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Aralia elata is an Araliaceae woody plant species found in Northeastern Asia. To understand how genetic pools are distributed for A.elata clones, we were to analyze the population structure of A.elata cultivars and identify how these are correlated with thorn-related phenotype which determines the utility of A.elata. We found that the de novo assembled genome of 'Yeongchun' shared major genomic compartments with the public A.elata genome assembled from the wild-type from China. To identify the population structure of the 32 Korean and Japanese cultivars, we identified 44 SSR markers and revealed three main sub-clusters using ΔK analysis with one isolated cultivar. Machine-learning based clustering with thorn-related phenotype correlated moderately with population structure based on SSR analysis suggested multi-layered genetic regulation of thorn-related phenotypes. Thus, we revealed genetic lineage of A.elata and uncovered isolated cultivar which can provide new genetic material for further breeding.
Assuntos
Aralia , Genoma de Planta , Repetições de Microssatélites , Fenótipo , Aralia/genética , Melhoramento Vegetal , Aprendizado de MáquinaRESUMO
BACKGROUND: Because human fetal ventral mesencephalic tissue grafts provide promising results in ameliorating Parkinson's disease-implicated motor dysfunctions, human fetal midbrain-derived dopamine neuronal precursor cells are considered good candidates for cell-based therapy for Parkinson's disease in that large quantities of cells can be supplied through a good manufacturing practice-compliant system. OBJECTIVE: We conducted a prospective, phase I/IIa, dose-escalation, open-label "first-in-human" clinical trial with fetal neural precursor cells to assess their safety and therapeutic efficacy in patients with idiopathic Parkinson's disease. METHODS: Fifteen patients were assigned to receive three different doses of cells (4 × 106 , 12 × 106 , and 40 × 106 cells) and completed a 12-month follow-up. The primary outcome was safety, by measuring the presence of grade 3 or higher cells according to National Cancer Institute guidelines and any contaminated cells. Secondary outcomes assessed motor and neurocognitive function, as well as the level of dopamine transporters, by positron emission tomography-computed tomography. RESULTS: Although a pronation-supination and hand/arm movement performance was remarkably enhanced in all three groups (all P < 0.05), the medium- and high-dose-treated groups exhibited significant improvement in Unified Parkinson's Disease Rating Scale Part III only up to 26.16% and 40%, respectively, at 12 months after transplantation without any serious clinical complications or graft-induced dyskinesia in all patients. However, the motor improvements did not correlate with increase in the dopamine transporter on positron emission tomography images. CONCLUSIONS: Our results primarily demonstrate the safety and plausible dose-dependent efficacy of human fetal midbrain-derived dopamine neuronal precursor cells for idiopathic Parkinson's disease. © 2023 International Parkinson and Movement Disorder Society.
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Células-Tronco Neurais , Doença de Parkinson , Humanos , Doença de Parkinson/terapia , Doença de Parkinson/tratamento farmacológico , Dopamina , Estudos Prospectivos , Tomografia Computadorizada por Raios X , Mesencéfalo/diagnóstico por imagemRESUMO
Our previous paper showed that microRNAs (miRNAs) present within human placental or mesenchymal stem cell-derived extracellular vesicles (EVs) directly interacted with the RNA genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), inhibiting viral replication. In this paper, we analyzed whether these miRNAs could exert antiviral activity against other variants of SARS-CoV-2. We downloaded compete SARS-CoV-2 genome data submitted to the National Center for Biotechnology Information for each SARS-CoV-2 variant, aligned the data to the reference SARS-CoV-2 genome sequence, and then confirmed the presence of 3' untranslated region (UTR) mutations. We identified one type of 3' UTR mutation in the Alpha variant, four in the Beta variant, four in the Gamma variant, three in the Delta variant, and none in the Omicron variant. Our findings indicate that 3' UTR mutations rarely occur as persistent mutations. Interestingly, we further confirmed that this phenomenon could suppress virus replication in the same manner as the previously discovered interaction of placental-EV-derived miRNA with 3' UTRs of SARS-CoV-2. Because the 3' UTR of the SARS-CoV-2 RNA genome has almost no mutations, it is expected to be an effective therapeutic target regardless of future variants. Thus, a therapeutic strategy targeting the 3' UTR of SARS-CoV-2 is likely to be extremely valuable, and such an approach is also expected to be applied to all RNA-based virus therapeutics.
