eoe sub articles

EoE Sub Articles

1. Microglia differentiation
NOTE: An overview of the protocol is summarized in&#160;Figure 1.
<ol>
	<li>Induced pluripotent stem cell (iPSC) culture 
	NOTE: Further details describing iPSC culture techniques can be found elsewhere.
	<ol>
		<li>Thawing and maintenance
		<ol>
			<li>Prepare&#160;the iPSC medium, aliquot it, and store it at -20 &#176;C for up to 6 months. Thaw the aliquots overnight at 4 &#176;C and use t...

differentiating human-induced pluripotent stem cells into microglia-like cells

Source: Funes, S. et al., <a href="https://app.jove.com/v/64323">Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes.</a>&#160;J. Vis. Exp. (2022)
This video demonstrates the procedure of the differentiation of human pluripotent cells into microglia-like cells. The process involves forming embryoid bodies, transferring them to coated wells, and using specific growth factors to guide differentiation into primitive macrophage precursors, which finally differentiate into microglia-like cells.

<img alt="Figure 1" src="/files/ftp_upload/22611/22611_Figure 1.jpg" /> 
Figure 1: Schematic of the protocol to differentiate iPSCs into microglia-like cells.&#160;On day 0, once iPSCs have reached 80% confluency, the colonies are dissociated into single cells and cultured in EB medium to induce EB formation. On day 4, EBs are collected and plated in PMP medium to induce PMP generation. After 28 days, PMPs are collected and terminally differentiated into iMGs by usin...

Differentiating Human-Induced Pluripotent Stem Cells into Microglia-Like Cells

1. Microglia differentiation
NOTE: An overview of the protocol is summarized in&#160;Figure 1.

	Induced pluripotent stem cell (iPSC) culture
	NOTE: ...

Begin with a low-adherence multi-well plate containing human-induced pluripotent stem cells in an embryoid body or EB medium.
Centrifuge to settle the cells. Incubate to promote cell proliferation and aggregation, forming embryoid bodies.
The cell lineage regulators promote cell differentiation into myeloid progenitors.
Collect these EBs and transfer them to the edge of an extracellular matrix or ECM-coated plate.
Replace the medium with a primitive macrophage precursor or PMP differentiation medium and gently agitate. Incubate to facilitate the EBs&#39; attachment to the ECM.
Replace half of the medium and incubate.
The hematopoietic growth factors drive myeloid progenitors to differentiate into primitive macrophage precursors, which separate from the EBs and appear in the medium.
Collect these PMPs and spin down the cells. Resuspend them in a microglia differentiation medium.
Seed these cells in a laminin-coated multi-well plate and incubate.
PMPs utilize the differentiation factors and nutrients, differentiating into microglia-like cells, a type of brain immune cell.

differentiating-human-induced-pluripotent-stem-cells-into-microglia

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

iPSC Differentiation into Posterior Primitive Streak and Nephrogenic Intermediate Mesoderm

IPSC Differentiation into Posterior Primitive Streak and Nephrogenic Intermediate Mesoderm  

Begin by washing the induced pluripotent stem cells or iPSCs on the membrane matrix coated 6-well plate with two milliliters DPBS. Aspirate the DPBS using a pipette. Next, add one milliliter of the commercially available cell detachment solution to detach the iPSCs.Then place the cell into an incubator at 37 degrees for five minutes. Now pipette one milliliter of mTeSR onto the iPSCs, ensuring that the cells have separated from the plastic surface. Centrifuge these cells at 400g for three minutes at room temperature.Resuspend the cell pellet, then count the cell numbers using a hemocytometer. Next seed a range of cell densities on a 6-well plate that has been coated with a membrane matrix. Culture these with mTeSR supplemented with 10 micromolar Rho-kinase inhibitor.Culture the plates overnight in a carbon dioxide incubator set at 37 degrees Celsius. The next day aspirate the mTeSR and wash the cells with two milliliters of DPBS. Next, add two milliliters of stage 1 medium to the cells.Then place the cells in a 37 degrees Celsius incubator for four days. On day four, remove the stage 1 medium and wash the cells with two milliliters of DPBS. Then add two milliliters of stage 2 medium to the cells before incubating the cells as before.From day zero to day four, iPSCs rapidly expanded and took on rhomboid or triangular shapes. The confluence reached 90 to 100%and accumulated evenly until day seven. Upon suspension culture, the aggregates spontaneously form nephron structures after dissociating on day seven.

