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1.
Stem Cells ; 41(11): 1006-1021, 2023 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-37622655

RESUMO

Human induced pluripotent stem cells (iPSCs) and iPSC-derived neurons (iPSC-Ns) represent a differentiated modality toward developing novel cell-based therapies for regenerative medicine. However, the successful application of iPSC-Ns in cell-replacement therapies relies on effective cryopreservation. In this study, we investigated the role of ice recrystallization inhibitors (IRIs) as novel cryoprotectants for iPSCs and terminally differentiated iPSC-Ns. We found that one class of IRIs, N-aryl-D-aldonamides (specifically 2FA), increased iPSC post-thaw viability and recovery with no adverse effect on iPSC pluripotency. While 2FA supplementation did not significantly improve iPSC-N cell post-thaw viability, we observed that 2FA cryopreserved iPSC-Ns re-established robust neuronal network activity and synaptic function much earlier compared to CS10 cryopreserved controls. The 2FA cryopreserved iPSC-Ns retained expression of key neuronal specific and terminally differentiated markers and displayed functional electrophysiological and neuropharmacological responses following treatment with neuroactive agonists and antagonists. We demonstrate how optimizing cryopreservation media formulations with IRIs represents a promising strategy to improve functional cryopreservation of iPSCs and post-mitotic iPSC-Ns, the latter of which have been challenging to achieve. Developing IRI enabling technologies to support an effective cryopreservation and an efficiently managed cryo-chain is fundamental to support the delivery of successful iPSC-derived therapies to the clinic.


Assuntos
Gelo , Células-Tronco Pluripotentes Induzidas , Humanos , Gelo/efeitos adversos , Neurônios , Criopreservação , Crioprotetores/farmacologia , Crioprotetores/química
2.
J Neurochem ; 146(6): 735-752, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29877588

RESUMO

The blood-brain barrier (BBB) is a formidable obstacle to the delivery of therapeutics to the brain. Antibodies that bind transferrin receptor (TfR), which is enriched in brain endothelial cells, have been shown to cross the BBB and are being developed as fusion proteins to deliver therapeutic cargos to brain targets. Various antibodies have been developed for this purpose and their in vivo evaluation demonstrated that either low affinity or monovalent receptor binding re-directs their transcellular trafficking away from lysosomal degradation and toward improved exocytosis on the abluminal side of the BBB. However, these studies have been performed with antibodies that recognize different TfR epitopes and have different binding characteristics, preventing inter-study comparisons. In this study, the efficiency of transcytosis in vitro and intracellular trafficking in endosomal compartments were evaluated in an in vitro BBB model for affinity variants (Kd from 5 to174 nM) of the rat TfR-binding antibody, OX26. Distribution in subcellular fractions of the rat brain endothelial cells was determined using both targeted quantitative proteomics-selected reaction monitoring and fluorescent imaging with markers of early- and late endosomes. The OX26 variants with affinities of 76 and 108 nM showed improved trancytosis (Papp values) across the in vitro BBB model compared with a 5 nM OX26. Although ~40% of the 5 nM OX26 and ~35% of TfR co-localized with late-endosome/lysosome compartment, 76 and 108 nM affinity variants showed lower amounts in lysosomes and a predominant co-localization with early endosome markers. The study links bivalent TfR antibody affinity to mechanisms of sorting and trafficking away from late endosomes and lysosomes, resulting in improvement in their transcytosis efficiency. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14193.


Assuntos
Anticorpos/metabolismo , Barreira Hematoencefálica/metabolismo , Receptores da Transferrina/imunologia , Receptores da Transferrina/metabolismo , Transcitose/fisiologia , Animais , Anticorpos/farmacologia , Afinidade de Anticorpos/fisiologia , Encéfalo/citologia , Endossomos/efeitos dos fármacos , Endossomos/fisiologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Técnicas In Vitro , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Espectrometria de Massas , Ligação Proteica/fisiologia , Ratos , Frações Subcelulares/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7 , Proteína Vermelha Fluorescente
3.
Tissue Eng Part C Methods ; 30(7): 289-306, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38946589

