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1.
Nature ; 574(7779): 553-558, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645721

RESUMO

Age-associated chronic inflammation (inflammageing) is a central hallmark of ageing1, but its influence on specific cells remains largely unknown. Fibroblasts are present in most tissues and contribute to wound healing2,3. They are also the most widely used cell type for reprogramming to induced pluripotent stem (iPS) cells, a process that has implications for regenerative medicine and rejuvenation strategies4. Here we show that fibroblast cultures from old mice secrete inflammatory cytokines and exhibit increased variability in the efficiency of iPS cell reprogramming between mice. Variability between individuals is emerging as a feature of old age5-8, but the underlying mechanisms remain unknown. To identify drivers of this variability, we performed multi-omics profiling of fibroblast cultures from young and old mice that have different reprogramming efficiencies. This approach revealed that fibroblast cultures from old mice contain 'activated fibroblasts' that secrete inflammatory cytokines, and that the proportion of activated fibroblasts in a culture correlates with the reprogramming efficiency of that culture. Experiments in which conditioned medium was swapped between cultures showed that extrinsic factors secreted by activated fibroblasts underlie part of the variability between mice in reprogramming efficiency, and we have identified inflammatory cytokines, including TNF, as key contributors. Notably, old mice also exhibited variability in wound healing rate in vivo. Single-cell RNA-sequencing analysis identified distinct subpopulations of fibroblasts with different cytokine expression and signalling in the wounds of old mice with slow versus fast healing rates. Hence, a shift in fibroblast composition, and the ratio of inflammatory cytokines that they secrete, may drive the variability between mice in reprogramming in vitro and influence wound healing rate in vivo. This variability may reflect distinct stochastic ageing trajectories between individuals, and could help in developing personalized strategies to improve iPS cell generation and wound healing in elderly individuals.


Assuntos
Envelhecimento/metabolismo , Reprogramação Celular , Senescência Celular/fisiologia , Fibroblastos/metabolismo , Cicatrização , Animais , Linhagem Celular , Reprogramação Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mediadores da Inflamação/metabolismo , Judeus/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência de RNA , Transdução de Sinais/efeitos dos fármacos , Análise de Célula Única , Processos Estocásticos , Fatores de Tempo , Cicatrização/efeitos dos fármacos
2.
J Pharmacol Sci ; 140(4): 331-336, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31501056

RESUMO

Various chemicals, including pharmaceuticals, can induce acute or delayed neurotoxicity in humans. Because isolation of human primary neurons is extremely difficult, toxicity tests for these agents have been performed using in vivo or in vitro models. Human induced pluripotent stem cells (hiPSCs) can be used to establish hiPSC-derived neural stem/progenitor cells (hiPSC-NSPCs), which can then be used to obtain hiPSC-neurons. In this study, we differentiated hiPSC-NSPCs into neurons and evaluated the susceptibility of hiPSC-neurons and parental hiPSC-NSPCs to anticancer drugs in vitro by ATP assay and immunocytostaining. The hiPSC-neurons were more resistant to anticancer drugs than the parental hiPSC-NSPCs. In the toxicity tests, high-dose cisplatin reduced the levels of ELAVL3/4, a neuronal marker, in the hiPSC-neurons. These results suggest that our methodology is potentially applicable for efficient determination of the toxicity of any drug to hiPSC-neurons.


Assuntos
Antineoplásicos/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Células-Tronco Neurais
3.
J Pharmacol Sci ; 140(4): 345-349, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31521491

RESUMO

Although the cardiotoxicity of anti-cancer drugs is an important issue, the underlying mechanisms remain unknown. To develop a sensitive assay system for cardiotoxicity, we examined effects of anti-cancer drugs on contractile functions of human iPS cell-derived cardiomyocytes by using non-invasive motion field imaging analysis with extended drug exposure time. We succeeded in continuously measuring stable contractile function. The continued exposure revealed that the difference in cardiotoxicity between cardiotoxic doxorubicin and less toxic erlotinib was more evident after 8 days of treatment than with 3 days of treatment, suggesting that continued exposure improved the predictive power for cardiotoxicity of anti-cancer drugs.


