RESUMEN

The investigation of neurodegenerative diseases advanced significantly with the advent of cell-reprogramming technology, leading to the creation of new models of human illness. These models, derived from induced pluripotent stem cells (iPSCs), facilitate the study of sporadic as well as hereditary diseases and provide a comprehensive understanding of the molecular mechanisms involved with neurodegeneration. Through proteomics, a quantitative tool capable of identifying thousands of proteins from small sample volumes, researchers have attempted to identify disease mechanisms by detecting differentially expressed proteins and proteoforms in disease models, biofluids, and postmortem brain tissue. The integration of these two technologies allows for the identification of novel pathological targets within the realm of neurodegenerative diseases. Here, we highlight studies from the past 5 years on the contributions of iPSCs within neuroproteomic investigations, which uncover the molecular mechanisms behind these illnesses.

Asunto(s)

Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Reprogramación Celular , Enfermedades Neurodegenerativas/metabolismo

RESUMEN

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease among adults worldwide. It is characterised by the death of dopaminergic neurons in the substantia nigra pars compacta and, in some cases, presence of intracytoplasmic inclusions of α-synuclein, called Lewy bodies, a pathognomonic sign of the disease. Clinical diagnosis of PD is based on the presence of motor alterations. The treatments currently available have no neuroprotective effect. The exact causes of PD are poorly understood. Therefore, more precise preclinical models have been developed in recent years that use induced pluripotent stem cells (iPSC). In vitro studies can provide new information on PD pathogenesis and may help to identify new therapeutic targets or to develop new drugs.

Asunto(s)

Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Fármacos Neuroprotectores , Enfermedad de Parkinson , Adulto , Humanos , Enfermedad de Parkinson/tratamiento farmacológico , Células Madre Pluripotentes Inducidas/patología , Neuronas Dopaminérgicas , Fármacos Neuroprotectores/farmacología

RESUMEN

Turner syndrome (TS) is a genetic disorder in females with X Chromosome monosomy associated with highly variable clinical features, including premature primary gonadal failure leading to ovarian dysfunction and infertility. The mechanism of development of primordial germ cells (PGCs) and their connection with ovarian failure in TS is poorly understood. An in vitro model of PGCs from TS would be beneficial for investigating genetic and epigenetic factors that influence germ cell specification. Here we investigated the potential of reprogramming peripheral mononuclear blood cells from TS women (PBMCs-TS) into iPSCs following in vitro differentiation in hPGCLCs. All hiPSCs-TS lines demonstrated pluripotency state and were capable of differentiation into three embryonic layers (ectoderm, endoderm, and mesoderm). The PGCLCs-TS recapitulated the initial germline development period regarding transcripts and protein marks, including the epigenetic profile. Overall, our results highlighted the feasibility of producing in vitro models to help the understanding of the mechanisms associated with germ cell formation in TS.

Asunto(s)

Técnicas de Cultivo de Célula/métodos , Células Germinativas/patología , Células Madre Pluripotentes Inducidas/patología , Síndrome de Turner/patología , Biomarcadores/metabolismo , Estudios de Casos y Controles , Diferenciación Celular/genética , Línea Celular , Reprogramación Celular/genética , Análisis Citogenético , Cuerpos Embrioides/citología , Epigénesis Genética , Vectores Genéticos/metabolismo , Células Germinativas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Plásmidos/genética

RESUMEN

BACKGROUND: Kidney organoids have been broadly obtained from commercially available induced pluripotent stem cells (iPSCs); however, it has been a great challenge to efficiently produce renal organoid models from patients with autosomal dominant polycystic kidney disease (ADPKD) that recapitulate both embryogenesis and the mechanisms of cystogenesis. METHODS: Blood erythroid progenitors (EPs) from two ADPKD patients and one healthy donor (HC) was used as a comparative control to normalize the many technical steps for reprogramming EPs and for the organoids generation. EPs were reprogrammed by an episomal vector into iPSCs, which were differentiated into renal tubular organoids and then stimulated by forskolin to induce cysts formation. RESULTS: iPSCs derived from EPs exhibited all characteristics of pluripotency and were able to differentiate into all three germ layers. 3D tubular organoids were generated from single cells after 28 days in Matrigel. HC and ADPKD organoids did not spontaneously form cysts, but upon forskolin stimulation, cysts-like structures were observed in the ADPKD organoids but not in the HC-derived organoids. CONCLUSION: The findings of this study showed that kidney organoids were successfully generated from the blood EP cells of ADPKD patients and a healthy control donor. This approach should contribute as a powerful tool for embryonic kidney development model, which is able to recapitulate the very early pathophysiological mechanisms involved in cytogenesis.

