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1.
Yakugaku Zasshi ; 140(8): 993-1000, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32741873

RESUMO

The human genome consists of more than 20000 genes and is essential for all biological phenomena. To understand these biological phenomena, including diseases, and to be able to modify them, approaches that enable optical control of the genome may be useful. Recently, we developed an optogenetic tool, named photoactivatable Cas9 (PA-Cas9). We divided Cas9 nuclease from the CRISPR-Cas9 system into two fragments and connected photo-inducible dimerization proteins, named Magnet system, to the fragments, leading to the development of PA-Cas9 of which nuclease activity is switchable with light. PA-Cas9 allows direct editing of DNA sequences by light stimulation. Additionally, we developed a light-inducible, RNA-guided programmable system for endogenous gene activation based on the CRISPR-Cas9 system. We demonstrated that this optogenetic tool allows rapid and reversible targeted gene activation by light. Using this tool, we exemplified optical control of neuronal differentiation of human induced pluripotent stem cells (iPSCs). The CRISPR-Cas9-based, photoactivatable transcription system offers a simple and versatile approach to precise gene activation. In addition to the CRISPR-Cas9-based optogenetic tools, we developed a photoactivatable Cre-loxP system. This tool allows optical control of DNA recombination reaction in an internal organ even by external, noninvasive illumination using LED light source. To date, genome engineering technology and optogenetics technology have emerged separately as different applications. Our studies described above merge these emerging research fields together.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Engenharia Genética , Luz , Optogenética , Ativação Transcricional , Animais , Diferenciação Celular , DNA/genética , Edição de Genes , Genoma Humano , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Recombinação Genética
2.
Nat Commun ; 11(1): 3791, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32728089

RESUMO

Brain organoids are promising tools for disease modeling and drug development. For proper neuronal network formation excitatory and inhibitory neurons as well as glia need to co-develop. Here, we report the directed self-organization of human induced pluripotent stem cells in a collagen hydrogel towards a highly interconnected neuronal network at a macroscale tissue format. Bioengineered Neuronal Organoids (BENOs) comprise interconnected excitatory and inhibitory neurons with supportive astrocytes and oligodendrocytes. Giant depolarizing potential (GDP)-like events observed in early BENO cultures mimic early network activity of the fetal brain. The observed GABA polarity switch and reduced GDPs in >40 day BENO indicate progressive neuronal network maturation. BENOs demonstrate expedited complex network burst development after two months and evidence for long-term potentiation. The similarity of structural and functional properties to the fetal brain may allow for the application of BENOs in studies of neuronal plasticity and modeling of disease.


Assuntos
Encéfalo/citologia , Neurogênese , Plasticidade Neuronal/fisiologia , Organoides/fisiologia , Engenharia Tecidual/métodos , Potenciais de Ação/fisiologia , Encéfalo/crescimento & desenvolvimento , Técnicas de Cultura de Células , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Neurônios/fisiologia , Ácido gama-Aminobutírico/metabolismo
3.
Am J Hum Genet ; 107(2): 278-292, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32707085

RESUMO

Dominantly inherited disorders are not typically considered to be therapeutic candidates for gene augmentation. Here, we utilized induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) to test the potential of gene augmentation to treat Best disease, a dominant macular dystrophy caused by over 200 missense mutations in BEST1. Gene augmentation in iPSC-RPE fully restored BEST1 calcium-activated chloride channel activity and improved rhodopsin degradation in an iPSC-RPE model of recessive bestrophinopathy as well as in two models of dominant Best disease caused by different mutations in regions encoding ion-binding domains. A third dominant Best disease iPSC-RPE model did not respond to gene augmentation, but showed normalization of BEST1 channel activity following CRISPR-Cas9 editing of the mutant allele. We then subjected all three dominant Best disease iPSC-RPE models to gene editing, which produced premature stop codons specifically within the mutant BEST1 alleles. Single-cell profiling demonstrated no adverse perturbation of retinal pigment epithelium (RPE) transcriptional programs in any model, although off-target analysis detected a silent genomic alteration in one model. These results suggest that gene augmentation is a viable first-line approach for some individuals with dominant Best disease and that non-responders are candidates for alternate approaches such as gene editing. However, testing gene editing strategies for on-target efficiency and off-target events using personalized iPSC-RPE model systems is warranted. In summary, personalized iPSC-RPE models can be used to select among a growing list of gene therapy options to maximize safety and efficacy while minimizing time and cost. Similar scenarios likely exist for other genotypically diverse channelopathies, expanding the therapeutic landscape for affected individuals.


