanied by the molecular bioeffects of FUS+ MB in ALS iBECs including changes in expression of tight and adherens junction markers, and drug transporter and inflammatory mediators, with sporadic and C9orf72 ALS iBECs generating transient specific responses. Additionally, we demonstrated an effective increase in the delivery of anti-TDP-43 antibody with FUS+ MB in C9orf72 (2.7-fold) and sporadic (1.9-fold) ALS iBECs providing the first proof-of-concept evidence that FUS+ MB can be used to enhance the permeability of large molecule therapeutics across the BBB in a human ALS in vitro model. CONCLUSIONS: Together, this study describes the first characterisation of cellular and molecular responses of ALS iBECs to FUS+ MB and provides a fully-human platform for FUS+ MB-mediated drug delivery screening on an ALS BBB in vitro model.
Asunto(s)
Esclerosis Amiotrófica Lateral , Barrera Hematoencefálica , Proteínas de Unión al ADN , Microburbujas , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Humanos , Proteínas de Unión al ADN/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Células Endoteliales/metabolismo , Anticuerpos/administración & dosificación , Ondas Ultrasónicas , Células CultivadasRESUMEN
RATIONALE: Physiological hypertrophy in the developing heart has been considered the product of an increase in volume of preexisting fetal cardiomyocytes in the absence of myocyte formation. OBJECTIVE: In this study, we tested whether the mouse heart at birth has a pool of cardiac stem cells (CSCs) that differentiate into myocytes contributing to the postnatal expansion of the parenchymal cell compartment. METHODS AND RESULTS: We have found that the newborn heart contains a population of c-kit-positive CSCs that are lineage negative, self-renewing, and multipotent. CSCs express the Notch1 receptor and show the nuclear localization of its active fragment, N1ICD. In 60% of cases, N1ICD was coupled with the presence of Nkx2.5, indicating that the commitment of CSCs to the myocyte lineage is regulated by Notch1. Importantly, overexpression of N1ICD in neonatal CSCs significantly expanded the proportion of transit-amplifying myocytes. To establish whether these in vitro findings had a functional counterpart in vivo, the Notch pathway was blocked in newborn mice by administration of a gamma-secretase inhibitor. This intervention resulted in the development of a dilated myopathy and high mortality rates. Ventricular decompensation was characterized by a 62% reduction in amplifying myocytes, which resulted in a 54% decrease in myocyte number. After cessation of Notch blockade and recovery of myocyte regeneration, cardiac anatomy and function were largely restored. CONCLUSIONS: Notch1 signaling is a critical determinant of CSC growth and differentiation; when this cascade of events is altered, cardiomyogenesis is impaired, physiological cardiac hypertrophy is prevented, and a life-threatening myopathy supervenes.
Asunto(s)
Cardiomiopatía Dilatada/etiología , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiología , Receptor Notch1/antagonistas & inhibidores , Actinina/metabolismo , Actinas/metabolismo , Animales , Animales Recién Nacidos , Capilares/citología , Capilares/fisiología , Cardiomiopatía Dilatada/fisiopatología , Diferenciación Celular , División Celular , Corazón/crecimiento & desarrollo , Humanos , Recién Nacido , Ratones , Receptor Notch1/fisiología , Receptores Notch/antagonistas & inhibidores , Receptores Notch/fisiología , Células Madre/citología , Células Madre/fisiología , Factores de Transcripción/metabolismoRESUMEN
An analysis of the clonality of cardiac progenitor cells (CPCs) and myocyte turnover in vivo requires genetic tagging of the undifferentiated cells so that the clonal marker of individual mother cells is traced in the specialized progeny. CPC niches in the atria and apex of the mouse heart were infected with a lentivirus carrying EGFP, and the destiny of the tagged cells was determined 1-5 months later. A common integration site was identified in isolated CPCs, cardiomyocytes, endothelial cells (ECs), and fibroblasts, documenting CPC self-renewal and multipotentiality and the clonal origin of the differentiated cell populations. Subsequently, the degree of EGFP-lentiviral infection of CPCs was evaluated 2-4 days after injection, and the number of myocytes expressing the reporter gene was measured 6 months later. A BrdU pulse-chasing protocol was also introduced as an additional assay for the analysis of myocyte turnover. Over a period of 6 months, each EGFP-positive CPC divided approximately eight times generating 230 cardiomyocytes; this value was consistent with the number of newly formed cells labeled by BrdU. To determine whether, human CPCs (hCPCs) are self-renewing and multipotent, these cells were transduced with the EGFP-lentivirus and injected after acute myocardial infarction in immunosuppressed rats. hCPCs, myocytes, ECs, and fibroblasts collected from the regenerated myocardium showed common viral integration sites in the human genome. Thus, our results indicate that the adult heart contains a pool of resident stem cells that regulate cardiac homeostasis and repair.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Células 3T3 , Animales , Secuencia de Bases , Linaje de la Célula , Células Clonales/citología , Células Clonales/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunohistoquímica , Lentivirus/genética , Ratones , Datos de Secuencia Molecular , Miocardio/citología , Miocardio/metabolismo , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Ratas , Ratas Endogámicas F344 , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
The study of neurodegenerative diseases using pluripotent stem cells requires new methods to assess neurodevelopment and neurodegeneration of specific neuronal subtypes. The cholinergic system, characterized by its use of the neurotransmitter acetylcholine, is one of the first to degenerate in Alzheimer's disease and is also affected in frontotemporal dementia. We developed a differentiation protocol to generate basal forebrain-like cholinergic neurons (BFCNs) from induced pluripotent stem cells (iPSCs) aided by the use of small molecule inhibitors and growth factors. Ten iPSC lines were successfully differentiated into BFCNs using this protocol. The neuronal cultures were characterised through RNA and protein expression, and functional analysis of neurons was confirmed by whole-cell patch clamp. We have developed a reliable protocol using only small molecule inhibitors and growth factors, while avoiding transfection or cell sorting methods, to achieve a BFCN culture that expresses the characteristic markers of cholinergic neurons.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Neuronas Colinérgicas/efectos de los fármacos , Medios de Cultivo/farmacología , Cuerpos Embrioides/efectos de los fármacos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Cultivo Primario de Células/métodos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Prosencéfalo Basal/metabolismo , Prosencéfalo Basal/patología , Benzamidas/farmacología , Factor Neurotrófico Derivado del Encéfalo/farmacología , Línea Celular , Neuronas Colinérgicas/citología , Neuronas Colinérgicas/metabolismo , Medios de Cultivo/química , Dioxoles/farmacología , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Factor 2 de Diferenciación de Crecimiento/farmacología , Proteínas Hedgehog/farmacología , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Modelos Biológicos , Factor de Crecimiento Nervioso/farmacología , Técnicas de Placa-Clamp , Pirazoles/farmacología , Pirimidinas/farmacología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Peripheral dermal fibroblasts (DF) from a healthy 56â¯year old female were obtained from the Centre for Healthy Brain Ageing (CHeBA) Biobank, University of New South Wales, under the material transfer agreement with the University of Wollongong. DFs were reprogrammed via mRNA-delivered transcription factors into induced pluripotent stem cells (iPSCs). The generated iPSCs were confirmed to be pluripotent, capable of three germ layer differentiation and are thus a useful resource for creating iPSC-derived healthy human cells of any lineage. Resource table.
Asunto(s)
Reprogramación Celular/fisiología , Fibroblastos/citología , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/citología , Piel/citología , Células Cultivadas , Reprogramación Celular/genética , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Persona de Mediana EdadRESUMEN
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.