Human iPSC-Derived Macrophages

AC Immune 3.4.17 IBA1Phalloidin3

iPSC-derived macrophages

Axol iPSC-Derived Macrophages are derived from iPSCs that have undergone an intermediate differentiation to monocytes prior to directed terminal differentiation to macrophages. This provides a highly pure and consistently reproducible population of macrophages. To support the growth and maintenance of Axol iPSC-Derived Macrophages, we offer a chemically defined, serum-free, fully optimized Macrophage Maintenance Medium.

One of the key functions of macrophages is to clean up! More specifically, by engulfing and destroying foreign substances, cell debris and microbes. Our iPSC-Derived Macrophages are highly phagocytic and express the ionized calcium-binding adapter molecule 1 (IBA1), which is typically expressed by macrophages upon activation.

When recruited to the site of injury and infection macrophages have the ability to differentiate to specific macrophage subtypes dependent on their location within the body. Within the lungs, pulmonary macrophages are a good example of tissue-specific macrophages, these cells function to protect the lungs by removing inhaled and deposited particles. iPSC-derived macrophages could be used in co-culture to model and investigate tissue-specific macrophages.

Axol iPSC-Derived Macrophages are shipped live providing an easy-to-use, assay-ready system for modeling inflammatory and immune responses.


Human iPSC-derived macrophages

Cat. No. Product Name Quantity Price
ax0601 Human iPSC-Derived Macrophages (Male) 96-well plate $2400
ax0606 Human iPSC-Derived Macrophages (Female) 96-well plate $2400

Macrophage culture media and reagents

Cat. No. Product Name Product Description Quantity Price
ax0600 Macrophage Maintenance Medium Medium for the maintenance of iPSC-Derived Macrophages 250 ml $160

Phase Contrast

Phase contrast images show the maturation of Axol Human iPSC-Derived Macrophages over one week post-differentiation from ipsc-derived monocytes.

Phase contrast images of Axol Human iPSC-Derived Macrophages

Phase contrast images of Axol Human iPSC-Derived Macrophages at days 3 (10x) and 7 (10x and 40x) post-differentiation. Human iPSC-derived monocytes are seeded using Macrophage Maintenance Medium (ax0600) into 96-well plates at a density of 50,000 cells/cm2. During maturation the spherical monocytes adhere strongly to the culture surface and as they differentiate to macrophages their morphology becomes rounded and elongated. Axol iPSC-Derived Macrophages show numerous large vesicles indicative of phagocytic entities.


Immunocytochemistry

Axol iPSC-Derived Macrophages show expression of the ionized calcium-binding adapter molecule 1 (IBA1), which is typically expressed by macrophages/microglia upon activation.

Expression of IBA1 in Axol iPSC macrophages confirmed using ICC

Expression of the IBA1 protein was confirmed in Axol iPSC-Derived Macrophage using immunocytochemistry (A and B). iPSC-Macrophages were cultured in Macrophage Maintenance Medium (ax0600) for 1 (A) and 4 (B) days post differentiation before staining for IBA1 and phalloidin. Phalloidin staining (red) confirmed the presence of F-actin filaments (B) and both images were counterstained with DAPI.


Flow Cytometry

Axol iPSC-Derived Macrophages show strong positivity for CD45, CD14 and CD11b and minimal staining for HLA-DR.

Flow cytometry show expression of CD45, CD14, CD11b in Axol iPS cell macrophages

Flow cytometry for key cell surface markers of innate immunity. Axol iPSC-Derived Macrophages (ax0606) were harvested at day 7 using cold 1x PBS (containing 5 mM EDTA). The markers used were CD45 (APC fluorophore, ImmunoTools), CD14 (PE fluorophore, ImmunoTools), CD11b (APC fluorophore, ImmunoTools). Axol macrophages are strongly positive for CD14, CD11b and CD45 . HLA-DR staining is minimal/negative as expected for unstimulated cells.


Phagocytosis Assay

Cytochalasin D (CytoD) inhibits phagocytosis in Axol iPSC-Derived Macrophages

Cytochalasin D inhibits phagocytosis in Axol iPSC-Derived Macrophages

Phagocytosis inhibition assay in Axol iPSC-Derived Macrophages. The macrophages were incubated with blue FluoSpheres and then treated with 5 μM CytoD or left untreated for control conditions. The macrophages were imaged live after 30, 60 and 90 mins. The DAPI beads can be seen in large vesicles of the macrophages in the untreated culture. In comparison, the CytoD-treated culture shows smaller macrophages with the DAPI beads in the medium. Data provided by Dr Will Stebbeds and Dr Graham Smith (Charles River Laboratories).

Axol IPSC derived Macrophage phagocytosis assay1
Axol IPSC derived Macrophage phagocytosis assay bar graph

Phagocytosis inhibition assay in Axol iPSC-Derived Macrophages. The macrophages were co-incubated with 1:100 or 1:250 dilution of blue FluoSpheres. The cultures were then treated with 5 μM CytoD or left untreated for control conditions. The macrophages were imaged live and showed that CytoD successfully inhibited bead uptake with no difference with the dilution of FluoSpheres. In the untreated condition the iPSC-derived macrophages have engulfed the blue FluoSpheres which can be seen by their disperse presence in the vesicles of the macrophages. CytoD treated has prevented phagocytosis resulting in blue FluoSpheres on the periphery of the cells. Data provided by Dr Will Stebbeds and Dr Graham Smith (Charles River Laboratories).

Stimulation and inhibition of phagocytosis in Axol iPSC-Derived Macrophages

phagocytosis LPS stimulationICC
phagocytosis LPS stimulation
phagocytosis CytoDinhibition curve

Activation and inhibition of phagocytosis in iPSC-Derived Macrophages iPSC-Macrophages were treated with 10 mM CytoD or 10ng/mL lipopolysaccharide (LPS) at day 7 of maturation from iPSC-monocytes to assess for further activation of phagocytic activity or inhibition of phagocytosis with CytoD. Treatment with 10ng/ml LPS shows a significant increase in phagocytosis; whereas treatment with CytoD shows a significant inhibition of phagocytosis (A). **** p<0·0001 compared to untreated; *** p<0·001 compared to untreated. Treatment with varying concentrations of CytoD revealed a dose response relationship with FluoroSpheres (B).


Posters

Visit poster F-2078 at ISSCR in Boston, MA in June - In vitro characterization of human induced pluripotent stem cell-derived macrophages

Development of high content approaches for investigating microglia function in neurodegenerative disorders