RESUMO

In the advent of tissue engineering and regenerative medicine, the demand for innovative approaches to biofabricate complex vascular structures is increasing. We describe a single-step 3D bioprinting method leveraging Aspect Biosystems RX1 technology, which integrates the crosslinking step at a flow-focusing junction, to biofabricate immortalized adult rat brain endothelial cell (SV-ARBEC)-encapsulated alginate-collagen type I hydrogel rings. This single-step biofabrication process involves the strategic layer-by-layer assembly of hydrogel rings, encapsulating SV-ARBECs in a spatially controlled manner while optimizing access to media and nutrients. The spatial arrangement of the SV-ARBECs within the rings promotes spontaneous angiogenic network formation and the constrained deposition of cells within the hydrogel matrix facilitates tissue-like organized vascular-like network development. This approach provides a platform that can be adapted to many different endothelial cell types and leveraged to better understand the mechanisms driving angiogenesis and vascular-network formation in 3D bioprinted constructs supporting the development of more complex tissue and disease models for advancing drug discovery, tissue engineering, and regenerative medicine applications.


Assuntos
Alginatos , Bioimpressão , Colágeno Tipo I , Células Endoteliais , Hidrogéis , Neovascularização Fisiológica , Impressão Tridimensional , Alginatos/química , Alginatos/farmacologia , Animais , Ratos , Neovascularização Fisiológica/efeitos dos fármacos , Bioimpressão/métodos , Hidrogéis/química , Colágeno Tipo I/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Engenharia Tecidual/métodos , Alicerces Teciduais/química
4.
Mol Ther Oncol ; 32(1): 200775, 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38596311

RESUMO

Chimeric antigen receptor (CAR) T cell therapies targeting B cell-restricted antigens CD19, CD20, or CD22 can produce potent clinical responses for some B cell malignancies, but relapse remains common. Camelid single-domain antibodies (sdAbs or nanobodies) are smaller, simpler, and easier to recombine than single-chain variable fragments (scFvs) used in most CARs, but fewer sdAb-CARs have been reported. Thus, we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein, sdAb-cDNA library construction, and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. Changing CD28- to CD8-transmembrane design increased CAR persistence and expression in vitro. CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro, although only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies, a lead CD22sdCAR-T was selected, achieving comparable complete responses to a benchmark short linker m971-scFv CAR-T in high-dose experiments. Finally, immunohistochemistry and flow cytometry confirm tissue and cellular-level specificity of the lead CD22-sdAb. This presents a complete report on preclinical development of a novel CD22sdCAR therapeutic.

5.
Biochem Cell Biol ; 91(5): 271-86, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24032676

RESUMO

There is a need for improved therapy for acquired brain injury, which has proven resistant to treatment by numerous drugs in clinical trials and continues to represent one of the leading causes of disability worldwide. Research into cell-based therapies for the treatment of brain injury is growing rapidly, but the ideal cell source has yet to be determined. Subpopulations of cells found in amniotic fluid, which is readily obtained during routine amniocentesis, can be easily expanded in culture, have multipotent differentiation capacity, are non-tumourigenic, and avoid the ethical complications associated with embryonic stem cells, making them a promising cell source for therapeutic purposes. Beneficial effects of amniotic fluid cell transplantation have been reported in various models of nervous system injury. However, evidence that amniotic fluid cells can differentiate into mature, functional neurons in vivo and incorporate into the existing circuitry to replace lost or damaged neurons is lacking. The mechanisms by which amniotic fluid cells improve outcomes after experimental nervous system injury remain unclear. However, studies reporting the expression and release of neurotrophic, angiogenic, and immunomodulatory factors by amniotic fluid cells suggest they may provide neuroprotection and (or) stimulate endogenous repair and remodelling processes in the injured nervous system. In this paper, we address recent research related to the neuronal differentiation of amniotic fluid-derived cells, the therapeutic efficacy of these cells in animal models of nervous system injury, and the possible mechanisms mediating the positive outcomes achieved by amniotic fluid cell transplantation.