Assuntos
Células-Tronco Pluripotentes Induzidas/fisiologia , Degeneração Macular/genética , Mutação/genética , Alelos , Bestrofinas/genética , Cálcio/metabolismo , Linhagem Celular , Canalopatias/genética , Proteínas do Olho/genética , Edição de Genes/métodos , Terapia Genética/métodos , Genótipo , Células HEK293 , Humanos , Epitélio Pigmentado da Retina/fisiologia
4.
PLoS One ; 15(6): e0234441, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32525941

RESUMO

Human iPSC-derived hepatocytes hold great promise as a cell source for cell therapy and drug screening. However, the culture method for highly-quantified hepatocytes has not yet been established. Herein, we have developed an encapsulation and 3D cultivation method for iPSC-hepatocytes in core-shell hydrogel microfibers (a.k.a. cell fiber). In the fiber-shaped 3D microenvironment consisting of abundant extracellular matrix (ECM), the iPSC-hepatocytes exhibited many hepatic characteristics, including the albumin secretion, and the expression of the hepatic marker genes (ALB, HNF4α, ASGPR1, CYP2C19, and CYP3A4). Furthermore, we found that the fibers were mechanically stable and can be applicable to hepatocyte transplantation. Three days after transplantation of the microfibers into the abdominal cavity of immunodeficient mice, human albumin was detected in the peripheral blood of the transplanted mice. These results indicate that the iPSC-hepatocyte fibers are promising either as in vitro models for drug screening or as implantation grafts to treat liver failure.


Assuntos
Técnicas de Cultura de Células/métodos , Encapsulamento de Células/métodos , Hepatócitos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Cavidade Abdominal/cirurgia , Alginatos/química , Animais , Técnicas de Cultura de Células/instrumentação , Diferenciação Celular , Encapsulamento de Células/instrumentação , Terapia Baseada em Transplante de Células e Tecidos/métodos , Perfilação da Expressão Gênica , Hepatócitos/transplante , Humanos , Hidrogéis/química , Falência Hepática/terapia , Masculino , Camundongos , Esferoides Celulares
5.
Nat Commun ; 11(1): 2997, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561714

RESUMO

PIEZO2 is the essential transduction channel for touch discrimination, vibration, and proprioception. Mice and humans lacking Piezo2 experience severe mechanosensory and proprioceptive deficits and fail to develop tactile allodynia. Bradykinin, a proalgesic agent released during inflammation, potentiates PIEZO2 activity. Molecules that decrease PIEZO2 function could reduce heightened touch responses during inflammation. Here, we find that the dietary fatty acid margaric acid (MA) decreases PIEZO2 function in a dose-dependent manner. Chimera analyses demonstrate that the PIEZO2 beam is a key region tuning MA-mediated channel inhibition. MA reduces neuronal action potential firing elicited by mechanical stimuli in mice and rat neurons and counteracts PIEZO2 sensitization by bradykinin. Finally, we demonstrate that this saturated fatty acid decreases PIEZO2 currents in touch neurons derived from human induced pluripotent stem cells. Our findings report on a natural product that inhibits PIEZO2 function and counteracts neuronal mechanical sensitization and reveal a key region for channel inhibition.


Assuntos
Ácidos Graxos/administração & dosagem , Canais Iônicos/antagonistas & inibidores , Mecanotransdução Celular/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Propriocepção/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Algoritmos , Animais , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Canais Iônicos/genética , Canais Iônicos/metabolismo , Mecanotransdução Celular/genética , Mecanotransdução Celular/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Neurônios/fisiologia , Propriocepção/genética , Propriocepção/fisiologia , Ratos , Tato/efeitos dos fármacos , Tato/fisiologia
6.
PLoS One ; 15(6): e0233860, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479513

RESUMO

The generation of laminated and light responsive retinal organoids from induced pluripotent stem cells (iPSCs) provides a powerful tool for the study of retinal diseases and drug discovery and a robust platform for cell-based therapies. The aim of this study is to investigate whether retinal organoids can retain their morphological and functional characteristics upon storage at room temperature (RT) conditions and shipment by air using a commercially available container that maintains the environment at ambient temperature. Morphological analysis and measurements of neuroepithelial thickness revealed no differences between control, RT incubated and shipped organoids. Similarly immunohistochemical analysis showed no differences in cell type composition and position within the laminated retinal structure. All groups showed a similar response to light, suggesting that the biological function of retinal organoids was not affected by RT storage or shipment. These findings provide an advance in transport of ready-made retinal organoids, increasing their availability to many research and pharma labs worldwide and facilitating cross-collaborative research.


