RESUMEN
The blood-brain barrier (BBB) constitutes the interface between the blood and the brain tissue. Its primary function is to maintain the tightly controlled microenvironment of the brain. Models of the BBB are useful for studying the development and maintenance of the BBB as well as diseases affecting it. Furthermore, BBB models are important tools in drug development and support the evaluation of the brain-penetrating properties of novel drug molecules. Currently used in vitro models of the BBB include immortalized brain endothelial cell lines and primary brain endothelial cells of human and animal origin. Unfortunately, many cell lines and primary cells do not recreate physiological restriction of transport in vitro. Human-induced pluripotent stem cell (iPSC)-derived brain endothelial cells have proven a promising alternative source of brain endothelial-like cells that replicate tight cell layers with low paracellular permeability. Given the possibility to generate large amounts of human iPSC-derived brain endothelial cells they are a feasible alternative when modelling the BBB in vitro. iPSC-derived brain endothelial cells form tight cell layers in vitro and their barrier properties can be enhanced through coculture with other cell types of the BBB. Currently, many different models of the BBB using iPSC-derived cells are under evaluation to study BBB formation, maintenance, disruption, drug transport and diseases affecting the BBB. This review summarizes important functions of the BBB and current efforts to create iPSC-derived BBB models in both static and dynamic conditions. In addition, it highlights key model requirements and remaining challenges for human iPSC-derived BBB models in vitro.
Asunto(s)
Transporte Biológico/fisiología , Barrera Hematoencefálica/metabolismo , Encéfalo/citología , Células Endoteliales/citología , Células Madre Pluripotentes Inducidas/citología , Animales , Barrera Hematoencefálica/patología , Técnicas de Cocultivo/métodos , HumanosRESUMEN
Cell-based models of the blood-brain barrier (BBB) are important for increasing the knowledge of BBB formation, degradation and brain exposure of drug substances. Human models are preferred over animal models because of interspecies differences in BBB structure and function. However, access to human primary BBB tissue is limited and has shown degeneration of BBB functions in vitro. Human induced pluripotent stem cells (iPSCs) can be used to generate relevant cell types to model the BBB with human tissue. We generated a human iPSC-derived model of the BBB that includes endothelial cells in coculture with pericytes, astrocytes and neurons. Evaluation of barrier properties showed that the endothelial cells in our coculture model have high transendothelial electrical resistance, functional efflux and ability to discriminate between CNS permeable and non-permeable substances. Whole genome expression profiling revealed transcriptional changes that occur in coculture, including upregulation of tight junction proteins, such as claudins and neurotransmitter transporters. Pathway analysis implicated changes in the WNT, TNF, and PI3K-Akt pathways upon coculture. Our data suggest that coculture of iPSC-derived endothelial cells promotes barrier formation on a functional and transcriptional level. The information about gene expression changes in coculture can be used to further improve iPSC-derived BBB models through selective pathway manipulation. Stem Cells 2018;36:1816-12.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Transcriptoma/fisiología , Diferenciación Celular , HumanosRESUMEN
Microphysiological systems (MPS) are gaining broader application in the pharmaceutical industry but have primarily been leveraged in early discovery toxicology and pharmacology studies with small molecules. The adoption of MPS offers a promising avenue to reduce animal use, improve in-vitro-to-in-vivo translation of pharmacokinetics/pharmacodynamics and toxicity correlation, and provide mechanistic understanding of model species suitability. While MPS have demonstrated utility in these areas with small molecules and biologics, MPS models in cell therapy development have not been fully explored, let alone validated. Distinguishing features of MPS, including long-term viability and physiologically relevant expression of functional enzymes, receptors, and pharmacological targets make them attractive tools for nonclinical characterization. However, there is currently limited published evidence of MPS being utilized to study the disposition, metabolism, pharmacology, and toxicity profiles of cell therapies. This review provides an industry perspective on the nonclinical application of MPS on cell therapies, first with a focus on oncology applications followed by examples in regenerative medicine.
Microphysiological systems (MPS) are advanced cell models, applied in the pharmaceutical industry to characterize novel therapies. While their application in studies of small molecule therapies has been very successful, the use of these models to study cell therapies has been limited. Cell therapies consist of cells and are living drugs, often with complex biological mechanisms of action, which can be very challenging to study. However, MPS have several features that make them attractive for studying cell therapies, including possibilities for longer-term studies and the ability to mimic physiologically relevant biological functions. MPS can mimic complex biological systems and processes, as such, the adoption of MPS offers a promising avenue to reduce the use of animals in the characterization of novel therapies. This review provides an industry perspective on current challenges and highlights opportunities for using MPS in the development of cell therapies.