Assuntos
Líquido Amniótico/citologia , Lesões Encefálicas/terapia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células-Tronco Multipotentes/transplante , Amniocentese , Animais , Diferenciação Celular , Humanos , Camundongos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Neurônios/citologia , Acidente Vascular Cerebral/terapia , Engenharia Tecidual/métodos
6.
Fluids Barriers CNS ; 20(1): 36, 2023 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-37237379

RESUMO

Blood brain barrier (BBB) models in vitro are an important tool to aid in the pre-clinical evaluation and selection of BBB-crossing therapeutics. Stem cell derived BBB models have recently demonstrated a substantial advantage over primary and immortalized brain endothelial cells (BECs) for BBB modeling. Coupled with recent discoveries highlighting significant species differences in the expression and function of key BBB transporters, the field is in need of robust, species-specific BBB models for improved translational predictability. We have developed a mouse BBB model, composed of mouse embryonic stem cell (mESC-D3)-derived brain endothelial-like cells (mBECs), employing a directed monolayer differentiation strategy. Although the mBECs showed a mixed endothelial-epithelial phenotype, they exhibited high transendothelial electrical resistance, inducible by retinoic acid treatment up to 400 Ω cm2. This tight cell barrier resulted in restricted sodium fluorescein permeability (1.7 × 10-5 cm/min), significantly lower than that of bEnd.3 cells (1.02 × 10-3 cm/min) and comparable to human induced pluripotent stem cell (iPSC)-derived BECs (2.0 × 10-5 cm/min). The mBECs expressed tight junction proteins, polarized and functional P-gp efflux transporter and receptor mediated transcytosis (RMT) receptors; collectively important criteria for studying barrier regulation and drug delivery applications in the CNS. In this study, we compared transport of a panel of antibodies binding species selective or cross-reactive epitopes on BBB RMT receptors in both the mBEC and human iPSC-derived BEC model, to demonstrate discrimination of species-specific BBB transport mechanisms.


Assuntos
Barreira Hematoencefálica , Células-Tronco Pluripotentes Induzidas , Humanos , Animais , Camundongos , Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Anticorpos/metabolismo , Transcitose
7.
J Neurosci Res ; 90(12): 2362-77, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22987726

RESUMO

Nitric oxide (NO) plays a key role in neurogenesis as a regulator of cell proliferation and differentiation. NO is synthesized from the amino acid L-arginine by nitric oxide synthases (NOS1, NOS2, and NOS3), which are encoded by separate genes and display different tissue distributions. We used an in vitro model of RA-induced neural differentiation of NT2 cells to examine which of the three NO-synthesizing enzymes is involved in this process. The results revealed a transient induction of NOS3 (known as the constitutively expressed endothelial nitric oxide synthase; eNOS) during the time course of the RA treatment. The peak of gene expression and the nuclear presence of NOS3 protein coincided with cell cycle exit of NT2-derived neuronal precursors. The subsequent analysis of cytosine methylation and histone H3 acetylation of the human NOS3 5' regulatory sequences indicated that epigenetic modifications, especially upstream of the proximal promoter (-734 to -989, relative to exon 2 TSS at +1), were also taking place. NOS1 was expressed only in the differentiated neurons (NT2-N), whereas NOS2 was not expressed at all in this cellular model. Thus, a burst of NO production, possibly required to inhibit neural cell proliferation, was generated by the transient expression of NOS3. This pattern of gene expression, in turn, required epigenetic remodeling of its regulatory region.


Assuntos
Proteínas do Tecido Nervoso/fisiologia , Neurogênese/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/fisiologia , Tretinoína/farmacologia , Regiões 5' não Traduzidas/genética , Acetilação , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Linhagem Celular Tumoral/citologia , Linhagem Celular Tumoral/efeitos dos fármacos , Núcleo Celular/enzimologia , Imunoprecipitação da Cromatina , Ilhas de CpG/genética , Metilação de DNA , Indução Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Histonas/metabolismo , Humanos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Neurogênese/fisiologia , Neuroglia/citologia , Neurônios/citologia , Óxido Nítrico/fisiologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo I/biossíntese , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/biossíntese , Óxido Nítrico Sintase Tipo III/genética , Ornitina/análogos & derivados , Ornitina/farmacologia , Processamento de Proteína Pós-Traducional , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Teratocarcinoma/patologia , Triazenos/farmacologia
8.
Methods Mol Biol ; 2454: 397-410, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-33881753