Assuntos
Organoides/transplante , Serviços Postais , Retina/citologia , Doenças Retinianas/terapia , Diferenciação Celular , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Luz , Organoides/efeitos dos fármacos , Organoides/fisiologia , Organoides/efeitos da radiação , Temperatura
7.
PLoS One ; 15(6): e0233980, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32511247

RESUMO

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS) that results in variable severities of neurodegeneration. The understanding of MS has been limited by the inaccessibility of the affected cells and the lengthy timeframe of disease development. However, recent advances in stem cell technology have facilitated the bypassing of some of these challenges. Towards gaining a greater understanding of the innate potential of stem cells from people with varying degrees of disability, we generated induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells derived from stable and progressive MS patients, and then further differentiated them into oligodendrocyte (OL) lineage cells. We analyzed differentiation under both homeostatic and inflammatory conditions via sustained exposure to low-dose interferon gamma (IFNγ), a prominent cytokine in MS. We found that all iPSC lines differentiated into mature myelinating OLs, but chronic exposure to IFNγ dramatically inhibited differentiation in both MS groups, particularly if exposure was initiated during the pre-progenitor stage. Low-dose IFNγ was not toxic but led to an early upregulation of interferon response genes in OPCs followed by an apparent redirection in lineage commitment from OL to a neuron-like phenotype in a significant portion of the treated cells. Our results reveal that a chronic low-grade inflammatory environment may have profound effects on the efficacy of regenerative therapies.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Esclerose Múltipla Crônica Progressiva/patologia , Oligodendroglia/citologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Homeostase , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/fisiologia , Inflamação/patologia , Interferon gama/farmacologia , Leucócitos Mononucleares/citologia , Regeneração
8.
Nat Commun ; 11(1): 2364, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398665

RESUMO

Human pluripotent stem cells (hPSCs) have the capacity to give rise to all differentiated cells of the adult. TGF-beta is used routinely for expansion of conventional hPSCs as flat epithelial colonies expressing the transcription factors POU5F1/OCT4, NANOG, SOX2. Here we report a global analysis of the transcriptional programme controlled by TGF-beta followed by an unbiased gain-of-function screening in multiple hPSC lines to identify factors mediating TGF-beta activity. We identify a quartet of transcriptional regulators promoting hPSC self-renewal including ZNF398, a human-specific mediator of pluripotency and epithelial character in hPSCs. Mechanistically, ZNF398 binds active promoters and enhancers together with SMAD3 and the histone acetyltransferase EP300, enabling transcription of TGF-beta targets. In the context of somatic cell reprogramming, inhibition of ZNF398 abolishes activation of pluripotency and epithelial genes and colony formation. Our findings have clear implications for the generation of bona fide hPSCs for regenerative medicine.


Assuntos
Autorrenovação Celular/genética , Regulação da Expressão Gênica/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Animais , Linhagem Celular , Reprogramação Celular/genética , Células-Tronco Embrionárias , Elementos Facilitadores Genéticos/genética , Mutação com Ganho de Função , Técnicas de Silenciamento de Genes , Humanos , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Dedos de Zinco
9.
Nat Commun ; 11(1): 1677, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32245952

RESUMO

Human stem cell-derived hepatocyte-like cells (HLCs) offer an attractive platform to study liver biology. Despite their numerous advantages, HLCs lack critical in vivo characteristics, including cell polarity. Here, we report a stem cell differentiation protocol that uses transwell filters to generate columnar polarized HLCs with clearly defined basolateral and apical membranes separated by tight junctions. We show that polarized HLCs secrete cargo directionally: Albumin, urea, and lipoproteins are secreted basolaterally, whereas bile acids are secreted apically. Further, we show that enterically transmitted hepatitis E virus (HEV) progeny particles are secreted basolaterally as quasi-enveloped particles and apically as naked virions, recapitulating essential steps of the natural infectious cycle in vivo. We also provide proof-of-concept that polarized HLCs can be used for pharmacokinetic and drug-drug interaction studies. This novel system provides a powerful tool to study hepatocyte biology, disease mechanisms, genetic variation, and drug metabolism in a more physiologically relevant setting.