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Brain-derived extracellular vesicles (BDEVs) are released from the central nervous system. Brain-related research and diagnostic techniques involving BDEVs have rapidly emerged as a means of diagnosing brain disorders because they are minimally invasive and enable repeatable measurements based on body fluids. However, EVs from various cells and organs are mixed in the blood, acting as potential obstacles for brain diagnostic systems using BDEVs. Therefore, it is important to screen appropriate brain EV markers to isolate BDEVs in blood. Here, we established a strategy for screening potential BDEV biomarkers. To collect various molecular data from the BDEVs, we propose that the sensitivity and specificity of the diagnostic system could be enhanced using machine learning and AI analysis. This BDEV-based diagnostic strategy could be used to diagnose various brain diseases and will help prevent disease through early diagnosis and early treatment.
Assuntos
Inteligência Artificial , Encefalopatias , Humanos , Biomarcadores , Encéfalo , Encefalopatias/diagnóstico , Diagnóstico PrecoceRESUMO
Extracellular vesicles (EVs) are cell-released, nanometer-scaled, membrane-bound materials and contain diverse contents including proteins, small peptides, and nucleic acids. Once released, EVs can alter the microenvironment and regulate a myriad of cellular physiology components, including cell-cell communication, proliferation, differentiation, and immune responses against viral infection. Among the cargoes in the vesicles, small non-coding micro-RNAs (miRNAs) have received attention in that they can regulate the expression of a variety of human genes as well as external viral genes via binding to the complementary mRNAs. In this study, we tested the potential of EVs as therapeutic agents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. First, we found that the mesenchymal stem-cell-derived EVs (MSC-EVs) enabled the rescue of the cytopathic effect of SARS-CoV-2 virus and the suppression of proinflammatory responses in the infected cells by inhibiting the viral replication. We found that these anti-viral responses were mediated by 17 miRNAs matching the rarely mutated, conserved 3'-untranslated regions (UTR) of the viral genome. The top five miRNAs highly expressed in the MSC-EVs, miR-92a-3p, miR-26a-5p, miR-23a-3p, miR-103a-3p, and miR-181a-5p, were tested. They were bound to the complemented sequence which led to the recovery of the cytopathic effects. These findings suggest that the MSC-EVs are a potential candidate for multiple variants of anti-SARS-CoV-2.
Assuntos
COVID-19/terapia , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/uso terapêutico , SARS-CoV-2/fisiologia , Regiões 3' não Traduzidas/genética , Animais , Antivirais/farmacologia , Sequência de Bases , Linhagem Celular , Sequência Conservada/genética , Feminino , Genoma Viral , Humanos , Modelos Biológicos , Mutação/genética , Placenta/metabolismo , Gravidez , RNA Viral/genética , SARS-CoV-2/genéticaRESUMO
Senescence in stem cells, which occurs as a consequence of chronic responses to the environment, defines the capacity of stem cells for proliferation and differentiation as well as their potential for tissue regeneration and homeostasis maintenance. Although stem cells reside under low oxygen pressure and the availability of oxygen is known to be a crucial determinant in their fate, the key modulators in stem cell aging and the underlying mechanism have yet to be unraveled. Human placenta-derived mesenchymal stem cells (hpMSCs) were cultured under hypoxia (3% O2 ) or normoxia (21% O2 ) to investigate the key factors that regulate stem cell senescence under hypoxic conditions. RNA sequencing results suggested that the expression of aminoacyl-tRNA synthetase-interacting multifunctional protein 3 (AIMP3, EEF1E1), an aging inducer, in the hpMSCs was dramatically repressed under hypoxia with concurrent suppression of the aging marker p16INK4a . The hpMSCs that overexpressed AIMP3 under hypoxic conditions displayed significantly decreased proliferation and fewer stem cell characteristics, whereas the downregulation of AIMP3 ameliorated the age-related senescence of MSCs. Consistent with the results of the hpMSCs, MSCs isolated from the adipose tissue of AIMP3-overexpressing mice exhibited decreased stem cell functions. Interestingly, AIMP3-induced senescence is negatively regulated by hypoxia-inducible factor 1α (HIF1α) and positively regulated by Notch3. Furthermore, we showed that AIMP3 enhanced mitochondrial respiration and suppressed autophagic activity, indicating that the AIMP3-associated modulation of metabolism and autophagy is a key mechanism in the senescence of stem cells and further suggesting a novel target for interventions against aging.