Assuntos
Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Cardiotoxicidade/etiologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Células Cultivadas , Doxorrubicina/efeitos adversos , Doxorrubicina/farmacologia , Cloridrato de Erlotinib/efeitos adversos , Cloridrato de Erlotinib/farmacologia , Humanos , Contração Miocárdica/efeitos dos fármacos
4.
Mater Sci Eng C Mater Biol Appl ; 104: 109904, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31499954

RESUMO

Brain extracellular matrix (ECM) is complex, heterogeneous and often poorly replicated in traditional 2D cell culture systems. The development of more physiologically relevant 3D cell models capable of emulating the native ECM is of paramount importance for the study of human induced pluripotent stem cell (iPSC)-derived neurons. Due to its structural similarity with hyaluronic acid, a primary component of brain ECM, alginate is a potential biomaterial for 3D cell culture systems. However, a lack of cell adhesion motifs within the chemical structure of alginate has limited its application in neural culture systems. This study presents a simple and accessible method of incorporating collagen fibrils into an alginate hydrogel by physical mixing and controlled gelation under physiological conditions and tests the hypothesis that such a substrate could influence the behaviour of human neurons in 3D culture. Regulation of the gelation process enabled the penetration of collagen fibrils throughout the hydrogel structure as demonstrated by transmission electron microscopy. Encapsulated human iPSC-derived neurons adhered to the blended hydrogel as evidenced by the increased expression of α1, α2 and ß1 integrins. Furthermore, immunofluorescence microscopy revealed that encapsulated neurons formed complex neural networks and matured into branched neurons expressing synaptophysin, a key protein involved in neurotransmission, along the neurites. Mechanical tuning of the hydrogel stiffness by modulation of the alginate ionic crosslinker concentration also influenced neuron-specific gene expression. In conclusion, we have shown that by tuning the physicochemical properties of the alginate/collagen blend it is possible to create different ECM-like microenvironments where complex mechanisms underpinning the growth and development of human neurons can be simulated and systematically investigated.


Assuntos
Alginatos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Colágeno/farmacologia , Hidrogéis/farmacologia , Neurogênese/efeitos dos fármacos , Neurônios/citologia , Adesão Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Junções Célula-Matriz/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fenótipo , Reologia
5.
Nat Commun ; 10(1): 4325, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541103

RESUMO

Cardiomyocytes from human induced pluripotent stem cells (hiPSC-CMs) are increasingly recognized as valuable for determining the effects of drugs on ion channels but they do not always accurately predict contractile responses of the human heart. This is in part attributable to their immaturity but the sensitivity of measurement tools may also be limiting. Measuring action potential, calcium flux or contraction individually misses critical information that is captured when interrogating the complete excitation-contraction coupling cascade simultaneously. Here, we develop an hypothesis-based statistical algorithm that identifies mechanisms of action. We design and build a high-speed optical system to measure action potential, cytosolic calcium and contraction simultaneously using fluorescent sensors. These measurements are automatically processed, quantified and then assessed by the algorithm. Multiplexing these three critical physical features of hiPSC-CMs allows identification of all major drug classes affecting contractility with detection sensitivities higher than individual measurement of action potential, cytosolic calcium or contraction.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Potenciais de Ação , Algoritmos , Cálcio/metabolismo , Biologia Computacional , Corantes Fluorescentes , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Canais Iônicos , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Imagem Óptica
6.
J Neuroinflammation ; 16(1): 164, 2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31395092