Assuntos
Técnicas de Cultura de Células/métodos , Polaridade Celular , Hepatócitos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Antivirais/farmacologia , Diferenciação Celular , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Interações Medicamentosas , Vírus da Hepatite A Humana/fisiologia , Vírus da Hepatite E/fisiologia , Hepatócitos/ultraestrutura , Hepatócitos/virologia , Humanos , Fígado/citologia , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Microscopia Eletrônica de Transmissão , Estudo de Prova de Conceito , Vírion/metabolismo , Liberação de Vírus , Replicação Viral
10.
Proc Natl Acad Sci U S A ; 117(16): 9094-9100, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32253308

RESUMO

Stem cell transplantation can improve behavioral recovery after stroke in animal models but whether stem cell-derived neurons become functionally integrated into stroke-injured brain circuitry is poorly understood. Here we show that intracortically grafted human induced pluripotent stem (iPS) cell-derived cortical neurons send widespread axonal projections to both hemispheres of rats with ischemic lesions in the cerebral cortex. Using rabies virus-based transsynaptic tracing, we find that at 6 mo after transplantation, host neurons in the contralateral somatosensory cortex receive monosynaptic inputs from grafted neurons. Immunoelectron microscopy demonstrates myelination of the graft-derived axons in the corpus callosum and that their terminals form excitatory, glutamatergic synapses on host cortical neurons. We show that the stroke-induced asymmetry in a sensorimotor (cylinder) test is reversed by transplantation. Light-induced inhibition of halorhodopsin-expressing, grafted neurons does not recreate the impairment, indicating that its reversal is not due to neuronal activity in the graft. However, we find bilateral decrease of motor performance in the cylinder test after light-induced inhibition of either grafted or endogenous halorhodopsin-expressing cortical neurons, located in the same area, and after inhibition of endogenous halorhodopsin-expressing cortical neurons by exposure of their axons to light on the contralateral side. Our data indicate that activity in the grafted neurons, probably mediated through transcallosal connections to the contralateral hemisphere, is involved in maintaining normal motor function. This is an example of functional integration of efferent projections from grafted neurons into the stroke-affected brain's neural circuitry, which raises the possibility that such repair might be achievable also in humans affected by stroke.


Assuntos
Células-Tronco Pluripotentes Induzidas/fisiologia , Infarto da Artéria Cerebral Média/terapia , Atividade Motora/fisiologia , Neurônios/transplante , Córtex Somatossensorial/fisiopatologia , Potenciais de Ação/fisiologia , Animais , Técnicas de Observação do Comportamento , Comportamento Animal/fisiologia , Diferenciação Celular/fisiologia , Linhagem Celular , Modelos Animais de Doenças , Humanos , Infarto da Artéria Cerebral Média/etiologia , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Neurônios/fisiologia , Optogenética , Técnicas de Patch-Clamp , Ratos , Recuperação de Função Fisiológica , Córtex Somatossensorial/citologia , Córtex Somatossensorial/patologia
11.
Curr Pharm Biotechnol ; 21(9): 807-828, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32321398

RESUMO

Drug-induced toxicity remains one of the leading causes of discontinuation of the drug candidate and post-marketing withdrawal. Thus, early identification of the drug candidates with the potential for toxicity is crucial in the drug development process. With the recent discovery of human- Induced Pluripotent Stem Cells (iPSC) and the establishment of the differentiation protocol of human iPSC into the cell types of interest, the differentiated cells from human iPSC have garnered much attention because of their potential applicability in toxicity evaluation as well as drug screening, disease modeling and cell therapy. In this review, we expanded on current information regarding the feasibility of human iPSC-derived cells for the evaluation of drug-induced toxicity with a focus on human iPSCderived hepatocyte (iPSC-Hep), cardiomyocyte (iPSC-CMs) and neurons (iPSC-Neurons). Further, we CSAHi, Consortium for Safety Assessment using Human iPS Cells, reported our gene expression profiling data with DNA microarray using commercially available human iPSC-derived cells (iPSC-Hep, iPSC-CMs, iPSC-Neurons), their relevant human tissues and primary cultured human cells to discuss the future direction of the three types of human iPSC-derived cells.