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Autofagia , Senescência Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células-Tronco Mesenquimais/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Camundongos , Camundongos TransgênicosRESUMO
Background: Treating aged animals with plasma of an early developmental stage (e.g, umbilical cord plasma) showed an impressive potential to slow age-associated degradation of neuronal and cognitive functions. Translating such findings to clinical realities, however, requires effective ways for assessing treatment efficacy; ideal methods should be minimally invasive, amenable for serial assays, cost-effective, and quantitative. Methods: We developed a new biosensor approach to monitor anti-aging therapy. We advanced two key sensor components: i) a blood-borne metabolite was identified as a surrogate aging-marker; and ii) a compact and cost-effective assay system was developed for on-site applications. We treated aged mice either with human umbilical cord plasma or saline; unbiased metabolite profiling on mouse plasma revealed arachidonic acid (AA) as a potent indicator associated with anti-aging effect. We next implemented a competitive magneto-electrochemical sensor (cMES) optimized for AA detection directly from plasma. The developed platform could detect AA directly from small volumes of plasma (0.5 µL) within 1.5 hour. Results: cMES assays confirmed a strong correlation between AA levels and anti-aging effect: AA levels, while decreasing with aging, increased in the plasma-treated aged mice which also showed improved learning and memory performance. Conclusions: The cMES platform will empower both pre- and clinical anti-aging research by enabling minimally invasive, longitudinal treatment surveillance; these capacities will accelerate the development of anti-aging therapies, improving the quality of individual lives.
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Envelhecimento , Ácido Araquidônico/sangue , Técnicas Biossensoriais/métodos , Transfusão de Sangue , Monitoramento de Medicamentos/métodos , Sangue Fetal , Metabolômica/métodos , Animais , Técnicas Eletroquímicas/métodos , Estudos Longitudinais , Magnetismo/métodos , Camundongos , Modelos Animais , Plasma/química , Resultado do TratamentoRESUMO
Humidifier disinfectants containing polyhexamethylene guanidine phosphate (PHMG-P) can induce pulmonary toxicity and has caused human casualties in South Korea since 2006. Thereby, the safety evaluation of household chemicals such as PHMG-P has garnered increased importance. However, many limitations, such as the lack of specialized facilities and animal welfare concerns associated with the use of murine models, persist. Zebrafish gills have high functional and structural similarity to mammalian lungs. Moreover, zebrafish are sensitive to toxic substances, resulting in changes in behavioral or ventilatory patterns. Based on these facts, in this study, we aimed to evaluate the pulmonary toxicity of PHMG-P in zebrafish. Zebrafish exposed to PHMG-P showed an increase in mRNA levels of inflammatory factors persisting for 28 days along with histopathologic changes in the gills. An exposure time-dependent alteration in infiltration of inflammatory cells and destruction of gill lamellae was observed. In addition, an increase in mRNA levels of fibrosis factors was observed in gills exposed to PHMG-P for 28 days, as assessed by collagen staining with Masson's trichrome. These results supported the cellular level results. Taken together, our results reveal pulmonary toxic effects of PHMG-P and suggest useful markers for evaluating pulmonary toxicity.