Asunto(s)

Células Precursoras Eritroides , Células Madre Pluripotentes Inducidas , Riñón , Organoides , Riñón Poliquístico Autosómico Dominante , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/patología , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Riñón/metabolismo , Riñón/patología , Organoides/metabolismo , Organoides/patología , Riñón Poliquístico Autosómico Dominante/metabolismo , Riñón Poliquístico Autosómico Dominante/patología

RESUMEN

In recent years, accumulating evidence has shown that the innate immune complement system is involved in several aspects of normal brain development and in neurodevelopmental disorders, including autism spectrum disorder (ASD). Although abnormal expression of complement components was observed in post-mortem brain samples from individuals with ASD, little is known about the expression patterns of complement molecules in distinct cell types in the developing autistic brain. In the present study, we characterized the mRNA and protein expression profiles of a wide range of complement system components, receptors and regulators in induced pluripotent stem cell (iPSC)-derived neural progenitor cells, neurons and astrocytes of individuals with ASD and neurotypical controls, which constitute in vitro cellular models that recapitulate certain features of both human brain development and ASD pathophysiology. We observed that all the analyzed cell lines constitutively express several key complement molecules. Interestingly, using different quantification strategies, we found that complement C4 mRNA and protein are expressed in significantly lower levels by astrocytes derived from ASD individuals compared to control astrocytes. As astrocytes participate in synapse elimination, and diminished C4 levels have been linked to defective synaptic pruning, our findings may contribute to an increased understanding of the atypically enhanced brain connectivity in ASD.

Asunto(s)

Astrocitos/patología , Trastorno del Espectro Autista/patología , Complemento C4/metabolismo , Células Madre Pluripotentes Inducidas/patología , Células-Madre Neurales/patología , Neuronas/patología , Astrocitos/metabolismo , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Células Cultivadas , Complemento C4/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células-Madre Neurales/metabolismo , Plasticidad Neuronal/fisiología , Neuronas/metabolismo

RESUMEN

Diabetes mellitus (DM) is one of the main causes of morbidity and mortality, with an increasing incidence worldwide. The impact of DM on public health in developing countries has triggered alarm due to the exaggerated costs of the treatment and monitoring of patients with this disease. Considerable efforts have been made to try to prevent the onset and reduce the complications of DM. However, because insulin-producing pancreatic ß-cells progressively deteriorate, many people must receive insulin through subcutaneous injection. Additionally, current therapies do not have consistent results regarding the prevention of chronic complications. Leveraging the approval of real-time continuous glucose monitors and sophisticated algorithms that partially automate insulin infusion pumps has improved glycemic control, decreasing the burden of diabetes management. However, these advances are facing physiologic barriers. New findings in molecular and cellular biology have produced an extraordinary advancement in tissue development for the treatment of DM. Obtaining pancreatic ß-cells from somatic cells is a great resource that currently exists for patients with DM. Although this therapeutic option has great prospects for patients, some challenges remain for this therapeutic plan to be used clinically. The purpose of this review is to describe the new techniques in cell biology and regenerative medicine as possible treatments for DM. In particular, this review highlights the origin of induced pluripotent cells (iPSCs) and how they have begun to emerge as a regenerative treatment that may mitigate the pathology of this disease.