Assuntos
Descoberta de Drogas , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/etiologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Neurônios/citologia , Neurônios/metabolismo
12.
Brain ; 143(3): 811-832, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32125365

RESUMO

Cyclin-dependent kinase-like 5 disorder is a severe neurodevelopmental disorder caused by mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene. It predominantly affects females who typically present with severe early epileptic encephalopathy, global developmental delay, motor dysfunction, autistic features and sleep disturbances. To develop a gene replacement therapy, we initially characterized the human CDKL5 transcript isoforms expressed in the brain, neuroblastoma cell lines, primary astrocytes and embryonic stem cell-derived cortical interneurons. We found that the isoform 1 and to a lesser extent the isoform 2 were expressed in human brain, and both neuronal and glial cell types. These isoforms were subsequently cloned into recombinant adeno-associated viral (AAV) vector genome and high-titre viral vectors were produced. Intrajugular delivery of green fluorescence protein via AAV vector serotype PHP.B in adult wild-type male mice transduced neurons and astrocytes throughout the brain more efficiently than serotype 9. Cdkl5 knockout male mice treated with isoform 1 via intrajugular injection at age 28-30 days exhibited significant behavioural improvements compared to green fluorescence protein-treated controls (1012 vg per animal, n = 10 per group) with PHP.B vectors. Brain expression of the isoform 1 transgene was more abundant in hindbrain than forebrain and midbrain. Transgene brain expression was sporadic at the cellular level and most prominent in hippocampal neurons and cerebellar Purkinje cells. Correction of postsynaptic density protein 95 cerebellar misexpression, a major fine cerebellar structural abnormality in Cdkl5 knockout mice, was found in regions of high transgene expression within the cerebellum. AAV vector serotype DJ efficiently transduced CDKL5-mutant human induced pluripotent stem cell-derived neural progenitors, which were subsequently differentiated into mature neurons. When treating CDKL5-mutant neurons, isoform 1 expression led to an increased density of synaptic puncta, while isoform 2 ameliorated the calcium signalling defect compared to green fluorescence protein control, implying distinct functions of these isoforms in neurons. This study provides the first evidence that gene therapy mediated by AAV vectors can be used for treating CDKL5 disorder.


Assuntos
Terapia Genética , Isoformas de Proteínas/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Adenoviridae , Animais , Encéfalo/metabolismo , Cálcio/metabolismo , Células Cultivadas , Proteína 4 Homóloga a Disks-Large/biossíntese , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Isoformas de Proteínas/genética , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Sinapses/metabolismo , Transfecção
13.
Nat Genet ; 52(4): 363-369, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203467

RESUMO

The genetic architecture of each individual comprises common and rare variants that, acting alone and in combination, confer risk of disease. The cell-type-specific and/or context-dependent functional consequences of the risk variants linked to brain disease must be resolved. Coupling human induced pluripotent stem cell (hiPSC)-based technology with CRISPR-based genome engineering facilitates precise isogenic comparisons of variants across genetic backgrounds. Although functional-validation studies are typically performed on one variant in isolation and in one cell type at a time, complex genetic diseases require multiplexed gene perturbations to interrogate combinations of genes and resolve physiologically relevant disease biology. Our aim is to discuss advances at the intersection of genomics, hiPSCs and CRISPR. A better understanding of the molecular mechanisms underlying disease risk will improve genetic diagnosis, drive phenotypic drug discovery and pave the way toward precision medicine.


Assuntos
Encefalopatias/genética , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genoma/genética , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Medicina de Precisão/métodos
14.
Phytomedicine ; 69: 153198, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32151917

RESUMO

BACKGROUND: Induced pluripotent stem cells (iPSCs) are regarded as the best potential cell source for cell-based regenerative medicine. To develop a safe and efficient iPSC-based cell therapy, it is very important to avoid possible teratoma formation, which can arise from undifferentiated iPSCs (USCs) remaining among differentiated cell products. Dried bark of Magnolia officinalis (Magnolia cortex, MC) has long been used in traditional medicine to treat gastrointestinal ailments and allergic diseases, and has shown have various pharmacological activities, including anti-bacterial, anti-inflammatory, and anti-cancer effects. However, its effects on iPSCs have not yet been examined. PURPOSE: In this study, we investigated the selective cytotoxic effects of ethanol extract of MC (EEMC) on undifferentiated iPSCs and elucidated the underlying apoptotic mechanisms in detail. We also investigated the inhibitory effects of EEMC on teratoma formation via in ovo experiments. RESULTS: We found that EEMC greatly reduced cell growth and induced apoptotic cell death in USCs, but not in differentiated or normal cells. EEMC caused G2/M cell cycle arrest, mitochondrial damage, and caspase activation of USCs, accompanied by p53 accumulation. In p53KO human iPSCs, EEMC had no cytotoxicity, reinforcing that EEMC-mediated apoptosis of USCs is p53-dependent. EEMC did not cause DNA damage in iPSC-derived differentiated cells. In ovo teratoma formation assay revealed that EEMC treatment before injection efficiently eliminated USCs and prevented teratoma formation. CONCLUSIONS: These results collectively indicate that EEMC has potent anti-teratoma activity, and therefore can be used for the development of safe iPSC-based therapy.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Magnolia/química , Extratos Vegetais/farmacologia , Teratoma/prevenção & controle , Animais , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Etanol/química , Hepatócitos/citologia , Hepatócitos/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Extratos Vegetais/química , Teratoma/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
15.
Anesthesiology ; 132(5): 1062-1079, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32149777