RESUMO

BACKGROUND: Astrocytes respond to central nervous system (CNS) injury and disease by transforming to a reactive astrogliosis cell state that can contribute to either CNS dysfunction or repair. Neuroinflammation is a powerful driver of a harmful A1 astrogliosis phenotype associated with in vitro neurotoxicity and histopathology in human neurodegenerative diseases. Here we report a protocol for the rapid development of a human cell culture model of neuroinflammatory astrogliosis using induced pluripotent stem cells (iPSCs). METHODS: Using RNA sequencing and in vitro cell assays, we measured transcriptional and cellular effects of chronic exposure of human iPSC-derived astrocytes to the cytokines TNFα (tumor necrosis factor alpha) or IL-1ß (interleukin-1 beta). RESULTS: We show TNFα and IL-1ß induce pro-inflammatory gene signatures but by widely different magnitudes. TNFα treatment results in 606 differential expressed genes, the suppression of glutamate-uptake, and increased phagocytic activity in astrocyte cultures. In contrast, IL-1ß effects are attenuated to 33 differential expressed genes and no significant effects on glutamate-uptake or increased phagocytic activity. CONCLUSION: Our approach demonstrates a rapid tool for modeling neuroinflammatory human astrocytic responses in nervous system trauma and disease. In particular, we reveal a model for robust TNFα-induced human astrogliosis suitable for the study of neurotoxic A1 astrocytes.


Assuntos
Astrócitos/metabolismo , Sangue Fetal/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Mediadores da Inflamação/metabolismo , Fagocitose/fisiologia , Astrócitos/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Sangue Fetal/citologia , Sangue Fetal/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Mediadores da Inflamação/farmacologia , Fagocitose/efeitos dos fármacos
7.
J Pharmacol Sci ; 140(4): 337-344, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31399314

RESUMO

Vitamin A (VA) is a fat-soluble micronutrient that plays essential roles in various biological processes, including cell growth, differentiation, and apoptosis. In the intestine, VA are known to promote mucosal homeostasis and immunity. However, the effect of VA in intestinal development has not been well elucidated. In the present study, we generated human intestine organoids from human induced pluripotent stem cells (iPSCs), and investigated the effect of the VA active metabolite all-trans retinoic acid (RA), on differentiation into intestinal organoids. As a result, RA increased the gene expression of a drug-metabolizing enzyme CYP3A4, as a functional molecule of intestinal mature development, in iPSC-derived intestinal organoids. In addition, RA increased transepithelial electrical resistance, an indicator of epithelial integrity, and decreased the permeability of monolayers to fluorescein isothiocyanate-labeled dextran in intestinal epithelial monolayers. Finally, RA increased the expression of ZO-1, a marker of tight junctions, which are essential for intestinal epithelial barrier function. Taken together, these results indicate that RA promotes barrier functions of iPSC-derived intestinal epithelial monolayers by increasing ZO-1 expression.


Assuntos
Células Epiteliais/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Tretinoína/farmacologia , Apoptose/efeitos dos fármacos , Células CACO-2 , Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Permeabilidade/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Vitamina A/farmacologia , Proteína da Zônula de Oclusão-1/metabolismo
8.
Mol Pharmacol ; 96(4): 475-484, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31399497

RESUMO

Topoisomerase II (TOP2) poisons are effective cytotoxic anticancer agents that stabilize the normally transient TOP2-DNA covalent complexes formed during the enzyme reaction cycle. These drugs include etoposide, mitoxantrone, and the anthracyclines doxorubicin and epirubicin. Anthracyclines also exert cell-killing activity via TOP2-independent mechanisms, including DNA adduct formation, redox activity, and lipid peroxidation. Here, we show that anthracyclines and another intercalating TOP2 poison, mitoxantrone, stabilize TOP2-DNA covalent complexes less efficiently than etoposide, and at higher concentrations they suppress the formation of TOP2-DNA covalent complexes, thus behaving as TOP2 poisons at low concentration and inhibitors at high concentration. We used induced pluripotent stem cell (iPSC)-derived human cardiomyocytes as a model to study anthracycline-induced damage in cardiac cells. Using immunofluorescence, our study is the first to demonstrate the presence of topoisomerase IIß (TOP2B) as the only TOP2 isoform in iPSC-derived cardiomyocytes. In these cells, etoposide robustly induced TOP2B covalent complexes, but we could not detect doxorubicin-induced TOP2-DNA complexes, and doxorubicin suppressed etoposide-induced TOP2-DNA complexes. In vitro, etoposide-stabilized DNA cleavage was attenuated by doxorubicin, epirubicin, or mitoxantrone. Clinical use of anthracyclines is associated with cardiotoxicity. The observations in this study have potentially important clinical consequences regarding the effectiveness of anticancer treatment regimens when TOP2-targeting drugs are used in combination. These observations suggest that inhibition of TOP2B activity, rather than DNA damage resulting from TOP2 poisoning, may play a role in doxorubicin cardiotoxicity. SIGNIFICANCE STATEMENT: We show that anthracyclines and mitoxantrone act as topoisomerase II (TOP2) poisons at low concentration but attenuate TOP2 activity at higher concentration, both in cells and in in vitro cleavage experiments. Inhibition of type II topoisomerases suppresses the action of other drugs that poison TOP2. Thus, combinations containing anthracyclines or mitoxantrone and etoposide may reduce the activity of etoposide as a TOP2 poison and thus reduce the efficacy of drug combinations.