Asunto(s)

Algoritmos , Diabetes Mellitus/terapia , Células Madre Pluripotentes Inducidas/trasplante , Trasplante de Células Madre , Enfermedad Crónica , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología

RESUMEN

Amyotrophic lateral sclerosis type 8 (ALS8) is an autosomal dominant form of ALS, which is caused by pathogenic variants in the VAPB gene. Here we investigated five ALS8 patients, classified as 'severe' and 'mild' from a gigantic Brazilian kindred, carrying the same VAPB mutation but displaying different clinical courses. Copy number variation and whole exome sequencing analyses in such individuals ruled out previously described genetic modifiers of pathogenicity. After deriving induced pluripotent stem cells (iPSCs) for each patient (N = 5) and controls (N = 3), motor neurons were differentiated, and high-throughput RNA-Seq gene expression measurements were performed. Functional cell death and oxidative metabolism assays were also carried out in patients' iPSC-derived motor neurons. The degree of cell death and mitochondrial oxidative metabolism were similar in iPSC-derived motor neurons from mild patients and controls and were distinct from those of severe patients. Similar findings were obtained when RNA-Seq from such cells was performed. Overall, 43 genes were upregulated and 66 downregulated in the two mild ALS8 patients when compared with severe ALS8 individuals and controls. Interestingly, significantly enriched pathways found among differentially expressed genes, such as protein translation and protein targeting to the endoplasmic reticulum (ER), are known to be associated with neurodegenerative processes. Taken together, the mitigating mechanisms here presented appear to maintain motor neuron survival by keeping translational activity and protein targeting to the ER in such cells. As ALS8 physiopathology has been associated with proteostasis mechanisms in ER-mitochondria contact sites, such differentially expressed genes appear to relate to the bypass of VAPB deficiency.

Asunto(s)

Esclerosis Amiotrófica Lateral/genética , Mitocondrias/genética , Degeneración Nerviosa/genética , Proteínas de Transporte Vesicular/genética , Anciano , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Diferenciación Celular/genética , Retículo Endoplásmico/genética , Femenino , Regulación de la Expresión Génica/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Masculino , Persona de Mediana Edad , Mitocondrias/metabolismo , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Degeneración Nerviosa/patología , Estrés Oxidativo/genética , RNA-Seq , Proteínas de Transporte Vesicular/deficiencia

RESUMEN

We describe generation of human induced pluripotent stem cell (hiPSC) lines of three unrelated idiopathic late onset Parkinson disease patients and two healthy controls above 60 years of age without neurological diseases nor Ashkenazi ancestry. Human iPSC were derived from peripheral blood-erythroblasts using integration free episomal plasmids carrying four reprogramming factors OCT4, SOX2, c-MYC, KLF4 and BCL-XL. The hiPSC lines were characterized according to established criteria.

Asunto(s)

Técnicas de Cultivo de Célula/métodos , Línea Celular/patología , Células Madre Pluripotentes Inducidas/patología , Enfermedad de Parkinson/patología , Anciano , Secuencia de Bases , Humanos , Factor 4 Similar a Kruppel , Persona de Mediana Edad , Reproducibilidad de los Resultados

RESUMEN

Induced pluripotent stem cell (iPSC) lines were generated from erythroblasts of two patients with amyotrophic lateral sclerosis (ALS) and two healthy individuals. One familial and one sporadic ALS patients were used, both with genetic alterations in VAPB gene. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (containing the reprogramming factors OCT3/4, KLF4, SOX2 and cMYC) was used to generate the iPSC cell lines. The four iPSCs express pluripotency markers, have normal karyotype and differentiated spontaneously in the three germ layers. The expression of Sendai virus was lost in all iPSC lines after 15 passages.

Asunto(s)

Esclerosis Amiotrófica Lateral/genética , Diferenciación Celular , Reprogramación Celular , Células Madre Pluripotentes Inducidas/patología , Leucocitos Mononucleares/patología , Mutación , Proteínas de Transporte Vesicular/genética , Adulto , Esclerosis Amiotrófica Lateral/patología , Células Cultivadas , Voluntarios Sanos , Heterocigoto , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Leucocitos Mononucleares/metabolismo , Masculino , Fenotipo

RESUMEN

Sickle cell disease (SCD) is one of the most prevalent and severe monogenetic disorders. Previously, we generated iPS cell lines from SCD patients. Here, we generated iPS cell lines from three age-, ethnicity- and gender-matched healthy individuals as control cell lines. Cell reprogramming was performed using erythroblasts expanded from PBMC by a non-integrative method. SCD-iPSC controls expressed pluripotency markers, presented a normal karyotype, were able to differentiate into the three germ layers in embryoid body spontaneous differentiation and confirmed to be integration-free. The cell lines generated here may be used as matched healthy controls for SCD studies.