RESUMO

BACKGROUND: Overactivation of ryanodine receptors and the resulting impaired calcium homeostasis contribute to Alzheimer's disease-related pathophysiology. This study hypothesized that exposing neuronal progenitors derived from induced pluripotent stems cells of patients with Alzheimer's disease to dantrolene will increase survival, proliferation, neurogenesis, and synaptogenesis. METHODS: Induced pluripotent stem cells obtained from skin fibroblast of healthy subjects and patients with familial and sporadic Alzheimer's disease were used. Biochemical and immunohistochemical methods were applied to determine the effects of dantrolene on the viability, proliferation, differentiation, and calcium dynamics of these cells. RESULTS: Dantrolene promoted cell viability and proliferation in these two cell lines. Compared with the control, differentiation into basal forebrain cholinergic neurons significantly decreased by 10.7% (32.9 ± 3.6% vs. 22.2 ± 2.6%, N = 5, P = 0.004) and 9.2% (32.9 ± 3.6% vs. 23.7 ± 3.1%, N = 5, P = 0.017) in cell lines from sporadic and familial Alzheimer's patients, respectively, which were abolished by dantrolene. Synapse density was significantly decreased in cortical neurons generated from stem cells of sporadic Alzheimer's disease by 58.2% (237.0 ± 28.4 vs. 99.0 ± 16.6 arbitrary units, N = 4, P = 0.001) or familial Alzheimer's disease by 52.3% (237.0 ± 28.4 vs.113.0 ± 34.9 vs. arbitrary units, N = 5, P = 0.001), which was inhibited by dantrolene in the familial cell line. Compared with the control, adenosine triphosphate (30 µM) significantly increased higher peak elevation of cytosolic calcium concentrations in the cell line from sporadic Alzheimer's patients (84.1 ± 27.0% vs. 140.4 ± 40.2%, N = 5, P = 0.049), which was abolished by the pretreatment of dantrolene. Dantrolene inhibited the decrease of lysosomal vacuolar-type H-ATPase and the impairment of autophagy activity in these two cell lines from Alzheimer's disease patients. CONCLUSIONS: Dantrolene ameliorated the impairment of neurogenesis and synaptogenesis, in association with restoring intracellular Ca homeostasis and physiologic autophagy, cell survival, and proliferation in induced pluripotent stem cells and their derived neurons from sporadic and familial Alzheimer's disease patients.


Assuntos
Doença de Alzheimer , Dantroleno/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Relaxantes Musculares Centrais/farmacologia , Neurogênese/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Adulto , Doença de Alzheimer/patologia , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Masculino , Pessoa de Meia-Idade , Neurogênese/fisiologia , Distribuição Aleatória , Sinapses/fisiologia
17.
Rev Mal Respir ; 37(3): 197-200, 2020 Mar.
Artigo em Francês | MEDLINE | ID: mdl-32146059

RESUMO

Chronic obstructive pulmonary disease (COPD) is a chronic lung disease leading to irreversible destruction of the terminal bronchioles. Although the precise patho-physiological mechanisms remain to be elucidated, the bronchial epithelium seems to play a pivotal role in the disease. Recent studies have highlighted a great heterogeneity among COPD patients, with various disease courses including, in about half the cases, an origin in childhood. Modelling of COPD is a major goal but currently available models are imperfect. Our work aims to create a new in vitro cellular model to study the pathology of the disease. The differentiation of human induced pluripotential stem cells (hiPSCs) in bronchial epithelium is a step towards a better understanding of the developmental origin and the identification of new therapeutic targets.