Assuntos
Antraciclinas/farmacologia , Adutos de DNA/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Etoposídeo/farmacologia , Mitoxantrona/farmacologia , Cardiotoxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Adutos de DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxorrubicina/efeitos adversos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células K562 , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Inibidores da Topoisomerase II/farmacologia
9.
Nihon Yakurigaku Zasshi ; 154(2): 72-77, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31406046

RESUMO

In drug disposition, the liver and small intestine are very important as tissues involving in drug metabolism, absorption, and excretion. Thus, in drug development studies, it is necessary to evaluate the pharmacokinetics in these tissues accurately including the contributions of drug-metabolizing enzymes and drug transporters. Currently, all kinds of evaluation systems have been used for the pharmacokinetic prediction; however, there are some issues in these systems. Therefore, the researches for the development of human induced pluripotent stem (iPS) cell-derived hepatocytes and enterocytes, as novel systems besides existing ones, are being advanced. Because human iPS cells have abilities of pluripotency and almost infinite proliferation, it is thought to be possible to stably provide the high-quality cells that have similar characteristics to human normal tissue cells by using human iPS cells. In this review, we describe current status of differentiation studies of human iPS cell-derived hepatocytes and enterocytes and the functional characteristics of these cells centered on pharmacokinetic functions.


Assuntos
Enterócitos/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Diferenciação Celular , Avaliação Pré-Clínica de Medicamentos , Enterócitos/citologia , Hepatócitos/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Intestino Delgado , Fígado , Farmacocinética
10.
Int J Mol Sci ; 20(15)2019 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-31382622

RESUMO

Cardiomyocytes from human induced pluripotent stem cells (hiPSC-CMs) are the most promising human source with preserved genetic background of healthy individuals or patients. This study aimed to establish a systematic procedure for exploring development of hiPSC-CM functional output to predict genetic cardiomyopathy outcomes and identify molecular targets for therapy. Biomimetic substrates with microtopography and physiological stiffness can overcome the immaturity of hiPSC-CM function. We have developed a custom-made apparatus for simultaneous optical measurements of hiPSC-CM action potential and calcium transients to correlate these parameters at specific time points (day 60, 75 and 90 post differentiation) and under inotropic interventions. In later-stages, single hiPSC-CMs revealed prolonged action potential duration, increased calcium transient amplitude and shorter duration that closely resembled those of human adult cardiomyocytes from fresh ventricular tissue of patients. Thus, the major contribution of sarcoplasmic reticulum and positive inotropic response to ß-adrenergic stimulation are time-dependent events underlying excitation contraction coupling (ECC) maturation of hiPSC-CM; biomimetic substrates can promote calcium-handling regulation towards adult-like kinetics. Simultaneous optical recordings of long-term cultured hiPSC-CMs on biomimetic substrates favor high-throughput electrophysiological analysis aimed at testing (mechanistic hypothesis on) disease progression and pharmacological interventions in patient-derived hiPSC-CMs.


Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Cálcio/metabolismo , Cardiomiopatias/tratamento farmacológico , Células-Tronco Pluripotentes Induzidas/metabolismo , Potenciais de Ação/efeitos dos fármacos , Biomimética , Cardiomiopatias/genética , Cardiomiopatias/patologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Acoplamento Excitação-Contração/efeitos dos fármacos , Humanos , Hidrogéis/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Especificidade por Substrato
11.
Drug Metab Pharmacokinet ; 34(4): 264-271, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31285099

RESUMO

Cholestatic drug-induced liver injury (DILI) is a type of hepatotoxicity. Its underlying mechanisms are dysfunction of bile salt export pump (BSEP) and multidrug resistance-associated protein 2/3/4 (MRP2/3/4), which play major roles in bile acid (BA) excretion into the bile canaliculi and blood, resulting in accumulation of BAs in hepatocytes. The sandwich-cultured hepatocyte (SCH) model can simultaneously analyze hepatic uptake and biliary excretion. Therefore, we investigated whether sandwich-cultured human induced pluripotent stem cell (iPS cell)-derived hepatocytes (SCHiHs) are suitable for evaluating cholestatic DILI. Fluorescent N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5ß-cholestan-26-oyl)-2'-aminoethanesulfonate (tauro-nor-THCA-24-DBD, a BSEP substrate) was accumulated in bile canaliculi, which supports the presence of a functional bile canaliculi lumen. MRP2 was highly expressed in the Western blot analysis, whereas the mRNA expression of BSEP was hardly detectable. MRP3/4 mRNA levels were maintained. Of the 22 compounds known to cause DILI with BAs, 7 showed significant cytotoxicity. Most high-risk drugs were detected using the developed SCHiH system. However, a shortcoming was the considerably low expression level of BSEP, which prevented the detection of some relevant drugs whose risks should be detected in primary human hepatocytes.


Assuntos
Ácidos e Sais Biliares/farmacologia , Hepatócitos/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Hepatócitos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo
12.
Artigo em Chinês | MEDLINE | ID: mdl-31327193

RESUMO

Objective:The purpose of the present study was to explore the characteristics and differentiation of somatic cells in vitro undergoing a low pH treatment, so as to provide new therapeutic strategies for treating sensorineural hearing loss.Method: The human mature somatic cells were selected as the target cells, and the cells were treated with different pH values to observe the cell morphology. The cell characteristics were identified from alkaline phosphatase (AKP) activity, immunohistochemical staining and molecular biology, and the most suitable pH value was selected. In addition, a mouse model of the cochlear lesion was constructed using bilirubin. Subsequently, the characteristics and therapeutic effect of somatic cells undergoing low pH treatment were examined by morphology, AKP activity, immunofluorescence assay and Q-PCR.Result:The cell growth of the experimental group was significantly better than those in the control group. The activity of AKP in the experimental group was higher than that in the control group. The expression of Nanog and Oct4 was both positive in the two groups. When the cells were changed to neurobasol medium, the marker of Nestin was positive.Conclusion:The human somatic cells undergoing a low pH treatment showed the similar characteristics as those of induced pluripotent stem (iPS) cells; although the functions and therapeutic effect of these altered human somatic cells need to be further studied.


Assuntos
Ácidos/farmacologia , Diferenciação Celular , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Animais , Humanos , Concentração de Íons de Hidrogênio , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Camundongos
13.
PLoS One ; 14(7): e0219592, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31356639

RESUMO

Chromosome abnormalities induces profound alterations in gene expression, leading to various disease phenotypes. Recent studies on yeast and mammalian cells have demonstrated that aneuploidy exerts detrimental effects on organismal growth and development, regardless of the karyotype, suggesting that aneuploidy-associated stress plays an important role in disease pathogenesis. However, whether and how this effect alters cellular homeostasis and long-term features of human disease are not fully understood. Here, we aimed to investigate cellular stress responses in human trisomy syndromes, using fibroblasts and induced pluripotent stem cells (iPSCs). Dermal fibroblasts derived from patients with trisomy 21, 18 and 13 showed a severe impairment of cell proliferation and enhanced premature senescence. These phenomena were accompanied by perturbation of protein homeostasis, leading to the accumulation of protein aggregates. We found that treatment with sodium 4-phenylbutyrate (4-PBA), a chemical chaperone, decreased the protein aggregates in trisomy fibroblasts. Notably, 4-PBA treatment successfully prevented the progression of premature senescence in secondary fibroblasts derived from trisomy 21 iPSCs. Our study reveals aneuploidy-associated stress as a potential therapeutic target for human trisomies, including Down syndrome.