RESUMO

The development of translational and predictive models in vitro for assessing blood-brain barrier (BBB) delivery has become an important requirement in preclinical testing of CNS-targeting therapeutics. Here we describe a directed monolayer differentiation strategy to generate a population of brain endothelial-like cells (BECs) from human induced pluripotent stem cell (iPSC) with robust BBB properties. To generate BBB permeability assays, the BECs are seeded as a monolayer on a semipermeable Transwell insert placed inside a companion plate to generate a two-compartment Transwell model. The BECs provide a BBB-like separation between the luminal (blood) and abluminal (brain) compartments to assess BBB permeability of CNS-targeting therapeutics.


Assuntos
Barreira Hematoencefálica , Células-Tronco Pluripotentes Induzidas , Encéfalo , Células Cultivadas , Células Endoteliais , Humanos , Permeabilidade
9.
Methods Mol Biol ; 2549: 345-357, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35218529

RESUMO

Automated high-throughput immunoassays are emerging as reliable analytic techniques for the quantitative detection of proteins from a variety of sample types. Herein, we describe a method using the Protein Simple Wes capillary-based automated immunoassays platform for the quantification of His- and HA-tagged antibody transcytosis across an in vitro transwell blood-brain barrier (BBB) model. Compared to conventional ELISA, fluorescence, and Mass Spec-based detection approaches, Wes provides comparable datasets with additional information regarding size, aggregation, and potential degradation of samples before and after BBB transcytosis. In this chapter, we have benchmarked our Wes technique against ELISA and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), using known BBB crossing (FC5) and non-crossing (A20.1) single domain antibodies.


Assuntos
Barreira Hematoencefálica , Células Endoteliais , Anticorpos/química , Barreira Hematoencefálica/metabolismo , Cromatografia Líquida , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Imunoensaio , Espectrometria de Massas em Tandem , Transcitose
10.
Fluids Barriers CNS ; 19(1): 38, 2022 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-35650594

RESUMO

Human blood brain barrier (BBB) models derived from induced pluripotent stem cells (iPSCs) have become an important tool for the discovery and preclinical evaluation of central nervous system (CNS) targeting cell and gene-based therapies. Chimeric antigen receptor (CAR)-T cell therapy is a revolutionary form of gene-modified cell-based immunotherapy with potential for targeting solid tumors, such as glioblastomas. Crossing the BBB is an important step in the systemic application of CAR-T therapy for the treatment of glioblastomas and other CNS malignancies. In addition, even CAR-T therapies targeting non-CNS antigens, such as the well-known CD19-CAR-T therapies, are known to trigger CNS side-effects including brain swelling due to BBB disruption. In this study, we used iPSC-derived brain endothelial-like cell (iBEC) transwell co-culture model to assess BBB extravasation of CAR-T based immunotherapies targeting U87MG human glioblastoma (GBM) cells overexpressing the tumor-specific mutated protein EGFRvIII (U87vIII). Two types of anti-EGFRvIII targeting CAR-T cells, with varying tonic signaling profiles (CAR-F263 and CAR-F269), and control Mock T cells were applied on the luminal side of BBB model in vitro. CAR-F263 and CAR-F269 T cells triggered a decrease in transendothelial electrical resistance (TEER) and an increase in BBB permeability. CAR-T cell extravasation and U87vIII cytotoxicity were assessed from the abluminal compartment using flow cytometry and Incucyte real-time viability imaging, respectively. A significant decrease in U87vIII cell viability was observed over 48 h, with the most robust cytotoxicity response observed for the constitutively activated CAR-F263. CAR-F269 T cells showed a similar cytotoxic profile but were approximately four fold less efficient at killing the U87vIII cells compared to CAR-F263, despite similar transmigration rates. Visualization of CAR-T cell extravasation across the BBB was further confirmed using BBTB-on-CHIP models. The described BBB assay was able to discriminate the cytotoxic efficacies of different EGFRvIII-CARs and provide a measure of potential alterations to BBB integrity. Collectively, we illustrate how BBB models in vitro can be a valuable tool in deciphering the mechanisms of CAR-T-induced BBB disruption, accompanying toxicity and effector function on post-barrier target cells.


