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1.
J Vis Exp ; (208)2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38912821

RESUMO

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem cells (iPSCs) under specific conditions. Synapse development and maturation in ROs have been studied immunocytochemically and functionally. However, the direct evidence of the synaptic contact ultrastructure is limited, containing both special ribbon synapses and conventional chemical synapses. Transmission electron microscopy (TEM) is characterized by high resolution and a respectable history elucidating retinal development and synapse maturation in humans and various species. It is a powerful tool to explore synaptic structure in ROs and is widely used in the research field of ROs. Therefore, to better explore the structure of RO synaptic contacts at the nanoscale and obtain high-quality microscopic evidence, we developed a simple and repeatable method of RO TEM sample preparation. This paper describes the protocol, reagents used, and detailed steps, including RO fixation preparation, post fixation, embedding, and visualization.


Assuntos
Microscopia Eletrônica de Transmissão , Organoides , Retina , Organoides/ultraestrutura , Organoides/citologia , Retina/citologia , Retina/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Animais , Sinapses/ultraestrutura
2.
Cells ; 10(11)2021 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-34831042

RESUMO

BACKGROUND: Liver transplantation remains the only viable therapy for liver failure but has a severely restricted utility. Here, we aimed to decellularize rat livers to form acellular 3D bio-scaffolds suitable for seeding with induced pluripotent cells (iPSCs) as a tool to investigate the role of Wnt/ß-catenin signaling in liver development and generation. METHODS: Dissected rat livers were randomly divided into three groups: I (control); II (decellularized scaffolds) and III (recellularized scaffolds). Liver decellularization was established via an adapted perfusion procedure and assessed through the measurement of extracellular matrix (ECM) proteins and DNA content. Liver recellularization was assessed through histological examination and measurement of transcript levels of Wnt/ß-catenin pathway, hepatogenesis, liver-specific microRNAs and growth factors essential for liver development. Adult rat liver decellularization was confirmed by the maintenance of ECM proteins and persistence of growth factors essential for liver regeneration. RESULTS: iPSCs seeded rat decellularized livers displayed upregulated transcript expression of Wnt/ß-catenin pathway-related, growth factors, and liver specification genes. Further, recellularized livers displayed restored liver-specific functions including albumin secretion and urea synthesis. CONCLUSION: This establishes proof-of-principle for the generation of three-dimensional liver organ scaffolds as grafts and functional re-establishment.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Fígado/citologia , Alicerces Teciduais/química , Regulação para Cima , Via de Sinalização Wnt , Albuminas/metabolismo , Animais , Diferenciação Celular , Hepatócitos/citologia , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Masculino , Ratos , Ureia/metabolismo , alfa-Fetoproteínas/metabolismo , beta Catenina/metabolismo
3.
Elife ; 102021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34590578

RESUMO

Astrocytes are essential cells of the central nervous system, characterized by dynamic relationships with neurons that range from functional metabolic interactions and regulation of neuronal firing activities, to the release of neurotrophic and neuroprotective factors. In Parkinson's disease (PD), dopaminergic neurons are progressively lost during the course of the disease, but the effects of PD on astrocytes and astrocyte-to-neuron communication remain largely unknown. This study focuses on the effects of the PD-related mutation LRRK2 G2019S in astrocytes generated from patient-derived induced pluripotent stem cells. We report the alteration of extracellular vesicle (EV) biogenesis in astrocytes and identify the abnormal accumulation of key PD-related proteins within multivesicular bodies (MVBs). We found that dopaminergic neurons internalize astrocyte-secreted EVs and that LRRK2 G2019S EVs are abnormally enriched in neurites and fail to provide full neurotrophic support to dopaminergic neurons. Thus, dysfunctional astrocyte-to-neuron communication via altered EV biological properties may participate in the progression of PD.


Assuntos
Astrócitos/enzimologia , Comunicação Celular , Neurônios Dopaminérgicos/enzimologia , Exossomos/enzimologia , Células-Tronco Pluripotentes Induzidas/enzimologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Células-Tronco Neurais/enzimologia , Doença de Parkinson/enzimologia , Animais , Astrócitos/ultraestrutura , Atrofia , Estudos de Casos e Controles , Linhagem Celular , Neurônios Dopaminérgicos/patologia , Endocitose , Exossomos/genética , Exossomos/ultraestrutura , Humanos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Células-Tronco Neurais/ultraestrutura , Biogênese de Organelas , Doença de Parkinson/genética , Doença de Parkinson/patologia
4.
Sci Rep ; 11(1): 18819, 2021 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-34552130