JoVE Journal

Developmental Biology

﻿Differentiation of hiPSCs into Endothelial Cells

Differentiation of hiPSCs into Endothelial Cells  

To begin, on day zero, pre-warm serum-free differentiation medium or SFD medium containing Mix 1 cytokine to 37 degrees Celsius. Then, under a sterile tissue culture hood, add the Mix 1 cytokine-supplemented medium to each well of a cell repellent six-well plate. To prepare the cells, aspirate the culture medium from a six-well plate containing human-induced pluripotent STEM cells or human IPS cells.Wash them with DPBS followed by aspirating the DPBS. Then, add the dissociation reagent to the cells and incubate them at room temperature for a minute. After aspirating this dissociation reagent, incubate the cells for another three minutes at room temperature.Then, tap the plate containing the cells 10 times on each side to detach the cell clusters. Add one milliliter of pre-warmed Mix 1 cytokine-supplemented SFD medium to the cells per well. Using a pasture pipette, transfer the cell clusters from each well to one well of the cell repellent plate containing the medium with Mix 1 cytokine.After introducing the plate into the incubator, move it back and forth, as well as side to side, to disperse the content evenly and incubate it for embryoid bodies, or EB, formation. The next day, swirl the cell repellent plate containing the EBs to collect them into the center of the wells. Using a pasteur pipette, transfer the EB suspension from the wells to a 15-milliliter centrifuge tube.Wait five to 10 minutes for the EBs to settle at the bottom of the tube. Meanwhile, wash the cell repellent plates with sterile water or DPBS to remove single cells or debris. Add two milliliters of Mix 1 cytokine-supplemented SFD medium to each well of the plate.Now, gently aspirate the supernatant from the centrifuge tube without dislodging the EBs. Resuspend the EBs in the calculated volume of SFD medium containing Mix 1 cytokine. Add one milliliter of EB suspension to each well of the washed cell repellent plate already containing two milliliters of medium.Incubate the plate after dispersing the EBs by gently moving the plate as mentioned before. After gathering the EBs at the center of the wells, add the chiron on the side of each well, avoiding direct contact with the cells, Incubate the wells as demonstrated previously. On days three and six of differentiation, collect the EBs and perform media changes as demonstrated previously for day one.Use SFD medium supplemented with Mix 3 cytokines on day three and use SFD medium supplemented with Mix 4 cytokines on day six. After collecting and settling the EBs from the cell repellent plate into a 15-milliliter centrifuge tube, as demonstrated previously, aspirate the supernatant, then add one milliliter of cell dissociation reagent per well of EB collected in the tube to resuspend the EBs. Next, transfer one milliliter of this EB suspension to each well of the cell repellent plate washed with water.After incubating the plate for 10 minutes in the incubator, use a P1000 pipette to dissociate the EBs in each well by gently pipetting up and down no more than 10 times. Then, add five milliliters of washing buffer per well of dissociated EBs. Collect the cells into a 50-milliliter centrifuge tube by passing them through a 40-micron strainer.After counting the cells in the filtered suspension, spin the cells down for 10 minutes at 300g. Resuspend the cells in 300 microliters of washing buffer by gently pipetting a few times, ensuring no clumps are present. Proceed to isolate the CD34-positive cells using a CD34 microbead kit per the manufacturer's instructions.EBs of good quality showed a defined edge by day two of the differentiation and appeared clear and bright when observed under a microscope. The presence of darker areas indicated cell death within the EBs. Following chiron treatment with varying concentrations on day two, quantifying the number of CD34-positive cells at day eight of differentiation revealed the response of the cell line to the treatment.Flow cytometry showed that the CD34-positive enrichment using the magnetic beads provided about 85%CD34-positive cells.

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key players in health and disease, acting as phagocytes during immune defense and mediating trophic, maintenance, and repair functions. Although it has been possible to study some of the molecular processes involved in human macrophage function, it has proved difficult to apply genetic engineering techniques to primary human macrophages. This has significantly hampered our ability to interrogate the complex genetic pathways involved in macrophage biology and to generate models for specific disease states. An off-the-shelf source of human macrophages that is amenable to the vast arsenal of genetic manipulation techniques would, therefore, provide a valuable tool in this field. We present an optimized protocol that allows for the generation of macrophages from human induced pluripotent stem cells (iPSCs) in vitro. These iPSC-derived macrophages (iPSC-DMs) express human macrophage cell surface markers, including CD45, 25F9, CD163, and CD169, and our live-cell imaging functional assay demonstrates that they exhibit robust phagocytic activity. Cultured iPSC-DMs can be activated to different&#160;macrophage states that display altered gene expression and phagocytic activity by the addition of LPS and IFNg, IL4, or IL10. Thus, this system provides a platform to generate human macrophages carrying genetic alterations that model specific human disease and a source of cells for drug screening or cell therapy to treat these diseases.

This protocol describes the robust generation of macrophages from human induced pluripotent stem cells, and methods for their subsequent characterization. Cell surface marker expression, gene expression, and functional assays are used to assess the phenotype and function of these iPSC-derived macrophages.

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key ...

Differentiating Human-Induced Pluripotent Stem Cells into Microglia-Like Cells

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