eoe sub articles

EoE Sub Articles

1. Preparation of materials required for iPSC culture and BEC differentiation.
<ol>
	<li>Matrix coating of tissue culture (TC) plastic for IMR90-4 iPSC culture
	<ol>
		<li>Aliquot basement membrane matrix gel (e.g., Matrigel) into 2.5 mg aliquots and store at -20 &#176;C. 
		NOTE: Work quickly when handling the matrix gel and aliquot on ice, as it forms a gel above 4 &#176;C and cannot be aliquoted once it has solidified.</li>
		<li>For coating TC plastic, quickly add one aliquot ...

generating brain endothelial cells from induced pluripotent stem cells

Source: Endres, L. M., et al., <a href="https://app.jove.com/v/61400">Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells.</a> J. Vis. Exp. (2020)
This video demonstrates a protocol to induce the differentiation of induced pluripotent stem cells into brain endothelial cells.

Generating Brain Endothelial Cells from Induced Pluripotent Stem Cells

1. Preparation of materials required for iPSC culture and BEC differentiation.

	Matrix coating of tissue culture (TC) plastic for IMR90-4 iPSC culture
	
	...

Take an induced pluripotent stem cell, or iPSC, culture in maintenance media containing rho-kinase inhibitors.
Growth factors keep cells undifferentiated, while rho-kinase inhibitors enhance cell survival.
Replace the media with fresh maintenance media, removing rho-kinase inhibitors. Incubate, promoting colony formation.
Regularly replace with unconditioned media, which provides a controlled environment, triggering iPSC differentiation into neural progenitors and then brain endothelial cells, followed by cell proliferation.
Replace with endothelial cell media containing retinoic acid and fibroblast growth factors. Incubate.
Fibroblast growth factors facilitate cell proliferation, while retinoic acid promotes brain endothelial cell maturation.
Add proteolytic enzymes to detach the cells. Collect the cells and centrifuge. Discard the supernatant.
Resuspend the cells in endothelial media and seed them onto a multi-well membrane insert coated with collagen and fibronectin.
The well contains endothelial media. Incubate for selective brain endothelial cell adhesion.
Replace the media with non-supplemented endothelial media for further experiments.

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Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

JoVE Journal

Developmental Biology

Differentiation of Human Induced Pluripotent Stem Cells to Brain Microvascular Endothelial Cell-Like Cells with a Mature Immune Phenotype

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous system (CNS). Due to the limited access to disease-related BBB samples, it is still not well understood whether BBB malfunction is causative for disease development or rather a consequence of the neuroinflammatory or neurodegenerative process. Human induced pluripotent stem cells (hiPSCs) therefore provide a novel opportunity to establish in vitro BBB models from healthy donors and patients, and thus to study disease-specific BBB characteristics from individual patients. Several differentiation protocols have been established for deriving brain microvascular endothelial cell (BMEC)-like cells from hiPSCs. Consideration of the specific research question is mandatory for the correct choice of the respective BMEC-differentiation protocol. Here, we describe the extended endothelial cell culture method (EECM), which is optimized to differentiate hiPSCs into BMEC-like cells with a mature immune phenotype, allowing the study of immune cell-BBB interactions. In this protocol, hiPSCs are first differentiated into endothelial progenitor cells (EPCs) by activating Wnt/&#946;-catenin signaling. The resulting culture, which contains smooth muscle-like cells (SMLCs), is then sequentially passaged to increase the purity of endothelial cells (ECs) and to induce BBB-specific properties. Co-culture of EECM-BMECs with these SMLCs or conditioned medium from SMLCs allows for the reproducible, constitutive, and cytokine-regulated expression of EC adhesion molecules. Importantly, EECM-BMEC-like cells establish barrier properties comparable to primary human BMECs, and due to their expression of all EC adhesion molecules, EECM-BMEC-like cells are different from other hiPSC-derived in vitro BBB models. EECM-BMEC-like cells are thus the model of choice for investigating the potential impact of disease processes at the level of the BBB, with an impact on immune cell interaction in a personalized fashion.

Here, we describe a protocol, the extended endothelial cell culture method (EECM), that allows differentiation of pluripotent stem cells to brain microvascular endothelial cell (BMEC)-like cells. These cells show endothelial cell adhesion molecule expression and are thus a human blood-brain barrier model suitable to study immune cell interactions in vitro.

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous ...

Generating Brain Endothelial Cells from Induced Pluripotent Stem Cells

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