Abstract TP281: Transcriptome Analysis of the Human Brain Microvascular Endothelial Cells During and After Oxygen-Glucose Deprivation Stress as a Tool to Identify Novel Targets

Background: The blood-brain barrier (BBB) constitutes an important component of the neurovascular unit (NVU) by providing a physical and a chemical barrier critical for the brain homeostasis. The disruption of the BBB during cerebral ischemia constitutes a key event of the disease, resulting in its opening and ultimately the formation of cerebral edema. Therefore, targeting such BBB disruption by restoring the barrier function could mitigate such insult. In this study, we investigated change in gene expression profile of the human BBB in vitro using an induced pluripotent stem cell (iPSC) based model. Methods: BMECs derived from two human iPSC lines (CTR90F and CTR65M) were exposed to acute OGD stress (1% O2, no glucose) for 6h, followed by reoxygenation (21% O2, 1g/L glucose) for 18h and compared to control (21% O2, 1g/L glucose, 24h) Total RNA was extracted for cDNA microarray analysis to the University of Texas Southwestern Genomic Core Facility using a ClariomD® chip. Fold-change >2 was considered statistically significant (P<0.05). Results: OGD/reoxygenation resulted in almost 3700 genes differentially expressed, including over 600 coding genes. Most of such differential expression occurred during the OGD phase. Reoxygenation yielded to quasi-similar expression profile than controls. Several original pathways have been identified and include nuclear receptors, PI3K/Akt signaling pathways, microRNAs, and adhesion signaling pathways. Conclusions: Our preliminary findings suggest that our iPSC-derived model of the BBB based on BMECs showed an overlap of several genes commonly identified as hypoxic/ischemic responsive genes, but also highlighted novel signaling pathways that have little or no literature on their function at the BBB. We are currently refining our list of genes and focusing our attention on genes that have shown the highest enrichment or depletion.

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An in vitro bioengineered model of the human arterial neurovascular unit to study neurodegenerative diseases

10.21203/rs.3.rs-33897/v1 ◽

2020 ◽

Author(s):

Jerome Robert ◽

Nicholas Weilinger ◽

Li-Ping Zao ◽

Stefano Cataldi ◽

Emily Button ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Cortical Neurons ◽

Neurovascular Unit ◽

Induced Pluripotent Stem Cell ◽

Cell Types ◽

Experimental Models ◽

Flow Conditions ◽

Anatomy And Physiology ◽

Induced Pluripotent

Abstract Introduction: The neurovascular unit (NVU) – the interaction between the neurons and the cerebrovasculature – is increasingly important to interrogate through human-based experimental models. Although advanced models of cerebral capillaries have been developed in the last decade, there is currently noin vitro3-dimensional (3D) perfusible model of the human cortical arterial NVU. Method: We used a tissue-engineering technique to develop a scaffold-directed, perfusible, 3D human NVU that is cultured in native-like flow conditions that mimics the anatomy and physiology of cortical penetrating arteries. Results: This system, composed of primary human vascular cells (endothelial cells, smooth muscle cells and astrocytes) and induced pluripotent stem cell (iPSC) derived neurons, demonstrates a physiological multilayer organization of the involved cell types. It also reproduces key characteristics of cortical neurons and astrocytes, as well as the formation of a selective and functional endothelial barrier. We further provide proof-of-principle that our in vitro human arterial NVU may be suitable to study neurodegenerative diseases such as Alzheimer’s disease (AD), as we report both phosphorylated tau and beta-amyloid accumulation in our model over time. Finally, we show that our arterial NVU model enables the study of neuronal and glial fluid biomarkers. Conclusion: This model is a suitable tool to investigate arterial NVU functions such as neuronal electrophysiology in health and disease. Further the design of platform allows culture under native-like flow conditions for extended periods of time and yields sufficient tissue and media for downstream immunohistochemistry and biochemistry analyses.

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An in vitro bioengineered model of the human arterial neurovascular unit to study neurodegenerative diseases

10.21203/rs.3.rs-33897/v2 ◽

2020 ◽

Author(s):

Jerome Robert ◽

Nicholas Weilinger ◽

Li-Ping Zao ◽

Stefano Cataldi ◽

Emily Button ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Cortical Neurons ◽

Neurovascular Unit ◽

Induced Pluripotent Stem Cell ◽

Cell Types ◽

Experimental Models ◽

Flow Conditions ◽

Anatomy And Physiology ◽

Induced Pluripotent

Abstract Introduction: The neurovascular unit (NVU) – the interaction between the neurons and the cerebrovasculature – is increasingly important to interrogate through human-based experimental models. Although advanced models of cerebral capillaries have been developed in the last decade, there is currently no in vitro 3-dimensional (3D) perfusible model of the human cortical arterial NVU.Method: We used a tissue-engineering technique to develop a scaffold-directed, perfusible, 3D human NVU that is cultured in native-like flow conditions that mimics the anatomy and physiology of cortical penetrating arteries.Results: This system, composed of primary human vascular cells (endothelial cells, smooth muscle cells and astrocytes) and induced pluripotent stem cell (iPSC) derived neurons, demonstrates a physiological multilayer organization of the involved cell types. It reproduces key characteristics of cortical neurons and astrocytes and enables formation of a selective and functional endothelial barrier. We provide proof-of-principle data showing that this in vitro human arterial NVU may be suitable to study neurovascular components of neurodegenerative diseases such as Alzheimer’s disease (AD), as endogenously produced phosphorylated tau and beta-amyloid accumulate in the model over time. Finally, neuronal and glial fluid biomarkers relevant to neurodegenerative diseases are measurable in our arterial NVU model.Conclusion: This model is a suitable research tool to investigate arterial NVU functions in healthy and disease states. Further, the design of the platform allows culture under native-like flow conditions for extended periods of time and yields sufficient tissue and media for downstream immunohistochemistry and biochemistry analyses.