Assuntos
Glioblastoma , Receptores de Antígenos Quiméricos , Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Glioblastoma/patologia , Glioblastoma/terapia , Humanos , Imunoterapia , Receptores de Antígenos Quiméricos/metabolismo
11.
Stem Cell Rev Rep ; 18(1): 259-277, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34687385

RESUMO

Human induced pluripotent stem cell (iPSC)-derived neurons are of interest for studying neurological disease mechanisms, developing potential therapies and deepening our understanding of the human nervous system. However, compared to an extensive history of practice with primary rodent neuron cultures, human iPSC-neurons still require more robust characterization of expression of neuronal receptors and ion channels and functional and predictive pharmacological responses. In this study, we differentiated human amniotic fluid-derived iPSCs into a mixed population of neurons (AF-iNs). Functional assessments were performed by evaluating electrophysiological (patch-clamp) properties and the effect of a panel of neuropharmacological agents on spontaneous activity (multi-electrode arrays; MEAs). These electrophysiological data were benchmarked relative to commercially sourced human iPSC-derived neurons (CNS.4U from Ncardia), primary human neurons (ScienCell™) and primary rodent cortical/hippocampal neurons. Patch-clamp whole-cell recordings showed that mature AF-iNs generated repetitive firing of action potentials in response to depolarizations, similar to that of primary rodent cortical/hippocampal neurons, with nearly half of the neurons displaying spontaneous post-synaptic currents. Immunochemical and MEA-based analyses indicated that AF-iNs were composed of functional glutamatergic excitatory and inhibitory GABAergic neurons. Principal component analysis of MEA data indicated that human AF-iN and rat neurons exhibited distinct pharmacological and electrophysiological properties. Collectively, this study establishes a necessary prerequisite for AF-iNs as a human neuron culture model suitable for pharmacological studies.


Assuntos
Células-Tronco Pluripotentes Induzidas , Animais , Benchmarking , Fenômenos Eletrofisiológicos , Humanos , Neurônios , Ratos , Roedores
12.
Stem Cells ; 28(2): 247-57, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20039365

RESUMO

Regulatory mechanisms pertaining to the self-renewal of stem cells remain incompletely understood. Here, we show that functional interactions between small GTPase Rap1 and the adhesion molecule E-cadherin uniquely regulate the self-renewal of human embryonic stem cells (hESCs). Inhibition of Rap1 suppresses colony formation and self-renewal of hESCs, whereas overexpression of Rap1 augments hESC clonogenicity. Rap1 does not directly influence the expression of the pluripotency genes Oct4 and Nanog. Instead, it affects the endocytic recycling pathway involved in the formation and maintenance of E-cadherin-mediated cell-cell cohesion, which is essential for the colony formation and self-renewal of hESCs. Conversely, distinct from epithelial cells, disruption of E-cadherin mediated cell-cell adhesions induces lysosome delivery and degradation of Rap1. This in turn leads to a further downregulation of E-cadherin function and a subsequent reduction in hESC clonogenic capacity. These findings provide the first demonstration that the interplay between Rap1 and E-cadherin along the endocytic recycling pathway serves as a timely and efficient mechanism to regulate hESC self-renewal. Given the availability of specific activators for Rap1, this work provides a new perspective to enable better maintenance of human pluripotent stem cells.