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Guanidinas/toxicidade , Mediadores da Inflamação/metabolismo , Sistema Respiratório/patologia , Peixe-Zebra , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Brânquias/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Sistema Respiratório/efeitos dos fármacosRESUMO
Parkinson's disease (PD) is the second most common age-related neurodegenerative disease in the elderly and the patients suffer from uncontrolled movement disorders due to loss of dopaminergic (DA) neurons on substantia nigra pars compacta (SNpc). We previously reported that transplantation of human fetal midbrain-derived neural precursor cells restored the functional deficits of a 6-hydroxy dopamine (6-OHDA)-treated rodent model of PD but its low viability and ethical issues still remain to be solved. Albeit immune privilege and neural differentiation potentials suggest mesenchymal stem cells (MSCs) from various tissues including human placenta MSCs (hpMSCs) for an alternative source, our understanding of their therapeutic mechanisms is still limited. To expand our knowledge on the MSC-mediated PD treatment, we here investigated the therapeutic mechanism of hpMSCs and hpMSC-derived neural phenotype cells (hpNPCs) using a PD rat model. Whereas both hpMSCs and hpNPCs protected DA neurons in the SNpc at comparable levels, the hpNPC transplantation into 6-OHDA treated rats exhibited longer lasting recovery in motor deficits than either the saline or the hpMSC treated rats. The injected hpNPCs induced delta-like ligand (DLL)1 and neurotrophic factors, and influenced environments prone to neuroprotection. Compared with hpMSCs, co-cultured hpNPCs more efficiently protected primary neural precursor cells from midbrain against 6-OHDA as well as induced their differentiation into DA neurons. Further experiments with conditioned media from hpNPCs revealed that the secreted factors from hpNPCs modulated immune responses and neural protection. Taken together, both DLL1-mediated contact signals and paracrine factors play critical roles in hpNPC-mediated improvement. First showing here that hpMSCs and their neural derivative hpNPCs were able to restore the PD-associated deficits via dual mechanisms, neuroprotection and immunosuppression, this study expanded our knowledge of therapeutic mechanisms in PD and other age-related diseases.
Assuntos
Encéfalo/patologia , Inflamação/patologia , Células-Tronco Neurais/citologia , Neuroproteção , Doença de Parkinson/patologia , Placenta/citologia , Animais , Morte Celular , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Microambiente Celular , Corpo Estriado/patologia , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Feminino , Humanos , Imunomodulação , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos , Microglia/metabolismo , Atividade Motora , Células-Tronco Neurais/transplante , Neurturina/metabolismo , Oxidopamina , Doença de Parkinson/fisiopatologia , Gravidez , Ratos Sprague-DawleyRESUMO
Aging is an inevitable progressive decline in every physiological function and serves as a primary risk factor for cognitive decline and Alzheimer's disease. Thus, age-dependent impairments in cognitive function must be understood in association with general aging processes with an integrative approach in a systemic manner. An integrative aging gene network was constructed based on mutual molecular interactions using literature-curated interactome data and separated into functionally distinct modules. To investigate key surrogate biomarkers of the aging brain in the context of the general aging process, co-expression networks were built on post-mortem and Alzheimer's brain transcriptome data. In both the normal aging brain and the brain affected by Alzheimer's disease, the immune-related co-expression module was positively correlated with advancing age, whereas the synaptic transmission-related co-expression module was decreased with age. Importantly, the network topology-based analysis indicated that complement system genes were prioritized as a surrogate biomarker in evaluating the process of brain aging. Our public data-centered analysis coupled with experimental validation revealed that the complement system is likely to be a master regulator in initiating and regulating the immune system in the aging brain and could serve as reliable and surrogate biomarkers for the diagnosis of cognitive dysfunction.
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Envelhecimento/genética , Biomarcadores , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Conectoma , Redes Reguladoras de Genes , Transcriptoma , Animais , Biologia Computacional/métodos , Bases de Dados Genéticas , Feminino , Perfilação da Expressão Gênica , Humanos , Redes e Vias Metabólicas , Camundongos , Anotação de Sequência Molecular , Reprodutibilidade dos TestesRESUMO
PURPOSE: Loss of cholinergic neurons in the hippocampus is a hallmark of many dementias. Administration of stem cells as a therapeutic intervention for patients is under active investigation, but the optimal stem cell type and transplantation modality has not yet been established. In this study, we studied the therapeutic effects of human placenta-derived mesenchymal stem cells (pMSCs) in dementia rat model using either intracerebroventricular (ICV) or intravenous (IV) injections and analyzed their mechanisms of therapeutic action. MATERIALS AND METHODS: Dementia modeling was established by intraventricular injection of 192 IgG-saporin, which causes lesion of cholinergic neurons. Sixty-five male Sprague-Dawley rats were divided into five groups: control, lesion, lesion+ICV injection of pMSCs, lesion+IV injection of pMSCs, and lesion+donepezil. Rats were subjected to the Morris water maze and subsequent immunostaining analyses. RESULTS: Both ICV and IV pMSC administrations allowed significant cognitive recovery compared to the lesioned rats. Acetylcholinesterase activity was significantly rescued in the hippocampus of rats injected with pMSCs post-lesion. Choline acetyltransferase did not co-localize with pMSCs, showing that pMSCs did not directly differentiate into cholinergic cells. Number of microglial cells increased in lesioned rats and significantly decreased back to normal levels with pMSC injection. CONCLUSION: Our results suggest that ICV and IV injections of pMSCs facilitate the recovery of cholinergic neuronal populations and cognitive behavior. This recovery likely occurs through paracrine effects that resemble microglia function rather than direct differentiation of injected pMSCs into cholinergic neurons.