Asunto(s)

Anemia de Células Falciformes , Técnicas de Reprogramación Celular , Eritroblastos , Células Madre Pluripotentes Inducidas/metabolismo , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/metabolismo , Anemia de Células Falciformes/patología , Técnicas de Cultivo de Célula , Línea Celular , Eritroblastos/metabolismo , Eritroblastos/patología , Humanos , Células Madre Pluripotentes Inducidas/patología

RESUMEN

Chagas disease (ChD) is one of the most neglected tropical diseases, with cardiomyopathy being the main cause of death in Trypanosoma cruzi-infected patients. As the parasite actively replicates in cardiomyocytes (CMs), the heart remains a key target organ in the pathogenesis of ChD. Here we modeled ChD using human induced pluripotent stem cell-derived CMs (iPSC-CMs) to understand the complex interplay between the parasite and host cells. We showed that iPSC-CMs can get infected with the T. cruzi Y strain and that all parasite cycle stages can be identified in our model system. Importantly, characterization of T. cruzi-infected iPSC-CMs showed significant changes in their gene expression profile, cell contractility, and distribution of key cardiac markers. Moreover, these infected iPSC-CMs exhibited a pro-inflammatory profile as indicated by significantly elevated cytokine levels and cell-trafficking regulators. We believe our iPSC-CM model is a valuable platform to explore new treatment strategies for ChD.

Asunto(s)

Cardiomiopatía Chagásica/metabolismo , Células Madre Pluripotentes Inducidas , Modelos Biológicos , Miocitos Cardíacos , Trypanosoma cruzi/metabolismo , Cardiomiopatía Chagásica/patología , Cardiomiopatía Chagásica/terapia , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/parasitología , Células Madre Pluripotentes Inducidas/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/parasitología , Miocitos Cardíacos/patología

RESUMEN

Alzheimer's disease (AD) is a progressive, degenerative disorder that mainly results in memory loss and a cognitive disorder. Although the cause of AD is still unknown, a minor percentage of AD cases are produced by genetic mutations in the presenilin-1 (PSEN1) gene. Differentiated neuronal cells derived from induced pluripotent stem cells (iPSCs) of patients can recapitulate key pathological features of AD in vitro; however, iPSCs studies focused on the p.E280 A mutation, which afflicts the largest family in the world with familial AD, have not been carried out yet. Although a link between the loss of the Y (LOY) chromosome in peripheral blood cells and risk for AD has been reported, LOY-associated phenotype has not been previously studied in PSEN1 E280 A carriers. Here, we report the reprogramming of fibroblast cells into iPSCs from a familial AD patient with the PSEN1 E280 A mutation, followed by neuronal differentiation into neural precursor cells (NPCs), and the differentiation of NPCs into differentiated neurons that lacked a Y chromosome. Although the PSEN1 E280 A iPSCs and NPCs were successfully obtained, after 8 days of differentiation, PSEN1 E280 A differentiated neurons massively died reflected by release and/ or activation of death markers, and failed to reach complete neural differentiation compared to PSEN 1 wild type cells.