RESUMO

Nanostructured cell culture substrates featuring nanowire (NW) arrays have been applied to a variety of basic cell lines and rodent neurons to investigate cellular behavior or to stimulate cell responses. However, patient-derived human neurons-a prerequisite for studying e.g. neurodegenerative diseases efficiently-are rarely employed due to sensitive cell culture protocols and usually long culturing periods. Here, we present human patient induced pluripotent stem cell-derived neurons cultured on densely-spaced spiky silicon NW arrays (600 NWs/ 100 µm[Formula: see text] with NW lengths of 1 µm) which show mature electrophysiological characteristics after only 20 days of culturing. Exemplary neuronal growth and network formation on the NW arrays are demonstrated using scanning electron microscopy and immunofluorescence microscopy. The cells and neurites rest in a fakir-like settling state on the NWs only in contact with the very NW tips shown by cross-sectional imaging of the cell/NW interface using focused ion beam milling and confocal laser scanning microscopy. Furthermore, the NW arrays promote the cell culture by slightly increasing the share of differentiated neurons determined by the quantification of immunofluorescence microscopy images. The electrophysiological functionality of the neurons is confirmed with patch-clamp recordings showing the excellent capability to fire action potentials. We believe that the short culturing time to obtain functional human neurons generated from patient-derived neural progenitor cells and the robustness of this differentiation protocol to produce these neurons on densely-spaced spiky nanowire arrays open up new pathways for stem cell characterization and neurodegenerative disease studies.


Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/fisiologia , Nanofios , Células-Tronco Neurais/fisiologia , Silício , Humanos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Células-Tronco Neurais/ultraestrutura
5.
Cell Rep Med ; 2(7): 100345, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34337561

RESUMO

Hereditary sensory neuropathy type 1 (HSN1) is caused by mutations in the SPTLC1 or SPTLC2 sub-units of the enzyme serine palmitoyltransferase, resulting in the production of toxic 1-deoxysphingolipid bases (DSBs). We used induced pluripotent stem cells (iPSCs) from patients with HSN1 to determine whether endogenous DSBs are neurotoxic, patho-mechanisms of toxicity and response to therapy. HSN1 iPSC-derived sensory neurons (iPSCdSNs) endogenously produce neurotoxic DSBs. Complex gangliosides, which are essential for membrane micro-domains and signaling, are reduced, and neurotrophin signaling is impaired, resulting in reduced neurite outgrowth. In HSN1 myelinating cocultures, we find a major disruption of nodal complex proteins after 8 weeks, which leads to complete myelin breakdown after 6 months. HSN1 iPSC models have, therefore, revealed that SPTLC1 mutation alters lipid metabolism, impairs the formation of complex gangliosides, and reduces axon and myelin stability. Many of these changes are prevented by l-serine supplementation, supporting its use as a rational therapy.


Assuntos
Axônios/metabolismo , Gangliosídeos/metabolismo , Neuropatias Hereditárias Sensoriais e Autônomas/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Modelos Biológicos , Neuroglia/metabolismo , Serina/farmacologia , Envelhecimento/patologia , Axônios/efeitos dos fármacos , Axônios/ultraestrutura , Sequência de Bases , Caspase 3/metabolismo , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Humanos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/ultraestrutura , Bainha de Mielina/metabolismo , Fatores de Crescimento Neural/metabolismo , Neuroglia/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Proteína Nodal/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologia , Células Receptoras Sensoriais/ultraestrutura , Transdução de Sinais/efeitos dos fármacos , Esfingolipídeos/metabolismo , Transcriptoma/genética
6.
Sci Rep ; 11(1): 17028, 2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34426605

RESUMO

In order to circumvent the limited access and donor variability of human primary alveolar cells, directed differentiation of human pluripotent stem cells (hiPSCs) into alveolar-like cells, provides a promising tool for respiratory disease modeling and drug discovery assays. In this work, a unique, miniaturized 96-Transwell microplate system is described where hiPSC-derived alveolar-like cells were cultured at an air-liquid interface (ALI). To this end, hiPSCs were differentiated into lung epithelial progenitor cells (LPCs) and subsequently matured into a functional alveolar type 2 (AT2)-like epithelium with monolayer-like morphology. AT2-like cells cultured at the physiological ALI conditions displayed characteristics of AT2 cells with classical alveolar surfactant protein expressions and lamellar-body like structures. The integrity of the epithelial barriers between the AT2-like cells was confirmed by applying a custom-made device for 96-parallelized transepithelial electric resistance (TEER) measurements. In order to generate an IPF disease-like phenotype in vitro, the functional AT2-like cells were stimulated with cytokines and growth factors present in the alveolar tissue of IPF patients. The cytokines stimulated the secretion of pro-fibrotic biomarker proteins both on the mRNA (messenger ribonucleic acid) and protein level. Thus, the hiPSC-derived and cellular model system enables the recapitulation of certain IPF hallmarks, while paving the route towards a miniaturized medium throughput approach of pharmaceutical drug discovery.