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Demência/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Neuroglia/metabolismo , Placenta/metabolismo , Animais , Anticorpos Monoclonais , Diferenciação Celular , Modelos Animais de Doenças , Feminino , Hipocampo , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/patologia , Microglia , Placenta/citologia , Placenta/patologia , Gravidez , Ratos , Ratos Sprague-Dawley , Proteínas Inativadoras de Ribossomos Tipo 1 , SaporinasRESUMO
Human placenta amniotic membrane-derived mesenchymal stem cells (AMSCs) regulate immune responses, and this property can be exploited to treat stroke patients via cell therapy. We investigated the expression profile of AMSCs cultured under hypoxic conditions and observed interesting expression changes in various genes involved in immune regulation. CD200, an anti-inflammatory factor and positive regulator of TGF-ß, was more highly expressed under hypoxic conditions than normoxic conditions. Furthermore, AMSCs exhibited inhibition of pro-inflammatory cytokine expression in co-cultures with LPS-primed BV2 microglia, and this effect was decreased in CD200-silenced AMSCs. The AMSCs transplanted into the ischemic rat model of stroke dramatically inhibited the expression of pro-inflammatory cytokines and up-regulated CD200, as compared with the levels in the sham-treated group. Moreover, decreased microglia activation in the boundary region and improvements in behavior were confirmed in AMSC-treated ischemic rats. The results suggested that the highly expressed CD200 from the AMSCs in a hypoxic environment modulates levels of inflammatory cytokines and microglial activation, thus increasing the therapeutic recovery potential after hypoxic-ischemic brain injury, and further demonstrated the immunomodulatory function of AMSCs in a stroke model.
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Antígenos CD/metabolismo , Placenta/citologia , Transplante de Células-Tronco/métodos , Células-Tronco/metabolismo , Acidente Vascular Cerebral/terapia , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Encéfalo/patologia , Hipóxia Celular , Células Cultivadas , Citocinas/metabolismo , Feminino , Humanos , Imunomodulação/fisiologia , Masculino , Camundongos , Microglia/citologia , Microglia/metabolismo , Gravidez , Ratos Sprague-Dawley , Acidente Vascular Cerebral/fisiopatologiaRESUMO
Traumatic brain injury (TBI), a complicated form of brain damage, is a major cause of mortality in adults. Following mechanical and structural primary insults, a battery of secondary insults, including neurotransmitter-mediated cytotoxicity, dysregulation of calcium and macromolecule homeostasis, and increased oxidative stress, exacerbate brain injury and functional deficits. Although stem cell therapy is considered to be an alternative treatment for brain injuries, such as TBI and stroke, many obstacles remain. In particular, the time window for TBI treatment with either drugs or stem cells and their efficacy is still vague. Human placenta-derived mesenchymal stem cells (hpMSCs) have received extensive attention in stem cell therapy because they can be acquired in large numbers without ethical issues and because of their immune-modulating capacity and effectiveness in several diseases, such as Alzheimer's disease and stroke. Here, we tested the feasibility of hpMSCs for TBI treatment with an animal model and attempted to identify appropriate time points for cell treatments. Double injections at 4 and 24 h post-injury significantly reduced the infarct size and suppressed astrocyte and microglial activation around the injury. With reduced damage, double-injected mice showed enhanced anti-inflammatory- and TNF-α receptor 2 (TNFR2)-associated survival signals and suppressed pro-inflammatory and oxidative responses. In addition, double-treated TBI mice displayed restored sensory motor functions and reduced neurotoxic Aß42 plaque formation around the damaged areas. In this study, we showed the extended therapeutic potentials of hpMSCs and concluded that treatment within an appropriate time window is critical for TBI recovery.