Asunto(s)

Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Cromosomas Humanos Y , Células Madre Pluripotentes Inducidas/metabolismo , Fragmentos de Péptidos/metabolismo , Presenilina-1/genética , Enfermedad de Alzheimer/genética , Muerte Celular , Diferenciación Celular , Reprogramación Celular , Espacio Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Células Madre Pluripotentes Inducidas/patología , Mutación , Células-Madre Neurales/patología , Neuronas/patología

RESUMEN

BACKGROUND: Doxorubicin (Dox) is a chemotherapy drug with limited application due to cardiotoxicity that may progress to heart failure. This study aims to evaluate the role of cardiomyocytes derived from mouse embryonic stem cells (CM-mESCs) in the treatment of Dox-induced cardiomyopathy (DIC) in mice. METHODS: The mouse embryonic stem cell (mESC) line E14TG2A was characterized by karyotype analysis, gene expression using RT-PCR and immunofluorescence. Cells were transduced with luciferase 2 and submitted to cardiac differentiation. Total conditioned medium (TCM) from the CM-mESCs was collected for proteomic analysis. To establish DIC in CD1 mice, Dox (7.5 mg/kg) was administered once a week for 3 weeks, resulting in a cumulative Dox dose of 22.5 mg/kg. At the fourth week, a group of animals was injected intramyocardially with CM-mESCs (8 × 105 cells). Cells were tracked by a bioluminescence assay, and the body weight, echocardiogram, electrocardiogram and number of apoptotic cardiomyocytes were evaluated. RESULTS: mESCs exhibited a normal karyotype and expressed pluripotent markers. Proteomic analysis of TCM showed proteins related to the negative regulation of cell death. CM-mESCs presented ventricular action potential characteristics. Mice that received Dox developed heart failure and showed significant differences in body weight, ejection fraction (EF), end-systolic volume (ESV), stroke volume (SV), heart rate and QT and corrected QT (QTc) intervals when compared to the control group. After cell or placebo injection, the Dox + CM-mESC group showed significant increases in EF and SV when compared to the Dox + placebo group. Reduction in ESV and QT and QTc intervals in Dox + CM-mESC-treated mice was observed at 5 or 30 days after cell treatment. Cells were detected up to 11 days after injection. The Dox + CM-mESC group showed a significant reduction in the percentage of apoptotic cardiomyocytes in the hearts of mice when compared to the Dox + placebo group. CONCLUSIONS: CM-mESC transplantation improves cardiac function in mice with DIC.

Asunto(s)

Cardiomiopatías/terapia , Doxorrubicina/efectos adversos , Células Madre Embrionarias Humanas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/trasplante , Cardiomiopatías/inducido químicamente , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Línea Celular , Doxorrubicina/uso terapéutico , Células Madre Embrionarias Humanas/patología , Humanos , Células Madre Pluripotentes Inducidas/patología , Miocitos Cardíacos/patología

RESUMEN

Several methods have been used to study the neuropathogenesis of Down syndrome (DS), such as mouse aneuploidies, post mortem human brains, and in vitro cell culture of neural progenitor cells. More recently, induced pluripotent stem cell (iPSC) technology has offered new approaches in investigation, providing a valuable tool for studying specific cell types affected by DS, especially neurons and astrocytes. Here, we investigated the role of astrocytes in DS developmental disease and the impact of the astrocyte secretome in neuron mTOR signaling and synapse formation using iPSC derived from DS and wild-type (WT) subjects. We demonstrated for the first time that DS neurons derived from hiPSC recapitulate the hyperactivation of the Akt/mTOR axis observed in DS brains and that DS astrocytes may play a key role in this dysfunction. Our results bear out that 21 trisomy in astrocytes contributes to neuronal abnormalities in addition to cell autonomous dysfunctions caused by 21 trisomy in neurons. Further research in this direction will likely yield additional insights, thereby improving our understanding of DS and potentially facilitating the development of new therapeutic approaches.

Asunto(s)

Astrocitos/patología , Síndrome de Down/patología , Células Madre Pluripotentes Inducidas/patología , Neurogénesis , Neuronas/patología , Transducción de Señal , Sinapsis/patología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Apoptosis , Astrocitos/metabolismo , Proliferación Celular , Técnicas de Cocultivo , Humanos , Ratones , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Neuronas/metabolismo , Esferoides Celulares/patología