Assuntos
Ar , Técnicas de Cultura de Células , Células-Tronco Pluripotentes Induzidas/citologia , Miniaturização , Modelos Biológicos , Alvéolos Pulmonares/citologia , Biomarcadores/metabolismo , Diferenciação Celular , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Fenótipo , Alvéolos Pulmonares/ultraestrutura , Fibrose Pulmonar/patologia , Transcrição Gênica
7.
Cell Biol Int ; 45(11): 2238-2250, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34288224

RESUMO

Even though rats are popular model animals, the ultrastructure of their pluripotent cells, that is, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), remains unexplored, although fine structure of pluripotent stem cells of mice and humans and its changes during differentiation have been investigated well. In the present study, we carried out ultrastructural and morphometric analyses of three lines of rat ESCs and two lines of rat iPSCs. The rat pluripotent stem cells were found to have the main typical morphological features of pluripotent cells: large nuclei of irregular or nearly round shape, scanty cytoplasm with few membrane organelles, and a poorly developed Golgi apparatus and endoplasmic reticulum. The cytoplasm of the rat pluripotent cells contains clusters of glycogen, previously described in human ESCs. To identify possible differences between rat ESCs and iPSCs, we performed a morphometric analysis of cell parameters. The mean area of cells and nuclei, the nuclear/cytoplasmic ratio, distributions of glycogen and diversity of mitochondria showed marked variations among the lines of rat pluripotent stem cells and were more pronounced than variations between rat ESCs and iPSCs as separate types of pluripotent stem cells. We noted morphological heterogeneity of the mitochondrial population in the rat pluripotent stem cells. The cells contained three types of mitochondria differing in the structure of cristae and in matrix density, and our morphometric analysis revealed differences in cristae structure.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Mitocôndrias/metabolismo , Animais , Diferenciação Celular/fisiologia , Núcleo Celular/patologia , Células-Tronco Embrionárias/ultraestrutura , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Células-Tronco Pluripotentes/citologia , Ratos
8.
Int J Mol Sci ; 22(4)2021 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-33672445

RESUMO

Hereditary retinal dystrophies (HRD) represent a significant cause of blindness, affecting mostly retinal pigment epithelium (RPE) and photoreceptors (PRs), and currently suffer from a lack of effective treatments. Highly specialized RPE and PR cells interact mutually in the functional retina, therefore primary HRD affecting one cell type leading to a secondary HRD in the other cells. Phagocytosis is one of the primary functions of the RPE and studies have discovered that mutations in the phagocytosis-associated gene Mer tyrosine kinase receptor (MERTK) lead to primary RPE dystrophy. Treatment strategies for this rare disease include the replacement of diseased RPE with healthy autologous RPE to prevent PR degeneration. The generation and directed differentiation of patient-derived human-induced pluripotent stem cells (hiPSCs) may provide a means to generate autologous therapeutically-relevant adult cells, including RPE and PR. However, the continued presence of the MERTK gene mutation in patient-derived hiPSCs represents a significant drawback. Recently, we reported the generation of a hiPSC model of MERTK-associated Retinitis Pigmentosa (RP) that recapitulates disease phenotype and the subsequent creation of gene-corrected RP-hiPSCs using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9. In this study, we differentiated gene-corrected RP-hiPSCs into RPE and found that these cells had recovered both wild-type MERTK protein expression and the lost phagocytosis of fluorescently-labeled photoreceptor outer segments observed in uncorrected RP-hiPSC-RPE. These findings provide proof-of-principle for the utility of gene-corrected hiPSCs as an unlimited cell source for personalized cell therapy of rare vision disorders.


Assuntos
Edição de Genes , Células-Tronco Pluripotentes Induzidas/patologia , Fagocitose , Epitélio Pigmentado da Retina/patologia , Retinose Pigmentar/patologia , Diferenciação Celular/genética , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Mutação/genética , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/patologia , Segmento Externo das Células Fotorreceptoras da Retina/ultraestrutura , Epitélio Pigmentado da Retina/ultraestrutura , Retinose Pigmentar/genética , c-Mer Tirosina Quinase/genética , c-Mer Tirosina Quinase/metabolismo
9.
Am J Physiol Heart Circ Physiol ; 320(4): H1670-H1686, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33606581