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Lesões Encefálicas Traumáticas/reabilitação , Sobrevivência Celular/fisiologia , Inflamação/reabilitação , Transplante de Células-Tronco Mesenquimais/métodos , Animais , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Resultado do TratamentoRESUMO
Alpha-synuclein (α-SYN) is expressed during neuronal development and is mainly involved in the modulation of synaptic transmission. Missense mutations and amplifications of this gene have been associated with the pathogenesis of Parkinson's disease. Here, we evaluate whether α-SYN plays a detrimental role in the phenotypic and morphological regulation of neurons. We also identify the underlying mechanisms of this process in all-trans-retinoic acid (RA)-induced differentiated SH-SY5Y cells, which represents dopaminergic (DAergic) phenotype. Our results indicate that overexpression of wild-type or mutant A53T α-SYN attenuated the RA-induced upregulation of tyrosine hydroxylase and dopamine transporter as well as neurite outgrowth in SH-SY5Y cells. In addition, GSK-3ß inactivation and downstream ß-catenin stabilization were associated with RA-induced differentiation, which was attenuated by α-SYN. Moreover, protein phosphatase 2A was positively regulated by α-SYN and was implicated in the α-SYN-mediated interference with RA signaling. The results obtained from SH-SY5Y cells were verified in primary cultures of mesencephalic DAergic neurons from A53T α-SYN transgenic mice, which represent high levels of α-SYN and protein phosphatase 2A in the midbrain. The number and length of neurites in tyrosine hydroxylase-positive as well as Tau-positive cells from A53T α-SYN transgenic mice were significantly lower than those in littermate controls. The current results provide novel insight into the role of α-SYN in the regulation of neuronal differentiation, including DAergic neurons. Identifying the signaling pathway involved in the α-SYN-mediated dysregulation of neuronal differentiation could lead to a better understanding of the developmental processes underlying α-SYN-related pathologies and facilitate the discovery of specifically targeted therapeutics.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Neurônios/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Tretinoína/farmacologia , alfa-Sinucleína/metabolismo , beta Catenina/metabolismo , Linhagem Celular Tumoral , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Humanos , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
We have developed a good manufacturing practice for long-term cultivation of fetal human midbrain-derived neural progenitor cells. The generation of human dopaminergic neurons may serve as a tool of either restorative cell therapies or cellular models, particularly as a reference for phenotyping region-specific human neural stem cell lines such as human embryonic stem cells and human inducible pluripotent stem cells. We cultivated 3 different midbrain neural progenitor lines at 10, 12, and 14 weeks of gestation for more than a year and characterized them in great detail, as well as in comparison with Lund mesencephalic cells. The whole cultivation process of tissue preparation, cultivation, and cryopreservation was developed using strict serum-free conditions and standardized operating protocols under clean-room conditions. Long-term-cultivated midbrain-derived neural progenitor cells retained stemness, midbrain fate specificity, and floorplate markers. The potential to differentiate into authentic A9-specific dopaminergic neurons was markedly elevated after prolonged expansion, resulting in large quantities of functional dopaminergic neurons without genetic modification. In restorative cell therapeutic approaches, midbrain-derived neural progenitor cells reversed impaired motor function in rodents, survived well, and did not exhibit tumor formation in immunodeficient nude mice in the short or long term (8 and 30 weeks, respectively). We conclude that midbrain-derived neural progenitor cells are a promising source for human dopaminergic neurons and suitable for long-term expansion under good manufacturing practice, thus opening the avenue for restorative clinical applications or robust cellular models such as high-content or high-throughput screening. Stem Cells Translational Medicine 2017;6:576-588.