RESUMEN

Amyotrophic Lateral Sclerosis (ALS) is one of the most common adult-onset motor neuron disease causing a progressive, rapid and irreversible degeneration of motor neurons in the cortex, brain stem and spinal cord. No effective treatment is available and cell therapy clinical trials are currently being tested in ALS affected patients. It is well known that in ALS patients, approximately 50% of pericytes from the spinal cord barrier are lost. In the central nervous system, pericytes act in the formation and maintenance of the blood-brain barrier, a natural defense that slows the progression of symptoms in neurodegenerative diseases. Here we evaluated, for the first time, the therapeutic effect of human pericytes in vivo in SOD1 mice and in vitro in motor neurons and other neuronal cells derived from one ALS patient. Pericytes and mesenchymal stromal cells (MSCs) were derived from the same adipose tissue sample and were administered to SOD1 mice intraperitoneally. The effect of the two treatments was compared. Treatment with pericytes extended significantly animals survival in SOD1 males, but not in females that usually have a milder phenotype with higher survival rates. No significant differences were observed in the survival of mice treated with MSCs. Gene expression analysis in brain and spinal cord of end-stage animals showed that treatment with pericytes can stimulate the host antioxidant system. Additionally, pericytes induced the expression of SOD1 and CAT in motor neurons and other neuronal cells derived from one ALS patient carrying a mutation in FUS. Overall, treatment with pericytes was more effective than treatment with MSCs. Our results encourage further investigations and suggest that pericytes may be a good option for ALS treatment in the future. Graphical Abstract ᅟ.

Asunto(s)

Esclerosis Amiotrófica Lateral/terapia , Células Madre Pluripotentes Inducidas/patología , Neuronas Motoras/patología , Pericitos/trasplante , Superóxido Dismutasa-1/genética , Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/mortalidad , Esclerosis Amiotrófica Lateral/patología , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Tronco Encefálico/metabolismo , Tronco Encefálico/patología , Catalasa/genética , Catalasa/metabolismo , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Transgénicos , Neuronas Motoras/metabolismo , Mutación , Pericitos/citología , Pericitos/metabolismo , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Médula Espinal/metabolismo , Médula Espinal/patología , Superóxido Dismutasa-1/deficiencia , Análisis de Supervivencia

RESUMEN

Focal cortical dysplasia (FCD) is caused by numerous alterations, which can be divided into abnormalities of the cortical architecture and cytological variations; however, the exact etiology of FCD remains unknown. The generation of induced pluripotent stem cells (iPSCs) from the cells of patients with neurological diseases, and their subsequent tissue­specific differentiation, serves as an invaluable source for testing and studying the initial development and subsequent progression of diseases associated with the central nervous system. A total of 2 patients demonstrating seizures refractory to drug treatment, characterized as FCD Type IIb, were enrolled in the present study. Fibroblasts were isolated from residual skin fragments obtained from surgical treatment and from brain samples obtained during surgical resection. iPSCs were generated following exposure of fibroblasts to viral vectors containing POU class 5 homeobox 1 (OCT4), sex determining region Y­box 2 (SOX2), Kruppel­like factor 4 and c­MYC genes, and were characterized by immunohistochemical staining for the pluripotent markers homeobox protein NANOG, SOX2, OCT4, TRA1­60 and TRA1­81. The brain samples were tested with antibodies against protein kinase B (AKT), phosphorylated­AKT, mechanistic target of rapamycin (mTOR) and phosphorylated­mTOR. Analysis of the AKT/mTOR pathway revealed a statistically significant difference between the cerebral tissues of the two patients, which were of different ages (45 and 12 years old). Clones with the morphological features of embryonic cells were detected on the 13th day and were characterized following three subcultures. The positive staining characteristics of the embryonic cells confirmed the successful generation of iPSCs derived from the patients' fibroblasts. Therefore, the present study presents a method to obtain a useful cellular source that may help to understand embryonic brain development associated with FCD.