Assuntos
Caderinas/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Benzamidas/farmacologia , Western Blotting , Caderinas/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Humanos , Microscopia de Fluorescência , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Complexo Shelterina , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas de Ligação a Telômeros/genética
13.
Genetics ; 215(4): 1055-1066, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32554600

RESUMO

Dravet syndrome is a developmental epileptic encephalopathy caused by pathogenic variation in SCN1A To characterize the pathogenic substitution (p.H939R) of a local individual with Dravet syndrome, fibroblast cells from the individual were reprogrammed to pluripotent stem cells and differentiated into neurons. Sodium currents of these neurons were compared with healthy control induced neurons. A novel Scn1aH939R/+ mouse model was generated with the p.H939R substitution. Immunohistochemistry and electrophysiological experiments were performed on hippocampal slices of Scn1aH939R/+ mice. We found that the sodium currents recorded in the proband-induced neurons were significantly smaller and slower compared to wild type (WT). The resting membrane potential and spike amplitude were significantly depolarized in the proband-induced neurons. Similar differences in resting membrane potential and spike amplitude were observed in the interneurons of the hippocampus of Scn1aH939R/+ mice. The Scn1aH939R/+ mice showed the characteristic features of a Dravet-like phenotype: increased mortality and both spontaneous and heat-induced seizures. Immunohistochemistry showed a reduction in amount of parvalbumin and vesicular acetylcholine transporter in the hippocampus of Scn1aH939R/+ compared to WT mice. Overall, these results underline hyper-excitability of the hippocampal CA1 circuit of this novel mouse model of Dravet syndrome which, under certain conditions, such as temperature, can trigger seizure activity. This hyper-excitability is due to the altered electrophysiological properties of pyramidal neurons and interneurons which are caused by the dysfunction of the sodium channel bearing the p.H939R substitution. This novel Dravet syndrome model also highlights the reduction in acetylcholine and the contribution of pyramidal cells, in addition to interneurons, to network hyper-excitability.


Assuntos
Região CA1 Hipocampal/patologia , Modelos Animais de Doenças , Epilepsias Mioclônicas/patologia , Fibroblastos/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Interneurônios/patologia , Células Piramidais/patologia , Animais , Região CA1 Hipocampal/metabolismo , Eletrofisiologia , Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Interneurônios/metabolismo , Masculino , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Células Piramidais/metabolismo
14.
J Neurosci Res ; 86(8): 1680-93, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18293417

RESUMO

SOX2 is a key neurodevelopmental gene involved in maintaining the pluripotency of stem cells and proliferation of neural progenitors and astroglia. Two evolutionally conserved enhancers, SRR1 and SRR2, are involved in controlling SOX2 expression during neurodevelopment; however, the molecular mechanisms regulating their activity are not known. We have examined DNA methylation and histone H3 acetylation at both enhancers in NT2-D1 progenitors, neurons and astrocytes, to establish the role of epigenetic mechanisms in cell-type-specific SOX2 expression. This study showed that 1) unmethylated DNA and acetylated histones at both enhancers correlated with a high level of SOX2 expression in proliferating neural progenitors and 2) reversible modifications of the SRR1 element were observed during gene reexpression in astrocytes, whereas permanent epigenetic marks on the SRR2 enhancer were seen in neurons where the gene was silenced. Taken together, these results are clear illustrations of cell-type-specific epigenomes and suggest mechanisms by which they may be created and maintained.


Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/biossíntese , Elementos Facilitadores Genéticos/fisiologia , Epigênese Genética/fisiologia , Proteínas HMGB/biossíntese , Glicoproteínas de Membrana/fisiologia , Neurônios/citologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Peptídeos/fisiologia , Fatores de Transcrição/biossíntese , Acetilação , Astrócitos/citologia , Astrócitos/metabolismo , Sequência de Bases , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Neurônios/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Peptídeos/genética , Fatores de Transcrição SOXB1 , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
Fluids Barriers CNS ; 15(1): 15, 2018 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-29759080

RESUMO

Zika virus (ZIKV) is a flavivirus that is highly neurotropic causing congenital abnormalities and neurological damage to the central nervous systems (CNS). In this study, we used a human induced pluripotent stem cell (iPSC)-derived blood brain barrier (BBB) model to demonstrate that ZIKV can infect brain endothelial cells (i-BECs) without compromising the BBB barrier integrity or permeability. Although no disruption to the BBB was observed post-infection, ZIKV particles were released on the abluminal side of the BBB model and infected underlying iPSC-derived neural progenitor cells (i-NPs). AXL, a putative ZIKV cellular entry receptor, was also highly expressed in ZIKV-susceptible i-BEC and i-NPs. This iPSC-derived BBB model can help elucidate the mechanism by which ZIKV can infect BECs, cross the BBB and gain access to the CNS.