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An in vitro bioengineered model of the human arterial neurovascular unit to study neurodegenerative diseases

Molecular Neurodegeneration ◽

10.1186/s13024-020-00418-z ◽

2020 ◽

Vol 15 (1) ◽

Author(s):

Jerome Robert ◽

Nicholas L. Weilinger ◽

Li-Ping Cao ◽

Stefano Cataldi ◽

Emily B. Button ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Cortical Neurons ◽

Neurovascular Unit ◽

Induced Pluripotent Stem Cell ◽

Cell Types ◽

Experimental Models ◽

Flow Conditions ◽

Anatomy And Physiology ◽

Induced Pluripotent

Abstract Introduction The neurovascular unit (NVU) – the interaction between the neurons and the cerebrovasculature – is increasingly important to interrogate through human-based experimental models. Although advanced models of cerebral capillaries have been developed in the last decade, there is currently no in vitro 3-dimensional (3D) perfusible model of the human cortical arterial NVU. Method We used a tissue-engineering technique to develop a scaffold-directed, perfusible, 3D human NVU that is cultured in native-like flow conditions that mimics the anatomy and physiology of cortical penetrating arteries. Results This system, composed of primary human vascular cells (endothelial cells, smooth muscle cells and astrocytes) and induced pluripotent stem cell (iPSC) derived neurons, demonstrates a physiological multilayer organization of the involved cell types. It reproduces key characteristics of cortical neurons and astrocytes and enables formation of a selective and functional endothelial barrier. We provide proof-of-principle data showing that this in vitro human arterial NVU may be suitable to study neurovascular components of neurodegenerative diseases such as Alzheimer’s disease (AD), as endogenously produced phosphorylated tau and beta-amyloid accumulate in the model over time. Finally, neuronal and glial fluid biomarkers relevant to neurodegenerative diseases are measurable in our arterial NVU model. Conclusion This model is a suitable research tool to investigate arterial NVU functions in healthy and disease states. Further, the design of the platform allows culture under native-like flow conditions for extended periods of time and yields sufficient tissue and media for downstream immunohistochemistry and biochemistry analyses.

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Use of 3D culture systems to generate human induced pluripotent stem cell-derived [beta]-cells in vitro

Endocrine Abstracts ◽

10.1530/endoabs.57.003 ◽

2018 ◽

Author(s):

Fantuzzi Federica ◽

Toivonen Sanna ◽

Schiavo Andrea Alex ◽

Pachera Nathalie ◽

Rajaei Bahareh ◽

...

Keyword(s):

Stem Cell ◽

Beta Cells ◽

Pluripotent Stem Cell ◽

3D Culture ◽

Induced Pluripotent Stem Cell ◽

Culture Systems ◽

Induced Pluripotent

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Bestrophinopathies: perspectives on clinical disease, Bestrophin-1 function and developing therapies

Therapeutic Advances in Ophthalmology ◽

10.1177/2515841421997191 ◽

2021 ◽

Vol 13 ◽

pp. 251584142199719

Author(s):

Simranjeet Singh Grewal ◽

Joseph J. Smith ◽

Amanda-Jayne F. Carr

Keyword(s):

Pigment Epithelium ◽

Retinal Pigment ◽

Induced Pluripotent Stem Cell ◽

Underlying Disease ◽

Incomplete Penetrance ◽

Inherited Retinal Dystrophies ◽

Retinal Dystrophies ◽

High Acuity ◽

Induced Pluripotent

Bestrophinopathies are a group of clinically distinct inherited retinal dystrophies that typically affect the macular region, an area synonymous with central high acuity vision. This spectrum of disorders is caused by mutations in bestrophin1 ( BEST1), a protein thought to act as a Ca2+-activated Cl- channel in the retinal pigment epithelium (RPE) of the eye. Although bestrophinopathies are rare, over 250 individual pathological mutations have been identified in the BEST1 gene, with many reported to have various clinical expressivity and incomplete penetrance. With no current clinical treatments available for patients with bestrophinopathies, understanding the role of BEST1 in cells and the pathological pathways underlying disease has become a priority. Induced pluripotent stem cell (iPSC) technology is helping to uncover disease mechanisms and develop treatments for RPE diseases, like bestrophinopathies. Here, we provide a comprehensive review of the pathophysiology of bestrophinopathies and highlight how patient-derived iPSC-RPE are being used to test new genomic therapies in vitro.

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