Asunto(s)
Alternativas a las Pruebas en Animales , Tratamiento Basado en Trasplante de Células y Tejidos , Animales , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Humanos , Medicina Regenerativa/métodos , Sistemas MicrofisiológicosRESUMEN
Aim & methods: The Health and Environmental Sciences Institute Cell Therapy-TRAcking, Circulation & Safety Technical Committee launched an international, multisite study to evaluate the sensitivity and reproducibility of the highly efficient culture (HEC) assay, an in vitro assay to detect residual undifferentiated human pluripotent stem cells (hPSCs) in cell therapy products. Results: All facilities detected colonies of human induced pluripotent stem cells (hiPSCs) when five hiPSCs were spiked into 1 million hiPSC-derived cardiomyocytes. Spiking with a trace amount of hiPSCs revealed that repeatability accounts for the majority of reproducibility while the true positive rate was high. Conclusion: The results indicate that the HEC assay is highly sensitive and robust and can be generally applicable for tumorigenicity evaluation of hPSC-derived cell therapy products.
Asunto(s)
Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Humanos , Reproducibilidad de los Resultados , Academias e Institutos , BioensayoRESUMEN
BACKGROUND: Human induced pluripotent stem cells (hiPSC) hold great promise for use in cell therapy applications and for improved in vitro models of human disease. So far, most hiPSC differentiation protocols to astroglia use undefined, animal-containing culture matrices. Laminins, which play an essential role in the regulation of cell behavior, offer a source of defined, animal-free culture matrix. METHODS: In order to understand how laminins affect astroglia differentiation, recombinant human laminin-521 (LN521), was compared to a murine Engelbreth-Holm-Swarm sarcoma derived laminin (L2020). Astroglia expression of protein and mRNA together with glutamate uptake and protein secretion function, were evaluated. Finally, these astroglia were evaluated in a coculture model of the blood-brain barrier (BBB). RESULTS: Astroglia of good quality were generated from hiPSC on both LN521 and L2020. However, astroglia differentiated on human LN521 showed higher expression of several astroglia specific mRNAs and proteins such as GFAP, S100B, Angiopoietin-1, and EAAT1, compared to astroglia differentiated on murine L2020. In addition, glutamate uptake and ability to induce expression of junction proteins in endothelial cells were affected by the culture matrix for differentiation. CONCLUSION: Our results suggest that astroglia differentiated on LN521 display an improved phenotype and are suitable for coculture in a hiPSC-derived BBB model. This provides a starting point for a more defined and robust derivation of astroglia for use in BBB coculture models.
Asunto(s)
Astrocitos/fisiología , Barrera Hematoencefálica/fisiología , Diferenciación Celular , Técnicas de Cocultivo/métodos , Células Endoteliales/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Línea Celular Tumoral , Ácido Glutámico/metabolismo , Humanos , Laminina/fisiología , ARN Mensajero/metabolismoRESUMEN
Microphysiological systems (MPS) may be able to provide the pharmaceutical industry models that can reflect human physiological responses to improve drug discovery and translational outcomes. With lack of efficacy being the primary cause for drug attrition, developing MPS disease models would help researchers identify novel targets, study mechanisms in more physiologically-relevant depth, screen for novel biomarkers and test/optimize various therapeutics (small molecules, nanoparticles and biologics). Furthermore, with advances in inducible pluripotent stem cell technology (iPSC), pharmaceutical companies can access cells from patients to help recreate specific disease phenotypes in MPS platforms. Combining iPSC and MPS technologies will contribute to our understanding of the complexities of neurodegenerative diseases and of the blood brain barrier (BBB) leading to development of enhanced therapeutics.
Asunto(s)
Barrera Hematoencefálica , Descubrimiento de Drogas , Células Madre Pluripotentes Inducidas , Animales , Humanos , Modelos Biológicos , Investigación Biomédica TraslacionalRESUMEN
Plasminogen binding inhibitors (PBIs) reduce the risk of bleeding in hemorrhagic conditions. However, generic PBIs are also associated with an increased risk of seizures, an adverse effect linked to unwanted activities towards inhibitory neuronal receptors. Development of novel PBIs serve to remove compounds with such properties, but progress is limited by a lack of higher throughput methods with human translatability. Herein we apply human induced pluripotent stem cell (hiPSC) derived neurons in combination with dynamic mass redistribution (DMR) technology to demonstrate robust and reproducible modulation of both GABAA and glycine receptors. These cells respond to GABA (EC50 0.33⯱â¯0.18⯵M), glycine (EC50 11.0⯱â¯3.7⯵M) and additional ligands in line with previous reports from patch clamp technologies. Additionally, we identify and characterize a competitive antagonistic behavior of the prototype inhibitor and drug tranexamic acid (TXA). Finally, we demonstrate proof of concept for effective counter-screening of lead series compounds towards unwanted GABAA receptor activities. No activity was observed for a previously identified PBI candidate drug, AZD6564, whereas a discontinued analog, AZ13267257, could be characterized as a potent GABAA receptor agonist.
Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Inactivadores Plasminogénicos/farmacología , Receptores de GABA-A/metabolismo , Receptores de Glicina/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Glicina/farmacología , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Neuronas/efectos de los fármacos , Unión Proteica/fisiología , Receptores de Glicina/agonistas , Ácido Tranexámico/farmacología , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
In vivo studies of human brain cellular function face challenging ethical and practical difficulties. Animal models are typically used but display distinct cellular differences. One specific example is astrocytes, recently recognized for contribution to neurological diseases and a link to the genetic risk factor apolipoprotein E (APOE). Current astrocytic in vitro models are questioned for lack of biological characterization. Here, we report human induced pluripotent stem cell (hiPSC)-derived astroglia (NES-Astro) developed under defined conditions through long-term neuroepithelial-like stem (ltNES) cells. We characterized NES-Astro and astrocytic models from primary sources, astrocytoma (CCF-STTG1), and hiPSCs through transcriptomics, proteomics, glutamate uptake, inflammatory competence, calcium signaling response, and APOE secretion. Finally, we assess modulation of astrocyte biology using APOE-annotated compounds, confirming hits of the cholesterol biosynthesis pathway in adult and hiPSC-derived astrocytes. Our data show large diversity among astrocytic models and emphasize a cellular context when studying astrocyte biology.
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Astrocitos/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Células-Madre Neurales/fisiología , Neuronas/fisiología , Apolipoproteínas E/metabolismo , Astrocitos/metabolismo , Encéfalo/metabolismo , Encéfalo/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células-Madre Neurales/metabolismo , Neuronas/metabolismoRESUMEN
The RNA-binding and -processing protein TAR DNA-binding protein 43 (TDP-43) is heavily linked to the underlying causes and pathology of neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. In these diseases, TDP-43 is mislocalized, hyperphosphorylated, ubiquitinated, aggregated and cleaved. The importance of TDP-43 cleavage in the disease pathogenesis is still poorly understood. Here we detail the use of D-sorbitol as an exogenous stressor that causes TDP-43 cleavage in HeLa cells, resulting in a 35 kDa truncated product that accumulates in the cytoplasm within one hour of treatment. We confirm that the formation of this 35 kDa cleavage product is mediated by the activation of caspases. Inhibition of caspases blocks the cleavage of TDP-43, but does not prevent the accumulation of full-length protein in the cytoplasm. Using D-sorbitol as a stressor and caspase activator, we also demonstrate that the A90V variant of TDP-43, which lies adjacent to the caspase cleavage site within the nuclear localization sequence of TDP-43, confers partial resistance against caspase-mediated generation of the 35 kDa cleavage product.
Asunto(s)
Codón , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Variación Genética , Sustitución de Aminoácidos , Caspasas/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ADN/química , Células HeLa , Humanos , Mutación , Presión Osmótica , Estrés Oxidativo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas/efectos de los fármacos , Proteolisis , Sorbitol/farmacologíaRESUMEN
A connection between dementias and blood-brain barrier (BBB) dysfunction has been suggested, but previous studies have yielded conflicting results. We examined cerebrospinal fluid (CSF)/serum albumin ratio in a large cohort of patients diagnosed with Alzheimer's disease (AD, early onset [EAD, n = 130], late onset AD [LAD, n = 666]), vascular dementia (VaD, n = 255), mixed AD and VaD (MIX, n = 362), Lewy body dementia (DLB, n = 50), frontotemporal dementia (FTD, n = 56), Parkinson's disease dementia (PDD, n = 23), other dementias (other, n = 48), and dementia not otherwise specified (NOS, n = 271). We compared CSF/serum albumin ratio to 2 healthy control groups (n = 292, n = 20), between dementia diagnoses, and tested biomarker associations. Patients in DLB, LAD, VaD, MIX, other, and NOS groups had higher CSF/serum albumin ratio than controls. CSF/serum albumin ratio correlated with CSF neurofilament light in LAD, MIX, VaD, and other groups but not with AD biomarkers. Our data show that BBB leakage is common in dementias. The lack of association between CSF/serum albumin ratio and AD biomarkers suggests that BBB dysfunction is not inherent to AD but might represent concomitant cerebrovascular pathology.
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Demencia/diagnóstico , Albúmina Sérica , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Barrera Hematoencefálica/fisiopatología , Estudios de Cohortes , Estudios Transversales , Demencia/fisiopatología , Femenino , Humanos , Filamentos Intermedios , Masculino , Persona de Mediana EdadRESUMEN
Mutations in the gene TARDBP, which encodes TAR DNA-binding protein 43 (TDP-43), are a rare cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). While the majority of mutations are found in the C-terminal glycine-rich domain, an alanine to valine amino acid change at position 90 (A90V) in the bipartite nuclear localization signal (NLS) of TDP-43 has been described. This sequence variant has previously been shown to cause cytoplasmic mislocalization of TDP-43 and decrease protein solubility, leading to the formation of insoluble aggregates. Since the A90V mutation has been described both in patients as well as healthy controls, its pathogenic potential in ALS and FTD remains unclear. Here we compare properties of overexpressed A90V to the highly pathogenic M337V mutation. Though both mutations drive mislocalization of the protein to the cytoplasm to the same extent, M337V produces more significant damage in terms of protein solubility, levels of pathogenic phosphorylation, and formation of C-terminal truncated protein species. Furthermore, the M337V, but not the A90V mutant, leads to a downregulation of histone deacetylase 6 and Ras GTPase-activating protein-binding protein. We conclude that in the absence of another genetic or environmental 'hit' the A90V variant is not sufficient to cause the deleterious phenotypes associated with ALS and FTD, despite prominent cytoplasmic protein relocalization of TDP-43.