RESUMO

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) may provide an important bridge between animal models and the intact human myocardium. Fulfilling this potential is hampered by their relative immaturity, leading to poor physiological responsiveness. hiPSC-CMs grown in traditional two-dimensional (2D) culture lack a t-tubular system, have only rudimentary intracellular calcium-handling systems, express predominantly embryonic sarcomeric protein isoforms, and preferentially use glucose as an energy substrate. Culturing hiPSC-CM in a variety of three-dimensional (3D) environments and the addition of nutritional, pharmacological, and electromechanical stimuli have proven, to various degrees, to be beneficial for maturation. We present a detailed assessment of a novel model in which hiPSC-CMs and hiPSC-derived cardiac fibroblasts are cocultured in a 3D fibrin matrix to form engineered cardiac tissue constructs (hiPSC-ECTs). The hiPSC-ECTs are responsive to physiological stimuli, including stretch, frequency, and ß-adrenergic stimulation, develop a t-tubular system, and demonstrate calcium-handling and contractile kinetics that compare favorably with ventricular human myocardium. Furthermore, transcript levels of various genes involved in calcium-handling and contraction are increased. These markers of maturation become more robust over a relatively short period of time in culture (6 wk vs. 2 wk in hiPSC-ECTs). A comparison of the hiPSC-ECT molecular and performance variables with those of human cardiac tissue and other available engineered tissue platforms is provided to aid selection of the most appropriate platform for the research question at hand. Important and noteworthy aspects of this human cardiac model system are its reliance on "off-the-shelf" equipment, ability to provide detailed physiological performance data, and the ability to achieve a relatively mature cardiac physiology without additional nutritional, pharmacological, and electromechanical stimuli that may elicit unintended effects on function.NEW & NOTEWORTHY This study seeks to provide an in-depth assessment of contractile performance of human iPSC-derived cardiomyocytes cultured together with fibroblasts in a 3-dimensional-engineered tissue and compares performance both over time as cells mature, and with corresponding measures found in the literature using alternative 3D culture configurations. The suitability of 3D-engineered human cardiac tissues to model cardiac function is emphasized, and data provided to assist in the selection of the most appropriate configuration based on the target application.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Engenharia Tecidual , Agonistas Adrenérgicos beta/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Cinética , Contração Miocárdica , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/ultraestrutura , Fenótipo
10.
Am J Physiol Heart Circ Physiol ; 320(3): H954-H968, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33416449

RESUMO

Exosomes are an important mechanism of cell-cell interaction in the cardiovascular system, both in maintaining homeostasis and in stress response. Interindividual differences that alter content in exosomes may play a role in cardiovascular disease pathology. To study the effect of interindividual cardiomyocyte (CM) variation, we characterized exosomal content in phenotypically diverse human induced pluripotent stem cell-derived CMs (hiPSC-CMs). Cell lines were generated from six participants in the HyperGEN cohort: three with left ventricular hypertrophy (LVH) and three with normal left ventricular mass (LVM). Sequence analysis of the intracellular and exosomal RNA populations showed distinct expression pattern differences between hiPSC-CM lines derived from individuals with LVH and those with normal LVM. Functional analysis of hiPSC-endothelial cells (hiPSC-ECs) treated with exosomes from both hiPSC-CM groups showed significant variation in response, including differences in tube formation, migration, and proliferation. Overall, treatment of hiPSC-ECs with exosomes resulted in significant expression changes associated with angiogenesis and endothelial cell vasculogenesis. However, the hiPSC-ECs treated with exosomes from the LVH-affected donors exhibited significantly increased proliferation but decreased tube formation and migration, suggesting angiogenic dysregulation.NEW & NOTEWORTHY The intracellular RNA and the miRNA content in exosomes are significantly different in hiPSC-CMs derived from LVH-affected individuals compared with those from unaffected individuals. Treatment of endothelial cells with these exosomes functionally affects cellular phenotypes in a donor-specific manner. These findings provide novel insight into underlying mechanisms of hypertrophic cell signaling between different cell types. With a growing interest in stem cells and exosomes for cardiovascular therapeutic use, this also provides information important for regenerative medicine.


Assuntos
Diferenciação Celular , Exossomos/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Neovascularização Fisiológica , Doadores de Tecidos , Adulto , Idoso , Estudos de Casos e Controles , Movimento Celular , Proliferação de Células , Separação Celular , Células Cultivadas , Exossomos/genética , Exossomos/ultraestrutura , Feminino , Regulação da Expressão Gênica , Humanos , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/patologia , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Miócitos Cardíacos/ultraestrutura , Neovascularização Fisiológica/genética , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Transcriptoma
11.
Methods Mol Biol ; 2258: 151-169, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33340360

RESUMO

Self-assembling brain spheroids derived from human stem cells closely emulate the tangled connectivity of the human brain, recapitulate aspects of organized tissue structure, and are relatively easy to manipulate compared to other existing three-dimensional (3D) cellular models. However, current platforms generate heterogeneously sized and short-lived spheroids, which do not robustly and reproducibly model human brain development and diseases. Here, we present a method to generate large-scale arrays of homogeneously sized 3D brain spheroids derived from human-induced pluripotent stem cells (hiPSCs) or immortalized neural progenitor cells to recapitulate Alzheimer's disease (AD) pathology in vitro. When embedded in extracellular matrix, these brain spheroids develop extensive outward projection of neurites and form networks, which are mediated by thick bundles of dendrites. This array facilitates cost-effective, high-throughput drug screening and mechanistic studies to better understand human brain development and neurodegenerative conditions, such as AD .