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Proliferação de Células , Neurônios Dopaminérgicos/fisiologia , Mesencéfalo/embriologia , Células-Tronco Neurais/fisiologia , Neurogênese , Transtornos Parkinsonianos/cirurgia , Transplante de Células-Tronco/métodos , Animais , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Feminino , Idade Gestacional , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Atividade Motora , Células-Tronco Neurais/metabolismo , Oxidopamina , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/patologia , Transtornos Parkinsonianos/fisiopatologia , Fenótipo , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Medição de Risco , Transplante de Células-Tronco/efeitos adversos , Teratoma/etiologia , Teratoma/patologia , Fatores de TempoRESUMO
Abnormal angiogenesis is a primary cause of many eye diseases, including diabetic retinopathy, age-related macular degeneration, and retinopathy of prematurity. Mesenchymal stem cells (MSCs) are currently being investigated as a treatment for several such retinal diseases based on their neuroprotective and angiogenic potentials. In this study, we evaluated the role of systemically injected human placental amniotic membrane-derived MSCs (AMSCs) on pathological neovascularization of proliferative retinopathy. We determined that AMSCs secrete higher levels of transforming growth factor-ß (TGF-ß1) than other MSCs, and the secreted TGF-ß1 directly suppresses the proliferation of endothelial cells under pathological conditions in vitro. Moreover, in a mouse model of oxygen-induced retinopathy, intraperitoneally injected AMSCs migrated into the retina and suppressed excessive neovascularization of the vasculature via expression of TGF-ß1, and the antineovascular effect of AMSCs was blocked by treatment with TGF-ß1 siRNA. These findings are the first to demonstrate that TGF-ß1 secreted from AMSCs is one of the key factors to suppress retinal neovascularization in proliferative retinopathy and further elucidate the therapeutic function of AMSCs for the treatment of retinal neovascular diseases.
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Comunicação Parácrina , Placenta/citologia , Neovascularização Retiniana/terapia , Transplante de Células-Tronco , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Âmnio/citologia , Animais , Movimento Celular , Proliferação de Células , Retinopatia Diabética/patologia , Feminino , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Injeções Intraperitoneais , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos C57BL , Gravidez , Neovascularização Retiniana/patologiaRESUMO
Parkinson's disease (PD), primarily caused by selective degeneration of midbrain dopamine (mDA) neurons, is the most prevalent movement disorder, affecting 1-2% of the global population over the age of 65. Currently available pharmacological treatments are largely symptomatic and lose their efficacy over time with accompanying severe side effects such as dyskinesia. Thus, there is an unmet clinical need to develop mechanism-based and/or disease-modifying treatments. Based on the unique dual role of the nuclear orphan receptor Nurr1 for development and maintenance of mDA neurons and their protection from inflammation-induced death, we hypothesize that Nurr1 can be a molecular target for neuroprotective therapeutic development for PD. Here we show successful identification of Nurr1 agonists sharing an identical chemical scaffold, 4-amino-7-chloroquinoline, suggesting a critical structure-activity relationship. In particular, we found that two antimalarial drugs, amodiaquine and chloroquine stimulate the transcriptional function of Nurr1 through physical interaction with its ligand binding domain (LBD). Remarkably, these compounds were able to enhance the contrasting dual functions of Nurr1 by further increasing transcriptional activation of mDA-specific genes and further enhancing transrepression of neurotoxic proinflammatory gene expression in microglia. Importantly, these compounds significantly improved behavioral deficits in 6-hydroxydopamine lesioned rat model of PD without any detectable signs of dyskinesia-like behavior. These findings offer proof of principle that small molecules targeting the Nurr1 LBD can be used as a mechanism-based and neuroprotective strategy for PD.
Assuntos
Comportamento Animal/efeitos dos fármacos , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/agonistas , Doença de Parkinson/psicologia , Amodiaquina/metabolismo , Amodiaquina/farmacologia , Animais , Cloroquina/metabolismo , Cloroquina/farmacologia , Modelos Animais de Doenças , Ligantes , Neurogênese , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Oxidopamina/toxicidade , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/patologia , RatosRESUMO
Genes can be divided into TATA-containing genes and TATA-less genes according to the presence of TATA box elements at promoter regions. TATA-containing genes tend to be stress-responsive, whereas many TATA-less genes are known to be related to cell growth or "housekeeping" functions. In a previous study, we demonstrated that there are striking differences among four gene sets defined by the presence of TATA box (TATA-containing) and essentiality (TATA-less) with respect to number of associated transcription factors, amino acid usage, and functional annotation. Extending this research in yeast, we identified KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways that are statistically enriched in TATA-containing or TATA-less genes and evaluated the possibility that the enriched pathways are related to stress or growth as reflected by the individual functions of the genes involved. According to their enrichment for either of these two gene sets, we sorted KEGG pathways into TATA-containing-gene-enriched pathways (TEPs) and essential-gene-enriched pathways (EEPs). As expected, genes in TEPs and EEPs exhibited opposite results in terms of functional category, transcriptional regulation, codon adaptation index, and network properties, suggesting the possibility that the bipolar patterns in these pathways also contribute to the regulation of the stress response and to cell survival. Our findings provide the novel insight that significant enrichment of TATA-binding or TATA-less genes defines pathways as stress-responsive or growth-related.