Assuntos
Senescência Celular , Fibroblastos/patologia , Agregados Proteicos , Trissomia/patologia , Aneuploidia , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Glucose/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lactatos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Estresse Oxidativo/efeitos dos fármacos , Fenilbutiratos/farmacologia , Agregados Proteicos/efeitos dos fármacos , RNA/metabolismo , Trissomia/genética
14.
Cells ; 8(6)2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31234430

RESUMO

The mitochondrial genetic disorder, Leber's hereditary optic neuropathy (LHON), is caused by a mutation in MT-ND4 gene, encoding NADH dehydrogenase subunit 4. It leads to the progressive death of retinal ganglion cells (RGCs) and causes visual impairment or even blindness. However, the precise mechanisms of LHON disease penetrance and progression are not completely elucidated. Human-induced pluripotent stem cells (hiPSCs) offer unique opportunities to investigate disease-relevant phenotypes and regulatory mechanisms underlying LHON pathogenesis at the cellular level. In this study, we successfully generated RGCs by differentiation of LHON patient-specific hiPSCs. We modified the protocol of differentiation to obtain a more enriched population of single-cell RGCs for LHON study. Based on assessing morphology, expression of specific markers and electrophysiological activity, we found that LHON-specific hiPSC-derived were more defective in comparison with normal wild-type RGCs. Based on our previous study, whereby by using microarray analysis we identified that the components of glutamatergic synapse signaling pathway were significantly downregulated in LHON-specific RGCs, we focused our study on glutamate-associated α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. We found that the protein expression levels of the subunits of the AMPA receptor, GluR1 and GluR2, and their associated scaffold proteins were decreased in LHON-RGCs. By performing the co-immunoprecipitation assay, we found several differences in the efficiencies of interaction between AMPA subunits and scaffold proteins between normal and LHON-specific RGCs.


Assuntos
Ácido Glutâmico/farmacologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Atrofia Óptica Hereditária de Leber/metabolismo , Atrofia Óptica Hereditária de Leber/patologia , Receptores de AMPA/metabolismo , Células Ganglionares da Retina/metabolismo , Transdução de Sinais , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Células Ganglionares da Retina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/metabolismo
15.
Biofabrication ; 11(4): 045011, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31247598

RESUMO

Neurological disorders affect millions of Americans and this number is expected to rise with the aging population. Development of drugs to treat these disorders may be facilitated by improved in vitro models that faithfully reproduce salient features of the relevant brain regions. Current 3D culture methods face challenges with reliably reproducing microarchitectural features of brain morphology such as cortical or hippocampal layers. In this work, polydimethylsiloxane (PDMS) mini-wells were used to create low aspect ratio, adherent 3D constructs where neurons self-assemble into layers. Layer self-assembly was determined to depend on the size of the PDMS mini-well. Layer formation occurred in cultures composed of primary rat cortical neurons or human induced pluripotent stem cell-derived neurons and astrocytes and was robust and reproducible. Layered 3D constructs were found to have spontaneous neural activity characterized by long bursts similar to activity in the developing cortex. The responses of layered 3D cultures to drugs were more similar to in vivo data than those of 2D cultures. 3D constructs created with this method may be thus suitable as in vitro models for drug discovery for neurological disorders.