Assuntos
Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/virologia , Zika virus/metabolismo , Permeabilidade Capilar/fisiologia , Técnicas de Cultura de Células , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/virologia , Microvasos/metabolismo , Microvasos/virologia , Infecção por Zika virus/metabolismo , Infecção por Zika virus/virologia
16.
Sci Rep ; 8(1): 1873, 2018 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-29382846

RESUMO

We have developed a renewable, scalable and transgene free human blood-brain barrier model, composed of brain endothelial cells (BECs), generated from human amniotic fluid derived induced pluripotent stem cells (AF-iPSC), which can also give rise to syngeneic neural cells of the neurovascular unit. These AF-iPSC-derived BECs (i-BEC) exhibited high transendothelial electrical resistance (up to 1500 Ω cm2) inducible by astrocyte-derived molecular cues and retinoic acid treatment, polarized expression of functional efflux transporters and receptor mediated transcytosis triggered by antibodies against specific receptors. In vitro human BBB models enable pre-clinical screening of central nervous system (CNS)-targeting drugs and are of particular importance for assessing species-specific/selective transport mechanisms. This i-BEC human BBB model discriminates species-selective antibody- mediated transcytosis mechanisms, is predictive of in vivo CNS exposure of rodent cross-reactive antibodies and can be implemented into pre-clinical CNS drug discovery and development processes.


Assuntos
Anticorpos/farmacologia , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Receptores de Superfície Celular/metabolismo , Transcitose/fisiologia , Animais , Astrócitos/citologia , Astrócitos/fisiologia , Transporte Biológico , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Masculino , Neurônios/citologia , Neurônios/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/antagonistas & inibidores
17.
ScientificWorldJournal ; 7: 1950-64, 2007 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-18167610

RESUMO

The current progress using the human embryonic stem cell (hESC) model system has provided much insight into the early origins of the hematopoietic and endothelial lineages, particularly the elusive hemangioblast. Recently, the cellular hierarchy and molecular regulation controlling hematopoietic commitment have been further elucidated. These findings not only provide new insights into early human development, but also advance the knowledge required to develop techniques capable of generating a given cell type for potential clinical applications. This review will focus on the latest advances using the hESC model system, capitalizing on the well-established mouse embryonic stem cell model system, as a means to investigate the lineage commitment events underlying the early embryonic development of human hematopoietic and endothelial cells.


Assuntos
Células-Tronco Embrionárias/fisiologia , Células Endoteliais/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Animais , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Células Endoteliais/citologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Modelos Animais , Neovascularização Fisiológica/fisiologia
18.
Stem Cell Rev Rep ; 10(2): 251-68, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24415130

RESUMO

Brain injury continues to be one of the leading causes of disability worldwide. Despite decades of research, there is currently no pharmacologically effective treatment for preventing neuronal loss and repairing the brain. As a result, novel therapeutic approaches, such as cell-based therapies, are being actively pursued to repair tissue damage and restore neurological function after injury. In this study, we examined the neuroprotective potential of amniotic fluid (AF) single cell clones, engineered to secrete glial cell derived neurotrophic factor (AF-GDNF), both in vitro and in a surgically induced model of brain injury. Our results show that pre-treatment with GDNF significantly increases cell survival in cultures of AF cells or cortical neurons exposed to hydrogen peroxide. Since improving the efficacy of cell transplantation depends on enhanced graft cell survival, we investigated whether AF-GDNF cells seeded on polyglycolic acid (PGA) scaffolds could enhance graft survival following implantation into the lesion cavity. Encouragingly, the AF-GDNF cells survived longer than control AF cells in serum-free conditions and continued to secrete GDNF both in vitro and following implantation into the injured motor cortex. AF-GDNF implantation in the acute period following injury was sufficient to activate the MAPK/ERK signaling pathway in host neural cells in the peri-lesion area, potentially boosting endogenous neuroprotective pathways. These results were complemented with promising trends in beam walk tasks in AF-GDNF/PGA animals during the 7 day timeframe. Further investigation is required to determine whether significant behavioural improvement can be achieved at a longer timeframe.