Asunto(s)

Epilepsia/patología , Células Madre Pluripotentes Inducidas/patología , Malformaciones del Desarrollo Cortical de Grupo I/patología , Células Cultivadas , Reprogramación Celular , Niño , Epilepsia/metabolismo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Malformaciones del Desarrollo Cortical de Grupo I/metabolismo , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo

RESUMEN

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder in which the MECP2 (methyl CpG-binding protein 2) gene is mutated. Recent studies showed that RTT-derived neurons have many cellular deficits when compared to control, such as: less synapses, lower dendritic arborization and reduced spine density. Interestingly, treatment of RTT-derived neurons with Insulin-like Growth Factor 1 (IGF1) could rescue some of these cellular phenotypes. Given the critical role of IGF1 during neurodevelopment, the present study used human induced pluripotent stem cells (iPSCs) from RTT and control individuals to investigate the gene expression profile of IGF1 and IGF1R on different developmental stages of differentiation. We found that the thyroid hormone receptor (TRalpha 3) has a differential expression profile. Thyroid hormone is critical for normal brain development. Our results showed that there is a possible link between IGF1/IGF1R and the TRalpha 3 and that over expression of IGF1R in RTT cells may be the cause of neurites improvement in neural RTT-derived neurons.

Asunto(s)

Factor I del Crecimiento Similar a la Insulina/genética , Proteína 2 de Unión a Metil-CpG/genética , Receptores de Somatomedina/genética , Síndrome de Rett/genética , Receptores alfa de Hormona Tiroidea/genética , Diferenciación Celular/genética , Cuerpos Embrioides/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Trastornos del Neurodesarrollo , Plasticidad Neuronal/genética , Neuronas/metabolismo , Neuronas/patología , Receptor IGF Tipo 1 , Síndrome de Rett/metabolismo , Síndrome de Rett/fisiopatología , Columna Vertebral/crecimiento & desarrollo , Columna Vertebral/patología , Sinapsis/genética , Sinapsis/patología , Transcriptoma/genética

RESUMEN

Protein synthesis regulation via mammalian target of rapamycin complex 1 (mTORC1) signaling pathway has key roles in neural development and function, and its dysregulation is involved in neurodevelopmental disorders associated with autism and intellectual disability. mTOR regulates assembly of the translation initiation machinery by interacting with the eukaryotic initiation factor eIF3 complex and by controlling phosphorylation of key translational regulators. Collybistin (CB), a neuron-specific Rho-GEF responsible for X-linked intellectual disability with epilepsy, also interacts with eIF3, and its binding partner gephyrin associates with mTOR. Therefore, we hypothesized that CB also binds mTOR and affects mTORC1 signaling activity in neuronal cells. Here, by using induced pluripotent stem cell-derived neural progenitor cells from a male patient with a deletion of entire CB gene and from control individuals, as well as a heterologous expression system, we describe that CB physically interacts with mTOR and inhibits mTORC1 signaling pathway and protein synthesis. These findings suggest that disinhibited mTORC1 signaling may also contribute to the pathological process in patients with loss-of-function variants in CB.

Asunto(s)

Trastorno Autístico/genética , Factor 3 de Iniciación Eucariótica/genética , Eliminación de Gen , Discapacidad Intelectual/genética , Complejos Multiproteicos/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Serina-Treonina Quinasas TOR/genética , Adolescente , Trastorno Autístico/metabolismo , Trastorno Autístico/fisiopatología , Estudios de Casos y Controles , Factor 3 de Iniciación Eucariótica/metabolismo , Regulación de la Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/fisiopatología , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/metabolismo , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Iniciación de la Cadena Peptídica Traduccional , Cultivo Primario de Células , Unión Proteica , Factores de Intercambio de Guanina Nucleótido Rho/deficiencia , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Transfección

RESUMEN

A utilização de células-tronco pluripotentes apresenta uma oportunidade promissora para o tratamento dedoenças degenerativas que, até o momento, não possuem cura. Contudo, a sua utilização encontra impassesrelacionados à ética e à biossegurança. Takahashi e Yamanaka (2006) elaboraram uma alternativa que permiteobter células-tronco pluripotentes a partir de células adultas por meio de indução com fatores de transcriçãoespecíficos (fatores Yamanaka). Tal feito é capaz de contornar os questionamentos relacionados à destruição deembriões e, também, torna possível a possibilidade de realizar transplantes autólogos, reduzindo os problemasrelacionados à biossegurança. A obtenção dessas células também é estudada na medicina veterinária, com o intuitode validar diversos modelos experimentais domésticos e de produção e, além disso, tem a intenção de ofereceralternativas para a conservação de um banco genético de espécies ameaçadas de extinção. Contudo, existem muitasdiferenças nos protocolos adotados e nos resultados obtidos entre as diferentes espécies, dificultando a eficácia dosestudos na área. Este trabalho apresenta uma revisão detalhada sobre o que foi efetivo ou não até o presentemomento na obtenção de células-tronco pluripotentes induzidas (iPSC) em canídeos, equinos, ruminantes e suínos.