Assuntos
Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Neurais/fisiologia , Neurogênese , Neurônios/fisiologia , Engenharia Tecidual , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Técnicas de Cultura de Células , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Microscopia Eletrônica de Varredura , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/ultraestrutura , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Organoides
12.
J Vis Exp ; (166)2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33346186

RESUMO

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical movement. The architecture of the NMJ structure influences the functions of the neuron, the muscle and the mutual interaction. Previous studies have reported many strategies by co-culturing the motor neurons and myotubes to generate NMJ in vitro with complex induction process and long culture period but have struggled to recapitulate mature NMJ morphology and function. Our in vitro NMJ induction system is constructed by differentiating human iPSC in a single culture dish. By switching the myogenic and neurogenic induction medium for induction, the resulting NMJ contained pre- and post- synaptic components, including motor neurons, skeletal muscle and Schwann cells in the one month culture. The functional assay of NMJ also showed that the myotubes contraction can be triggered by Ca++ then inhibited by curare, an acetylcholine receptor (AChR) inhibitor, in which the stimulating signal is transmitted through NMJ. This simple and robust approach successfully derived the complex structure of NMJ with functional connectivity. This in vitro human NMJ, with its integrated structures and function, has promising potential for studying pathological mechanisms and compound screening.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Junção Neuromuscular/citologia , Animais , Curare , Matriz Extracelular/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Junção Neuromuscular/ultraestrutura , Células de Schwann/citologia , Células de Schwann/metabolismo
13.
Nat Med ; 26(12): 1888-1898, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32989314

RESUMO

22q11.2 deletion syndrome (22q11DS) is a highly penetrant and common genetic cause of neuropsychiatric disease. Here we generated induced pluripotent stem cells from 15 individuals with 22q11DS and 15 control individuals and differentiated them into three-dimensional (3D) cerebral cortical organoids. Transcriptional profiling across 100 days showed high reliability of differentiation and revealed changes in neuronal excitability-related genes. Using electrophysiology and live imaging, we identified defects in spontaneous neuronal activity and calcium signaling in both organoid- and 2D-derived cortical neurons. The calcium deficit was related to resting membrane potential changes that led to abnormal inactivation of voltage-gated calcium channels. Heterozygous loss of DGCR8 recapitulated the excitability and calcium phenotypes and its overexpression rescued these defects. Moreover, the 22q11DS calcium abnormality could also be restored by application of antipsychotics. Taken together, our study illustrates how stem cell derived models can be used to uncover and rescue cellular phenotypes associated with genetic forms of neuropsychiatric disease.


Assuntos
Sinalização do Cálcio/genética , Córtex Cerebral/ultraestrutura , Síndrome de DiGeorge/diagnóstico , Neurônios/ultraestrutura , Adulto , Diferenciação Celular/genética , Córtex Cerebral/patologia , Síndrome de DiGeorge/patologia , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Masculino , Neurônios/patologia , Organoides/patologia , Organoides/ultraestrutura , Adulto Jovem
14.
Oxid Med Cell Longev ; 2020: 6821247, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32855765

RESUMO

Riboflavin transporter deficiency (RTD) is a childhood-onset neurodegenerative disorder characterized by progressive pontobulbar palsy, sensory and motor neuron degeneration, sensorineural hearing loss, and optic atrophy. As riboflavin (RF) is the precursor of FAD and FMN, we hypothesize that both mitochondrial and peroxisomal energy metabolism pathways involving flavoproteins could be directly affected in RTD, thus impacting cellular redox status. In the present work, we used induced pluripotent stem cells (iPSCs) from RTD patients to investigate morphofunctional features, focusing on mitochondrial and peroxisomal compartments. Using this model, we document the following RTD-associated alterations: (i) abnormal colony-forming ability and loss of cell-cell contacts, revealed by light, electron, and confocal microscopy, using tight junction marker ZO-1; (ii) mitochondrial ultrastructural abnormalities, involving shape, number, and intracellular distribution of the organelles, as assessed by focused ion beam/scanning electron microscopy (FIB/SEM); (iii) redox imbalance, with high levels of superoxide anion, as assessed by MitoSOX assay accompanied by abnormal mitochondrial polarization state, evaluated by JC-1 staining; (iv) altered immunofluorescence expression of antioxidant systems, namely, glutathione, superoxide dismutase 1 and 2, and catalase, as assessed by quantitatively evaluated confocal microscopy; and (v) peroxisomal downregulation, as demonstrated by levels and distribution of fatty acyl ß-oxidation enzymes. RF supplementation results in amelioration of cell phenotype and rescue of redox status, which was associated to improved ultrastructural features of mitochondria, thus strongly supporting patient treatment with RF, to restore mitochondrial- and peroxisomal-related aspects of energy dysmetabolism and oxidative stress in RTD syndrome.