Assuntos
Líquido Amniótico/citologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/fisiologia , Transplante de Células-Tronco , Células-Tronco/fisiologia , Animais , Lesões Encefálicas/patologia , Lesões Encefálicas/fisiopatologia , Lesões Encefálicas/terapia , Sobrevivência Celular , Células Cultivadas , Feminino , Expressão Gênica , Humanos , Peróxido de Hidrogênio/farmacologia , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Córtex Motor/patologia , Células-Tronco Neurais/fisiologia , Oxidantes/farmacologia , Estresse Oxidativo , Próteses e Implantes , Desempenho Psicomotor , Alicerces Teciduais
19.
Stem Cells Int ; 2012: 607161, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22792116

RESUMO

The usage of stem cells is a promising strategy for the repair of damaged tissue in the injured brain. Recently, amniotic fluid (AF) cells have received a lot of attention as an alternative source of stem cells for cell-based therapies. However, the success of this approach relies significantly on proper interactions between graft and host tissue. In particular, the reestablishment of functional brain networks requires formation of gap junctions, as a key step to provide sufficient intercellular communication. In this study, we show that AF cells express high levels of CX43 (GJA1) and are able to establish functional gap junctions with cortical cultures. Furthermore, we report an induction of Cx43 expression in astrocytes following injury to the mouse motor cortex and demonstrate for the first time CX43 expression at the interface between implanted AF cells and host brain cells. These findings suggest that CX43-mediated intercellular communication between AF cells and cortical astrocytes may contribute to the reconstruction of damaged tissue by mediating modulatory, homeostatic, and protective factors in the injured brain and hence warrants further investigation.

20.
Stem Cell Rev Rep ; 6(2): 199-214, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20221716

RESUMO

Recently, human amniotic fluid (AF) cells have attracted a great deal of attention as an alternative cell source for transplantation and tissue engineering. AF contains a variety of cell types derived from fetal tissues, of which a small percentage is believed to represent stem cell sub-population(s). In contrast to human embryonic stem (ES) cells, AF cells are not subject to extensive legal or ethical considerations; nor are they limited by lineage commitment characteristic of adult stem cells. However, to become therapeutically valuable, better protocols for the isolation of AF stem cell sub-populations need to be developed. This study was designed to examine the molecular components involved in self-renewal, neural commitment and differentiation of AF cells obtained at different gestational ages. Our results showed that, although morphologically heterogeneous, AF cells derived from early gestational periods ubiquitously expressed KERATIN 8 (K8), suggesting that the majority of these cells may have an epithelial origin. In addition, AF cells expressed various components of NOTCH signaling (ligands, receptors and target genes), a pathway involved in stem cell maintenance, determination and differentiation. A sub-population of K8 positive cells (<10%) co-expressed NESTIN, a marker detected in the neuroepithelium, neural stem cells and neural progenitors. Throughout the gestational periods, a much smaller AF cell sub-population (<1%) expressed pluripotency markers, OCT4a, NANOG and SOX2, from which SOX2 positive AF cells could be isolated through single cell cloning. The SOX2 expressing AF clones showed the capacity to give rise to a neuron-like phenotype in culture, expressing neuronal markers such as MAP2, NFL and NSE. Taken together, our findings demonstrated the presence of fetal cells with stem cell characteristics in the amniotic fluid, highlighting the need for further research on their biology and clinical applications.


Assuntos
Líquido Amniótico/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Western Blotting , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Feminino , Citometria de Fluxo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Queratina-8/genética , Queratina-8/metabolismo , Modelos Biológicos , Proteína Homeobox Nanog , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais
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