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The use of pluripotent stem cells is a promising opportunity for the treatment of degenerative diseases thathave no cure. The use of those is limited by ethics and biosafety arguments. Takahashi e Yamanaka (2006)developed an alternative that allows the obtainment of pluripotent stem cells from adult cells by induction withspecific transcription factors (Yamanaka factors). Such discovery created a way to avoid the questions aboutembryo destruction also, created the possibility of autologous transplantation decreasing problems related tobiosafety. The process for obtaining (iPSC) is also studied in Veterinary Medicine field in order to validate severalexperimental in domestic and production models. Above that, it is an alternative to create a genomic bank ofendangered species. Nervethless there are many differences on protocols and results obtained hindering theefficiency and effectiveness of the studies in this area. This work presents a detailed review about what was effectiveor not on the processes of obtaining iPSCs in canine, feline, equine, bovine and swine until this moment.

Asunto(s)

Animales , Animales Domésticos/embriología , Animales Domésticos/genética , Células Madre Pluripotentes Inducidas/clasificación , Células Madre Pluripotentes Inducidas/patología

RESUMEN

A utilização de células-tronco pluripotentes apresenta uma oportunidade promissora para o tratamento dedoenças degenerativas que, até o momento, não possuem cura. Contudo, a sua utilização encontra impassesrelacionados à ética e à biossegurança. Takahashi e Yamanaka (2006) elaboraram uma alternativa que permiteobter células-tronco pluripotentes a partir de células adultas por meio de indução com fatores de transcriçãoespecíficos (fatores Yamanaka). Tal feito é capaz de contornar os questionamentos relacionados à destruição deembriões e, também, torna possível a possibilidade de realizar transplantes autólogos, reduzindo os problemasrelacionados à biossegurança. A obtenção dessas células também é estudada na medicina veterinária, com o intuitode validar diversos modelos experimentais domésticos e de produção e, além disso, tem a intenção de ofereceralternativas para a conservação de um banco genético de espécies ameaçadas de extinção. Contudo, existem muitasdiferenças nos protocolos adotados e nos resultados obtidos entre as diferentes espécies, dificultando a eficácia dosestudos na área. Este trabalho apresenta uma revisão detalhada sobre o que foi efetivo ou não até o presentemomento na obtenção de células-tronco pluripotentes induzidas (iPSC) em canídeos, equinos, ruminantes e suínos.(AU)

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The use of pluripotent stem cells is a promising opportunity for the treatment of degenerative diseases thathave no cure. The use of those is limited by ethics and biosafety arguments. Takahashi e Yamanaka (2006)developed an alternative that allows the obtainment of pluripotent stem cells from adult cells by induction withspecific transcription factors (Yamanaka factors). Such discovery created a way to avoid the questions aboutembryo destruction also, created the possibility of autologous transplantation decreasing problems related tobiosafety. The process for obtaining (iPSC) is also studied in Veterinary Medicine field in order to validate severalexperimental in domestic and production models. Above that, it is an alternative to create a genomic bank ofendangered species. Nervethless there are many differences on protocols and results obtained hindering theefficiency and effectiveness of the studies in this area. This work presents a detailed review about what was effectiveor not on the processes of obtaining iPSCs in canine, feline, equine, bovine and swine until this moment.(AU)

Asunto(s)

Animales , Animales Domésticos/embriología , Animales Domésticos/genética , Células Madre Pluripotentes Inducidas/clasificación , Células Madre Pluripotentes Inducidas/patología