Differentiation of Human-Induced Pluripotent Stem Cells into Macrophages

Published: July 31, 2023

Abstract

Source: Lopez-Yrigoyen, M., et al. Production and Characterization of Human Macrophages from Pluripotent Stem Cells. 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.

Protocol

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

Representative Results

Figure 1: Graphic summary of iPSC differentiation to mature functional macrophages. Diagram drawn with Biorender. Figure 2: iPSC differentiation towards macrophages and iPSC-DM number and morphology. (A) Bright Field images obt…

Declarações

The authors have nothing to disclose.

Materials

CellMask Deep Red Plasma Membrane Stain Thermofisher C10046 Cryopreservation media
Cryostor CS10 Sigma C2874
CTS CELLstart Substrate Invitrogen A1014201 Stem cell substrate
Porcine Skin Gelatin Sigma G9136
Recombinant Human BMP4 Protein R&D 314-BP-010
Recombinant Human IL3 Preprotech 0200-03-10
Recombinant Human MCSF (carrier-free) 100ug Biolegend 574806
Recombinant Human VEGF Protein R&D 293-VE-010
SCF (C-Kit Ligand) Recombinant Human Protein Thermofisher PHC2111
StemPro hESC SFM Thermofisher A1000701
StemPro EZPassage Disposable Stem Cell Passaging Tool Thermofisher 23181010
Ultralow attachment plates: Cell culture multi-well plate, 6 well, cell star cell repellent surface Greiner 657970
2-Mercaptoethanol (50 mM)  Invitrogen  31350010
DPBS, calcium, magnesium (500ml) Thermofisher  14040091
GlutaMAX Supplement  Thermofisher  35050061

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Differentiation of Human-Induced Pluripotent Stem Cells into Macrophages. J. Vis. Exp. (Pending Publication), e21516, doi: (2023).

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