Assuntos
Técnicas de Cultura de Células/métodos , Neurônios/citologia , Animais , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Ácido Cinurênico/farmacologia , Neurônios/efeitos dos fármacos , Ratos Sprague-Dawley , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Tetrodotoxina/farmacologia
16.
Genes (Basel) ; 10(6)2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31197102

RESUMO

Deep-sequencing of the ABCA4 locus has revealed that ~10% of autosomal recessive Stargardt disease (STGD1) cases are caused by deep-intronic mutations. One of the most recurrent deep-intronic variants in the Belgian and Dutch STGD1 population is the c.4539+2001G>A mutation. This variant introduces a 345-nt pseudoexon to the ABCA4 mRNA transcript in a retina-specific manner. Antisense oligonucleotides (AONs) are short sequences of RNA that can modulate splicing. In this work, we designed 26 different AONs to perform a thorough screening to identify the most effective AONs to correct splicing defects associated with c.4539+2001G>A. All AONs were tested in patient-derived induced pluripotent stem cells (iPSCs) that were differentiated to photoreceptor precursor cells (PPCs). AON efficacy was assessed through RNA analysis and was based on correction efficacy, and AONs were grouped and their properties assessed. We (a) identified nine AONs with significant correction efficacies (>50%), (b) confirmed that a single nucleotide mismatch was sufficient to significantly decrease AON efficacy, and (c) found potential correlations between efficacy and some of the parameters analyzed. Overall, our results show that AON-based splicing modulation holds great potential for treating Stargardt disease caused by splicing defects in ABCA4.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Diferenciação Celular/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , /genética , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Íntrons/genética , Oligonucleotídeos Antissenso/genética , Células Fotorreceptoras/efeitos dos fármacos , Células Fotorreceptoras/patologia , Retina/efeitos dos fármacos , Retina/patologia , /tratamento farmacológico
17.
Pharmacol Res Perspect ; 7(3): e00487, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31149342

RESUMO

The phospholipid l-α-lysophosphatidylinositol (LPI), an endogenous ligand for GPR55, is elevated in patients with acute coronary syndrome, and a GPR55 antagonist cannabidiol (CBD) reduces experimental ischemia/reperfusion (I/R) injury. While LPI activates multiple signaling pathways, little is known about which ones are important in cardiomyocytes. In this study we explored whether activation of the Rho kinase/ROCK/p38 MAPK pathway is responsible for LPI-induced extension of I/R injury. Using a high-throughput screening method (dynamic mass redistribution; DMR), mouse- and human-induced pluripotent stem cell (iPSC) cardiomyocytes exposed to LPI were shown to exhibit a rapid, sustained, and concentration-dependent (1 nmol L-1-30 µmol L-1) cellular response. Y-27632 (ROCK inhibitor; 10 & 50 µmol L-1) and CBD (1 µmol L-1) both abolished the DMR response to LPI (10 µmol L-1). In murine iPSC cardiomyocytes, LPI-induced ROCK and p38 MAPK phosphorylation, both of which were prevented by Y-27632 and CBD, but did not induce JNK activation or cleavage of caspase-3. In hearts isolated from wild type (WT) mice subjected to 30 minutes global I/R, LPI (10 µmol L-1) administered via the coronary circulation increased infarct size when applied prior to ischemia onset, but not when given at the time of reperfusion. The exacerbation of tissue injury by LPI was not seen in hearts from GPR55-/- mice or in the presence of Y-27632, confirming that injury is mediated via the GPR55/ROCK/p38 MAPK pathway. These findings suggest that raised levels of LPI in the vicinity of a developing infarct may worsen the outcome of AMI.


Assuntos
Lisofosfolipídeos/efeitos adversos , Traumatismo por Reperfusão Miocárdica/induzido quimicamente , Receptores de Canabinoides/metabolismo , Quinases Associadas a rho/metabolismo , Amidas/farmacologia , Animais , Modelos Animais de Doenças , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Traumatismo por Reperfusão Miocárdica/metabolismo , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Receptores de Canabinoides/genética , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
18.
Int J Mol Sci ; 20(11)2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31146354