Assuntos
Modelos Animais de Doenças , Células-Tronco Pluripotentes Induzidas/fisiologia , Doença Pulmonar Obstrutiva Crônica/patologia , Mucosa Respiratória/patologia , Animais , Diferenciação Celular/fisiologia , Progressão da Doença , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Camundongos , Ratos , Mucosa Respiratória/citologia
18.
J Neurosci ; 40(6): 1186-1193, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-32024767

RESUMO

In vitro differentiation of pluripotent stem cells provides a systematic platform to study development and disease. Recent advances in brain organoid technology have created new opportunities to investigate the formation and function of the human brain, under physiological and pathological conditions. Brain organoids can be generated to model the cellular and structural development of the human brain, and allow the investigation of the intricate interactions between resident neural and glial cell types. Combined with new advances in gene editing, imaging, and genomic analysis, brain organoid technology can be applied to address questions pertinent to human brain development, disease, and evolution. However, the current iterations of brain organoids also have limitations in faithfully recapitulating the in vivo processes. In this perspective, we evaluate the recent progress in brain organoid technology, and discuss the experimental considerations for its utilization.Dual Perspectives Companion Paper: Integrating CRISPR Engineering and hiPSC-Derived 2D Disease Modeling Systems, by Kristina Rehbach, Michael B. Fernando, and Kristen J. Brennand.


Assuntos
Encéfalo/fisiologia , Organoides , Encéfalo/embriologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia
19.
Invest Ophthalmol Vis Sci ; 61(2): 9, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32049341

RESUMO

Purpose: Variant B precursor cysteine protease inhibitor cystatin C, a known recessive risk factor for developing exudative age-related macular degeneration (AMD), presents altered intracellular trafficking and reduced secretion from retinal pigment epithelial (RPE) cells. Because cystatin C inhibits multiple extracellular matrix (ECM)-degrading cathepsins, this study evaluated the role of this mutation in inducing ECM-related functional changes in RPE cellular behavior. Methods: Induced pluripotent stem cells gene-edited bi-allelically by CRISPR/Cas9 to express the AMD-linked cystatin C variant were differentiated to RPE cells and assayed for their ability to degrade fluorescently labeled ECM proteins. Cellular migration and adhesion on multiple ECM proteins, differences in transepithelial resistance and polarized protein secretion were tested. Vessel formation induced by gene edited cells-conditioned media was quantified using primary human dermal microvascular epithelial cells. Results: Variant B cystatin C-expressing induced pluripotent stem cells-derived RPE cells displayed a significantly higher rate of laminin and fibronectin degradation 3 days after seeding on fluorescently labeled ECM (P < 0.05). Migration on matrigel, collagen IV and fibronectin was significantly faster for edited cells compared with wild-type (WT) cells. Both edited and WT cells displayed polarized secretion of cystatin C, but transepithelial resistance was lower in gene-edited cells after 6 weeks culture, with significantly lower expression of tight junction protein claudin-3. Media conditioned by gene-edited cells stimulated formation of significantly longer microvascular tubes (P < 0.05) compared with WT-conditioned media. Conclusions: Reduced levels of cystatin C lead to changes in the RPE ability to degrade, adhere, and migrate supporting increased invasiveness and angiogenesis relevant for AMD pathology.


Assuntos
Cistatina C/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Degeneração Macular/patologia , Epitélio Pigmentado da Retina/citologia , Movimento Celular/fisiologia , Células Cultivadas , Cistatina C/genética , Cistatina C/metabolismo , Fibronectinas/metabolismo , Edição de Genes , Humanos , Laminina/metabolismo , Mutação Puntual/genética
20.
Nat Commun ; 11(1): 764, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034154

RESUMO

Our understanding of the signalling pathways regulating early human development is limited, despite their fundamental biological importance. Here, we mine transcriptomics datasets to investigate signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors, along with IGF1 ligand. Consequently, we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions, we derive several pluripotent stem cell lines that express pluripotency-associated genes, retain high viability and a normal karyotype, and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos, and in both primed and naïve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche.


Assuntos
Autorrenovação Celular/fisiologia , Células-Tronco Embrionárias Humanas/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Ativinas/metabolismo , Animais , Blastocisto/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura/química , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Endoderma/citologia , Endoderma/metabolismo , Membranas Extraembrionárias/citologia , Membranas Extraembrionárias/metabolismo , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma , Inativação do Cromossomo X/fisiologia
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