eoe sub articles

EoE Sub Articles

1. Stem Cell Maintenance
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	<li>Maintain H9 hESCs on a layer of growth factor reduced basement membrane matrix (see the Table of Materials, henceforth simply referred to as matrix) according to the manufacturer&#39;s instructions.
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		<li>To coat one 6-well plate or one 10 cm dish, combine 100 &#181;L of matrix with 5.9 mL of ice-cold Dulbecco&#39;s modified Eagle medium (DMEM)/F12 media. Wrap plates in paraffin film and store overnight at 4 &#176;C. Use them on the next day for...

a technique to generate brain organoids from human embryonic stem cells

Source: Boisvert, E. M., et al. <a href="https://app.jove.com/v/60379">A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells.</a> J. Vis. Exp &#160;(2020).
This video demonstrates a technique for generating cerebral organoids from human embryonic stem cells (hESCs). The adherent culture of hESC colonies is treated with enzymes to detach and break them into clusters. These clusters form three-dimensional spheres in suspension, which are then induced to differentiate into organoids that mimic the brain cortex with layers of neurons and glial cells.

A Technique to Generate Brain Organoids from Human Embryonic Stem Cells

1. Stem Cell Maintenance

	Maintain H9 hESCs on a layer of growth factor reduced basement membrane matrix (see the Table of Materials, henceforth simply ...

Take colonies of human embryonic stem cells, or hESCs, cultured on a supporting matrix.
Aspirate the medium and add enzymes that degrade the matrix.
Wash to remove the enzymes. Then, add a medium and pipette to dislodge the loosened colonies, breaking them into smaller clusters.
Transfer the clusters into a fresh medium and incubate, allowing the clusters to form spherical aggregates.
Allow the spheres to settle and remove any dead cells.
Incubate with a medium containing basic fibroblast growth factor, or bFGF, for cell proliferation.
Replenish with media containing gradually decreasing bFGF concentrations to facilitate cell differentiation.
Introduce a neural induction medium, promoting hESC differentiation into neural stem cells or NSCs, transforming the spheres into organoids exhibiting a neural rosette-like structure.
Add antibiotics to prevent the growth of any contaminating microbes during prolonged culture.
The NSCs differentiate into neurons and glia, progressively developing the organoids into a complex structure resembling a brain cortex.

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Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in vitro. Developing more efficient organ-like platforms that can model complex in vivo phenotypes, such as organoids and organs-on-a-chip, will enhance the ability to study human heart development and disease. This paper describes a protocol to generate highly complex human heart organoids (hHOs) by self-organization using human pluripotent stem cells and stepwise developmental pathway activation using small molecule inhibitors. Embryoid bodies (EBs) are generated in a 96-well plate with round-bottom, ultra-low attachment wells, facilitating suspension culture of individualized constructs.
The EBs undergo differentiation into hHOs by a three-step Wnt signaling modulation strategy, which involves an initial Wnt pathway activation to induce cardiac mesoderm fate, a second step of Wnt inhibition to create definitive cardiac lineages, and a third Wnt activation step to induce proepicardial organ tissues. These steps, carried out in a 96-well format, are highly efficient, reproducible, and produce large amounts of organoids per run. Analysis by immunofluorescence imaging from day 3 to day 11 of differentiation reveals first and second heart field specifications and highly complex tissues inside hHOs at day 15, including myocardial tissue with regions of atrial and ventricular cardiomyocytes, as well as internal chambers lined with endocardial tissue. The organoids also exhibit an intricate vascular network throughout the structure and an external lining of epicardial tissue. From a functional standpoint, hHOs beat robustly and present normal calcium activity as determined by Fluo-4 live imaging. Overall, this protocol constitutes a solid platform for in vitro studies in human organ-like cardiac tissues.

Here, we describe a protocol to create developmentally relevant human heart organoids (hHOs) efficiently using human pluripotent stem cells by self-organization. The protocol relies on the sequential activation of developmental cues and produces highly complex, functionally relevant human heart tissues.

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in ...

JoVE Journal

Biochemistry

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Developmental Biology

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental defects whose underlying mechanisms remain to be elucidated. A major roadblock is the lack of effective models recapitulating the early-onset neuronal impairment seen in the patients. Advances in the technology of induced pluripotent stem cells (iPSCs) enable the generation of three-dimensional (3D) brain organoids that can be used to investigate the impact of diseases on the development and organization of the nervous system. Researchers, including these authors, have recently introduced human brain organoids to model mitochondrial disorders. This paper reports a detailed protocol for the robust generation of human iPSC-derived brain organoids and their use in mitochondrial bioenergetic profiling and imaging analyses. These experiments will allow the use of brain organoids to investigate metabolic and developmental dysfunctions and may provide crucial information to dissect the neuronal pathology of mitochondrial diseases.

We describe a detailed protocol for the generation of human induced pluripotent stem cell-derived brain organoids and their use in modeling mitochondrial diseases.

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental ...

A Technique to Generate Brain Organoids from Human Embryonic Stem Cells

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