RESUMO

Modern diagnostic strategies for early recognition of cancer therapeutics-related cardiac dysfunction involve cardiac troponins measurement. Still, the role of other markers of cardiotoxicity is still unclear. The present study was designed to investigate dynamics of response of human cardiomyocytes derived from induced pluripotent stem cells (hiPCS-CMs) to doxorubicin with the special emphasis on their morphological changes in relation to expression and organization of troponins. The hiPCS-CMs were treated with doxorubicin concentrations (1 and 0.3 µM) for 48 h and followed for next up to 6 days. Exposure of hiPCS-CMs to 1 µM doxorubicininduced suppression of both cardiac troponin T (cTnT) and cardiac troponin I (cTnI) gene expression. Conversely, lower 0.3 µM doxorubicin concentration produced no significant changes in the expression of aforementioned genes. However, the intracellular topography, arrangement, and abundance of cardiac troponin proteins markedly changed after both doxorubicin concentrations. In particular, at 48 h of treatment, both cTnT and cTnI bundles started to reorganize, with some of them forming compacted shapes extending outwards and protruding outside the cells. At later intervals (72 h and onwards), the whole troponin network collapsed and became highly disorganized following, to some degree, overall changes in the cellular shape. Moreover, membrane permeability of cardiomyocytes was increased, and intracellular mitochondrial network rearranged and hypofunctional. Together, our results demonstrate complex effects of clinically relevant doxorubicin concentrations on hiPCS-CM cells including changes in cTnT and cTnI, but also in other cellular compartments contributing to the overall cytotoxicity of this class of cytostatics.


Assuntos
Antineoplásicos/toxicidade , Doxorrubicina/toxicidade , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Troponina/metabolismo , Antineoplásicos/farmacologia , Cardiotoxicidade , Linhagem Celular , Doxorrubicina/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo
19.
Neurochem Res ; 44(7): 1773-1779, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31102025

RESUMO

Spinal muscular atrophy (SMA) is an inherited disease characterized by progressive motor neuron death and subsequent muscle weakness and is caused by deletion or mutation of survival motor neuron (SMN) 1 gene. Protecting spinal motor neuron is an effective clinical strategy for SMA. The purpose of this study was to investigate the potential effect of an anti-epileptic drug levetiracetam on SMA. In the present study, we used differentiated spinal motor neurons (MNs) from SMA patient-derived induced pluripotent stem cells (SMA-iPSCs) to investigate the effect of levetiracetam. Levetiracetam promoted neurite elongation in SMA-iPSCs-MNs. TUNEL-positive spinal motor neurons were significantly reduced by levetiracetam in SMA-iPSCs-MNs. In addition, the expression level of cleaved-caspase 3 was decreased by levetiracetam in SMA-iPSCs-MNs. Furthermore, levetiracetam improved impaired mitochondrial function in SMA-iPSCs-MNs. On the other hand, levetiracetam did not affect the expression level of SMN protein in SMA-iPSCs-MNs. These findings indicate that levetiracetam has a neuroprotective effect for SMA.


Assuntos
Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Levetiracetam/uso terapêutico , Neurônios Motores/efeitos dos fármacos , Atrofia Muscular Espinal/prevenção & controle , Neuritos/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Apoptose/efeitos dos fármacos , Chaperonina 60/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/patologia , Neuritos/patologia
20.
Genes Cells ; 24(7): 473-484, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31099158

RESUMO

Induced pluripotent stem (iPS) cells hold great promise for regenerative medicine and the treatment of various diseases. Before proceeding to clinical trials, it is important to test the efficacy and safety of iPS cell-based treatments using experimental animals. The common marmoset is a new world monkey widely used in biomedical studies. However, efficient methods that could generate iPS cells from a variety of cells have not been established. Here, we report that marmoset cells are efficiently reprogrammed into iPS cells by combining RNA transfection and chemical compounds. Using this novel combination, we generate transgene integration-free marmoset iPS cells from a variety of cells that are difficult to reprogram using conventional RNA transfection method. Furthermore, we show this is similarly effective for human and cynomolgus monkey iPS cell generation. Thus, the addition of chemical compounds during RNA transfection greatly facilitates reprogramming and efficient generation of completely integration-free safe iPS cells in primates, particularly from difficult-to-reprogram cells.


Assuntos
Reprogramação Celular , Fibroblastos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Preparações Farmacêuticas/administração & dosagem , RNA/administração & dosagem , Transfecção/métodos , Idoso , Animais , Diferenciação Celular , Células Cultivadas , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/fisiologia , Platirrinos
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