eoe sub articles

EoE Sub Articles

1. Human iPSC Differentiation to Macrophages
NOTE: A schematic summary of the macrophage differentiation protocol is depicted in Figure 1.
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	<li>Preparing cell differentiation growth factors and other reagents
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		<li>Prepare human embryonic stem cell-serum-free media (hESC-SFM; see Table of Materials) by supplementing Dulbecco&#39;s modified Eagle medium-F12 (DMEM/F12) with hESC supplement, 1.8% w/v...

differentiation of human-induced pluripotent stem cells into macrophages

Source: Lopez-Yrigoyen, M., et al. <a href="https://app.jove.com/v/61038">Production and Characterization of Human Macrophages from Pluripotent Stem Cells.</a> J. Vis. Exp. (2020).
This video describes an in vitro method for generating macrophages from human induced pluripotent stem cells (iPSCs). Macrophage generation from iPSCs occurs in three stages: (i) aggregation of iPSCs in suspension to form three-dimensional embryoid bodies (EBs) that differentiate into mesodermal cells, (ii) formation of macrophage precursor cells from the differentiated EBs, and (iii) differentiation of the macrophage precursor cells into mature, functional macrophages.

<img alt="Figure 1" src="/files/ftp_upload/21516/21516_Figure1.jpg" /> 
Figure 1: Graphic summary of iPSC differentiation to mature functional macrophages. Diagram drawn with Biorender.
<img alt="Figure 2" src="/files/ftp_upload/21516/21516_Figure2.jpg" /> 
Figure 2: iPSC differentiation towards macrophages and iPSC-DM number and morphology. (A) Bright Field images obt...

Differentiation of Human-Induced Pluripotent Stem Cells into Macrophages

Source: Lopez-Yrigoyen, M., et al. Production and Characterization of Human Macrophages from Pluripotent Stem Cells. J. Vis. Exp. (2020).
This video ...

Adult somatic cells can be genetically reprogrammed to obtain induced pluripotent stem cells, iPSCs, that exhibit stem cell-like undifferentiated and proliferative states.&#160;
To differentiate human iPSCs into macrophages &#8212; white blood cells that phagocytose pathogens and cellular debris &#8212; begin with a culture plate with a non-cell adhesive surface.
Add media containing the growth factors bone morphogenetic protein, stem cell factor, and vascular endothelial growth factor. Seed the iPSCs and incubate.
The non-adhesive surface prevents iPSC attachment and maintains the cells in suspension, facilitating aggregation into three-dimensional embryoid bodies, EBs. The growth factors initiate iPSC differentiation within the EBs into mesodermal lineage cells.
Transfer the differentiated EBs to a centrifuge tube. Allow them to settle through gravity. Resuspend the EBs in fresh media containing interleukin-3, IL3, and macrophage colony-stimulating factor, CSF1. Plate the EBs on a gelatin-coated cell culture plate.
Gelatin &#8212; an extracellular matrix protein &#8212; facilitates EB attachment. IL3 stimulates the EBs to differentiate into hematopoietic stem-progenitor cells, giving rise to myeloid cells. CSF1 supports myeloid cell proliferation and their differentiation into macrophage precursors that get released and remain in suspension in the media.
Collect the suspension cells and centrifuge. Resuspend the cells in CSF1-supplemented media.
Plate the cells on uncoated culture plates.
CSF1 induces terminal differentiation of the macrophage precursors into fully-functional, adherent mature macrophages.

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Research

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Cell Differentiation Techniques

This video describes an in vitro method for generating macrophages from human induced pluripotent stem cells (iPSCs). Macrophage generation from iPSCs occurs in three stages: (i) aggregation of iPSCs in suspension to form three-dimensional embryoid bodies (EBs) that differentiate into mesodermal cells, (ii) formation of macrophage precursor cells from the differentiated EBs, and (iii) differentiation of the macrophage precursor cells into mature, functional macrophages.

Overview

Protocol

<img title="figure-representative results-58" alt="figure-representative results-58" src="/files/ftp_upload/21516/21516_Figure1.jpg" /> 
Figure 1: Graphic summary of iPSC differentiation to mature functional macrophages. Diagram drawn with Biorender.
<img title="figure-representative results-327" alt="figure-representative results-327" src="/files/ftp_upload/21516/21516_Figure2.jpg" /> 
Figure 2: iPSC differentiation towards macrophages and iPSC-DM number and morphology. (A) Bright Field images obt...

Differentiation of Human-Induced Pluripotent Stem Cells into Macrophages

In This Article

Overview

Protocol

Representative Results