Differentiation of Human-Induced Pluripotent Stem Cells into Macrophages

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.

1. Preparing cell differentiation growth factors and other reagents
   1. 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 bovine serum albumin (BSA), and 0.1 mM 2-mercaptoethanol.
   2. Prepare 0.1% w/v solution of porcine gelatin by dissolving the gelatin into sterile ....

Results

Figure 1: Graphic summary of iPSC differentiation to mature functional macrophages. Diagram drawn with Biorender.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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