Assuntos
Metabolismo Energético , Mitocôndrias/metabolismo , Peroxissomos/metabolismo , Receptores Acoplados a Proteínas G/deficiência , Riboflavina/metabolismo , Antioxidantes/metabolismo , Sequência de Bases , Benzimidazóis/metabolismo , Transporte Biológico , Carbocianinas/metabolismo , Forma Celular , Criança , Pré-Escolar , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Mitocôndrias/ultraestrutura , Oxirredução , Receptores Acoplados a Proteínas G/metabolismo
15.
Dis Model Mech ; 13(6)2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32423971

RESUMO

The basal lamina is a specialized sheet of dense extracellular matrix (ECM) linked to the plasma membrane of specific cell types in their tissue context, which serves as a structural scaffold for organ genesis and maintenance. Disruption of the basal lamina and its functions is central to many disease processes, including cancer metastasis, kidney disease, eye disease, muscular dystrophies and specific types of brain malformation. The latter three pathologies occur in the α-dystroglycanopathies, which are caused by dysfunction of the ECM receptor α-dystroglycan. However, opportunities to study the basal lamina in various human disease tissues are restricted owing to its limited accessibility. Here, we report the generation of embryoid bodies from human induced pluripotent stem cells that model the basal lamina. Embryoid bodies cultured via this protocol mimic pre-gastrulation embryonic development, consisting of an epithelial core surrounded by a basal lamina and a peripheral layer of ECM-secreting endoderm. In α-dystroglycanopathy patient embryoid bodies, electron and fluorescence microscopy reveal ultrastructural basal lamina defects and reduced ECM accumulation. By starting from patient-derived cells, these results establish a method for the in vitro synthesis of patient-specific basal lamina and recapitulate disease-relevant ECM defects seen in the α-dystroglycanopathies. Finally, we apply this system to evaluate an experimental ribitol supplement therapy on genetically diverse α-dystroglycanopathy patient samples.This article has an associated First Person interview with the first author of the paper.


Assuntos
Membrana Basal/metabolismo , Corpos Embrioides/metabolismo , Matriz Extracelular/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Síndrome de Walker-Warburg/metabolismo , Membrana Basal/efeitos dos fármacos , Membrana Basal/ultraestrutura , Estudos de Casos e Controles , Técnicas de Cultura de Células , Células Cultivadas , Criança , Pré-Escolar , Distroglicanas/genética , Distroglicanas/metabolismo , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/ultraestrutura , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/ultraestrutura , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/ultraestrutura , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Ribitol/farmacologia , Síndrome de Walker-Warburg/tratamento farmacológico , Síndrome de Walker-Warburg/genética , Síndrome de Walker-Warburg/patologia
16.
Cells ; 9(5)2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32369990

RESUMO

We present a hiPSC-based 3D in vitro system suitable to test neurotoxicity (NT). Human iPSCs-derived 3D neurospheres grown in 96-well plate format were characterized timewise for 6-weeks. Changes in complexity and homogeneity were followed by immunocytochemistry and transmission electron microscopy. Transcriptional activity of major developmental, structural, and cell-type-specific markers was investigated at weekly intervals to present the differentiation of neurons, astrocytes, and oligodendrocytes. Neurospheres were exposed to different well-known toxicants with or without neurotoxic effect (e.g., paraquat, acrylamide, or ibuprofen) and examined at various stages of the differentiation with an ATP-based cell viability assay optimized for 3D-tissues. Concentration responses were investigated after acute (72 h) exposure. Moreover, the compound-specific effect of rotenone was investigated by a panel of ER-stress assay, TUNEL assay, immunocytochemistry, electron microscopy, and in 3D-spheroid based neurite outgrowth assay. The acute exposure to different classes of toxicants revealed distinct susceptibility profiles in a differentiation stage-dependent manner, indicating that hiPSC-based 3D in vitro neurosphere models could be used effectively to evaluate NT, and can be developed further to detect developmental neurotoxicity (DNT) and thus replace or complement the use of animal models in various basic research and pharmaceutical applications.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/citologia , Síndromes Neurotóxicas/diagnóstico , Esferoides Celulares/citologia , Biomarcadores/metabolismo , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Modelos Biológicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Rotenona/toxicidade , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/ultraestrutura
17.
Cell Physiol Biochem ; 54(2): 271-286, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32233339