Assuntos
Genes Essenciais/genética , TATA Box/genética , Redes Reguladoras de Genes , Genes Fúngicos , Redes e Vias Metabólicas/genética , Saccharomyces cerevisiae/genética , Transcrição GênicaRESUMO
Deciphering the molecular basis of neuronal cell death is a central issue in the etiology of neurodegenerative diseases, such as Parkinson's and Alzheimer's. Dysregulation of p53 levels has been implicated in neuronal apoptosis. The role of histone deacetylase 3 (HDAC3) in suppressing p53-dependent apoptosis has been recently emphasized; however, the molecular basis of modulation of p53 function by HDAC3 remains unclear. Here we show that PTEN-induced putative kinase 1 (PINK1), which is linked to autosomal recessive early-onset familial Parkinson's disease, phosphorylates HDAC3 at Ser-424 to enhance its HDAC activity in a neural cell-specific manner. PINK1 prevents H2O2-induced C-terminal cleavage of HDAC3 via phosphorylation of HDAC3 at Ser-424, which is reversed by protein phosphatase 4c. PINK1-mediated phosphorylation of HDAC3 enhances its direct association with p53 and causes subsequent hypoacetylation of p53. Genetic deletion of PINK1 partly impaired the suppressive role of HDAC3 in regulating p53 acetylation and transcriptional activity. However, depletion of HDAC3 fully abolished the PINK1-mediated p53 inhibitory loop. Finally, ectopic expression of phosphomometic-HDAC3(S424E) substantially overcomes the defective action of PINK1 against oxidative stress in dopaminergic neuronal cells. Together, our results uncovered a mechanism by which PINK1-HDAC3 network mediates p53 inhibitory loop in response to oxidative stress-induced damage.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Histona Desacetilases/metabolismo , Proteínas Quinases/metabolismo , Acetilação/efeitos dos fármacos , Animais , Caspase 7/metabolismo , Morte Celular/genética , Linhagem Celular , Citoplasma/metabolismo , Neurônios Dopaminérgicos/patologia , Ativação Enzimática , Histona Desacetilases/genética , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , Especificidade de Órgãos , Fosforilação , Proteínas Quinases/genética , Proteólise , Proteína Supressora de Tumor p53/metabolismoRESUMO
Aristolochic acids are natural products found in Chinese herbs of the Aristolochiaceae family. Aristolochic acid I (AAI) is a potent carcinogen and was found to be toxic in animal and clinical studies. Apoptosis is a rapid, selective process of physiological cell deletion that regulates the balance between cell proliferation and cell death and is induced by various kinds of damage. However, the toxicity of AAI during ovarian maturation in the mouse is unclear and is the subject of the present investigation. We used Chinese hamster ovary-K1 (CHO-K1) cells and an AAI injection mouse model: MTT assay was used to assess AA toxicity to cells; ovary size and weight were measured to determine the toxicity of AA to mouse ovary; western blot was used to assess apoptosis; TUNEL assay was used to evaluate apoptotic cell death; and immunohistochemistry was used to examine the local expression of apoptotic proteins in ovary tissue. We found that AAI significantly inhibits the viability of CHO-K1 cells and strongly induces apoptotic cell death in CHO-K1 cells and in mouse ovary. In addition, we observed that AAI markedly increases the expression of pro-apoptotic proteins, including Bax, caspase-3, caspase-9, and poly(ADP) ribose polymerase (PARP). In contrast, anti-apoptotic proteins, such as Bcl-2 and survivin, were decreased by AAI treatment. Furthermore, we observed that ovary size and weight were significantly reduced and that the number of ovulated oocytes was markedly suppressed in AAI-treated mice. These results suggest that AAI strongly induces toxic damage during ovarian maturation by inhibiting Akt phosphorylation-mediated suppression of apoptosis.