RESUMO

BACKGROUND/AIMS: Pericytes (PCs) are multipotent vascular precursors that play a critical physiological role in the development and maintenance of blood vessel integrity. In this study, we aim to characterize PCs isolated from human abdominal adipose tissue and develop an integration-free induced pluripotent stem cells (iPSCs) using episomal vectors. METHODS: The ultrastructure of adipose tissue-derived PCs was determined using scanning and transmission electron microscopy. The expression of mesenchymal stem cells (MSCs) and pericyte markers were examined using flow cytometry and PCR analysis. PCs were induced to adipogenic, osteogenic and myogenic lineages, and their angiogenic potential was determined using tube formation assay. We further established pericyte reprogramming protocol using episomal vectors. RESULTS: Our data showed that human adipose tissue-derived PCs uniformly expressed MSCs, CD105 and CD73, and PCs markers, desmin, and alpha smooth muscle actin (α-SMA), while lacked the expression of HLA-DR and the hematopoietic markers CD34, CD11b and CD45. Ultrastructure analysis showed typical internal structure for the PCs with a characteristic prominent eccentric nuclei and cytoplasmic invaginations forming a caveolar system. Functional analysis showed efficient differentiation into adipocytes, osteocytes, and myocyte-like cells. Adipose tissue-derived PCs showed angiogenic potential using tube-forming assay. To determine further application of these cells for personalized therapy, we reprogrammed PCs into induced pluripotent stem cells (iPSCs) using episomal vectors. Reprogrammed cells gradually lost their fusiform shape, acquired the epithelial cell morphology and formed colonies. Furthermore, reprogrammed cells successfully expressed the pluripotency markers OCT4, Nanog, SSEA-4, and ß-catenin, an early reprogramming marker. CONCLUSION: The accessibility and abundance of human fat supports the application of adipose derived PCs as a novel and promising source of cell therapy and regenerative medicine.


Assuntos
Tecido Adiposo/citologia , Técnicas de Reprogramação Celular/métodos , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Pericitos/citologia , 5'-Nucleotidase/metabolismo , Actinas/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Tecido Adiposo/ultraestrutura , Linhagem da Célula , Células Cultivadas , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Desmina/metabolismo , Endoglina/metabolismo , Citometria de Fluxo , Proteínas Ligadas por GPI/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Células Musculares/citologia , Células Musculares/metabolismo , Desenvolvimento Muscular/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Osteócitos/citologia , Osteócitos/metabolismo , Osteogênese/genética , Pericitos/metabolismo , Pericitos/ultraestrutura , Antígenos Embrionários Estágio-Específicos/metabolismo , beta Catenina/metabolismo
18.
J Am Heart Assoc ; 9(6): e014345, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32131688

RESUMO

Background Induced pluripotent stem cells and their differentiated cardiomyocytes (iCMs) have tremendous potential as patient-specific therapy for ischemic cardiomyopathy following myocardial infarctions, but difficulties in viable transplantation limit clinical translation. Exosomes secreted from iCMs (iCM-Ex) can be robustly collected in vitro and injected in lieu of live iCMs as a cell-free therapy for myocardial infarction. Methods and Results iCM-Ex were precipitated from iCM supernatant and characterized by protein marker expression, nanoparticle tracking analysis, and functionalized nanogold transmission electron microscopy. iCM-Ex were then used in in vitro and in vivo models of ischemic injuries. Cardiac function in vivo was evaluated by left ventricular ejection fraction and myocardial viability measurements by magnetic resonance imaging. Cardioprotective mechanisms were studied by JC-1 (tetraethylbenzimidazolylcarbocyanine iodide) assay, immunohistochemistry, quantitative real-time polymerase chain reaction, transmission electron microscopy, and immunoblotting. iCM-Ex measured ≈140 nm and expressed CD63 and CD9. iCM and iCM-Ex microRNA profiles had significant overlap, indicating that exosomal content was reflective of the parent cell. Mice treated with iCM-Ex demonstrated significant cardiac improvement post-myocardial infarction, with significantly reduced apoptosis and fibrosis. In vitro iCM apoptosis was significantly reduced by hypoxia and exosome biogenesis inhibition and restored by treatment with iCM-Ex or rapamycin. Autophagosome production and autophagy flux was upregulated in iCM-Ex groups in vivo and in vitro. Conclusions iCM-Ex improve post-myocardial infarction cardiac function by regulating autophagy in hypoxic cardiomyoytes, enabling a cell-free, patient-specific therapy for ischemic cardiomyopathy.


Assuntos
Autofagia , Exossomos/transplante , Células-Tronco Pluripotentes Induzidas/transplante , Infarto do Miocárdio/terapia , Miocárdio/ultraestrutura , Miócitos Cardíacos/transplante , Animais , Apoptose , Proteínas Relacionadas à Autofagia/metabolismo , Hipóxia Celular , Linhagem Celular , Modelos Animais de Doenças , Exossomos/metabolismo , Exossomos/ultraestrutura , Feminino , Fibrose , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Camundongos SCID , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Recuperação de Função Fisiológica , Transdução de Sinais , Volume Sistólico , Função Ventricular Esquerda
19.
J Am Soc Nephrol ; 31(5): 962-982, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32198276

RESUMO

BACKGROUND: Mutations in CTNS-a gene encoding the cystine transporter cystinosin-cause the rare, autosomal, recessive, lysosomal-storage disease cystinosis. Research has also implicated cystinosin in modulating the mTORC1 pathway, which serves as a core regulator of cellular metabolism, proliferation, survival, and autophagy. In its severest form, cystinosis is characterized by cystine accumulation, renal proximal tubule dysfunction, and kidney failure. Because treatment with the cystine-depleting drug cysteamine only slows disease progression, there is an urgent need for better treatments. METHODS: To address a lack of good human-based cell culture models for studying cystinosis, we generated the first human induced pluripotent stem cell (iPSC) and kidney organoid models of the disorder. We used a variety of techniques to examine hallmarks of cystinosis-including cystine accumulation, lysosome size, the autophagy pathway, and apoptosis-and performed RNA sequencing on isogenic lines to identify differentially expressed genes in the cystinosis models compared with controls. RESULTS: Compared with controls, these cystinosis models exhibit elevated cystine levels, increased apoptosis, and defective basal autophagy. Cysteamine treatment ameliorates this phenotype, except for abnormalities in apoptosis and basal autophagy. We found that treatment with everolimus, an inhibitor of the mTOR pathway, reduces the number of large lysosomes, decreases apoptosis, and activates autophagy, but it does not rescue the defect in cystine loading. However, dual treatment of cystinotic iPSCs or kidney organoids with cysteamine and everolimus corrects all of the observed phenotypic abnormalities. CONCLUSIONS: These observations suggest that combination therapy with a cystine-depleting drug such as cysteamine and an mTOR pathway inhibitor such as everolimus has potential to improve treatment of cystinosis.


Assuntos
Cisteamina/uso terapêutico , Cistinose/tratamento farmacológico , Modelos Animais de Doenças , Everolimo/uso terapêutico , Células-Tronco Pluripotentes Induzidas/transplante , Organoides/transplante , Serina-Treonina Quinases TOR/antagonistas & inibidores , Sistemas de Transporte de Aminoácidos Neutros/deficiência , Sistemas de Transporte de Aminoácidos Neutros/genética , Animais , Autofagia/efeitos dos fármacos , Sistemas CRISPR-Cas , Linhagem Celular , Cisteamina/farmacologia , Cistina/sangue , Avaliação Pré-Clínica de Medicamentos , Quimioterapia Combinada , Everolimo/farmacologia , Edição de Genes , Xenoenxertos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Lisossomos/efeitos dos fármacos , Lisossomos/ultraestrutura , Camundongos , Camundongos SCID , Organoides/metabolismo , Fenótipo
20.
Curr Gene Ther ; 19(6): 395-403, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32072883

RESUMO

OBJECTIVE: In order to generate induced Pluripotent Stem Cells (iPSCs) more efficiently, it is crucial to identify somatic cells that are easily accessible and possibly require fewer factors for conversion into iPSCs. METHODS: Human epidermal melanocytes were transduced with lentiviral vectors carrying 3 transcription factors (OCT-4, KLF-4 and c-MYC, 3F) or 4 transcription factors (OCT-4, KLF-4, c-MYC and SOX-2, 4F). Once the clones had formed, assays related to stem cell pluripotency, including alkaline phosphatase staining, DNA methylation levels, expression of stem cell markers and ultrastructure analysis were carried out. The iPSCs obtained were then induced to differentiate into the cells representing the three embryonic layers in vitro. RESULTS: Seven days after the transduction of epidermal melanocytes with 3F or 4F, clones were formed that were positive for alkaline phosphatase staining. Fluorescent staining with antibodies against OCT-4 and SOX-2 was strongly positive, and the cells showed a high nucleus-cytoplasm ratio and active karyokinesis. No melanosomes were found in the cytoplasm by ultrastructural analysis. There were obvious differences in DNA methylation levels between the cloned cells and their parental cells. However, there was not a significant difference between 3F or 4F transfected clonal cells. Meanwhile, the iPSCs successfully differentiated into the three germ layer cells in vitro. CONCLUSION: Human epidermal melanocytes do not require ectopic SOX-2 expression for conversion into iPSCs, and may serve as an alternative source for deriving patient-specific iPSCs with fewer genetic elements.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Melanócitos/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Células Cultivadas , Reprogramação Celular , Criança , Metilação de DNA , Terapia Genética , Humanos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Lentivirus/genética , Masculino , Melanócitos/ultraestrutura , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução Genética
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