Related Experiment Video
Updated: Aug 14, 2025
Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages
Published on: June 30, 2016
Chicken CSF2 and IL-4-, and CSF2-dependent bone marrow cultures differentiate into macrophages over time
Dominika Borowska1, Samantha Sives1, Lonneke Vervelde1
1The Division of Infection and Immunity, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, United Kingdom.
Abstract:
Chicken bone marrow-derived macrophages (BMMΦ) and dendritic cells (BMDC) are utilized as models to study the mononuclear phagocytic system (MPS). A widely used method to generate macrophages and DC in vitro is to culture bone marrow cells in the presence of colony-stimulating factor-1 (CSF1) to differentiate BMMΦ and granulocyte-macrophage-CSF (GM-CSF, CSF2) and interleukin-4 (IL-4) to differentiate BMDC, while CSF2 alone can lead to the development of granulocyte-macrophage-CSF-derived DC (GMDC). However, in chickens, the MPS cell lineages and their functions represented by these cultures are poorly understood. Here, we decipher the phenotypical, functional and transcriptional differences between chicken BMMΦ and BMDC along with examining differences in DC cultures grown in the absence of IL-4 on days 2, 4, 6 and 8 of culture. BMMΦ cultures develop into a morphologically homogenous cell population in contrast to the BMDC and GMDC cultures, which produce morphologically heterogeneous cell cultures. At a phenotypical level, all cultures contained similar cell percentages and expression levels of MHCII, CD11c and CSF1R-transgene, whilst MRC1L-B expression decreased over time in BMMΦ. All cultures were efficiently able to uptake 0.5 µm beads, but poorly phagocytosed 1 µm beads. Little difference was observed in the kinetics of phagosomal acidification across the cultures on each day of analysis. Temporal transcriptomic analysis indicated that all cultures expressed high levels of CSF3R, MERTK, SEPP1, SPI1 and TLR4, genes associated with macrophages in mammals. In contrast, low levels of FLT3, XCR1 and CAMD1, genes associated with DC, were expressed at day 2 in BMDC and GMDC after which expression levels decreased. Collectively, chicken CSF2 + IL-4- and CSF2-dependent BM cultures represent cells of the macrophage lineage rather than inducing conventional DC.
Insights
Chicken bone marrow cultures stimulated with GM-CSF and IL-4 or GM-CSF alone primarily generate macrophage lineage cells, not conventional dendritic cells (DCs). This study clarifies chicken mononuclear phagocytic system (MPS) cell types for better research models.
Area of Science:
- Immunology
- Cell Biology
- Comparative Genomics
Background:
- Chicken bone marrow-derived macrophages (BMMΦ) and dendritic cells (BMDC) are crucial models for studying the mononuclear phagocytic system (MPS).
- Established methods use colony-stimulating factor-1 (CSF1) for BMMΦ and granulocyte-macrophage-CSF (GM-CSF, CSF2) with interleukin-4 (IL-4) for BMDC, but chicken MPS cell lineages remain poorly understood.
- Granulocyte-macrophage-CSF-derived DC (GMDC) can also be generated using CSF2 alone.
Purpose of the Study:
- To decipher phenotypical, functional, and transcriptional differences between chicken BMMΦ and BMDC.
- To examine differences in DC cultures grown with or without IL-4 over 8 days.
- To clarify the identity of chicken CSF2 + IL-4- and CSF2-dependent bone marrow cultures.
Main Methods:
- Culture of chicken bone marrow cells with CSF1, or CSF2 + IL-4, or CSF2 alone.
- Phenotypical analysis using cell morphology and expression of MHCII, CD11c, CSF1R, and MRC1L-B.
- Functional assays including bead uptake and phagosomal acidification.
- Temporal transcriptomic analysis to identify gene expression patterns.
Main Results:
- BMMΦ cultures yielded homogenous cell populations, while BMDC and GMDC cultures were heterogeneous.
- All cultures showed similar MHCII, CD11c, and CSF1R expression, but MRC1L-B decreased in BMMΦ over time.
- Phagocytic capacity for 0.5 µm beads was high, but low for 1 µm beads, with similar phagosomal acidification kinetics.
- Transcriptomic analysis revealed high expression of macrophage-associated genes (CSF3R, MERTK, SEPP1, SPI1, TLR4) in all cultures.
- Low expression of DC-associated genes (FLT3, XCR1, CAMD1) was observed in BMDC and GMDC at day 2, decreasing thereafter.
Conclusions:
- Chicken CSF2 + IL-4- and CSF2-dependent bone marrow cultures predominantly generate cells of the macrophage lineage.
- These cultures do not induce conventional dendritic cells (DCs) as typically defined in mammals.
- The study provides critical insights into chicken mononuclear phagocytic system (MPS) cell differentiation and function.
Related Concept Videos
01:15Differentiation of Common Myeloid Progenitor Cells
01:01Regulation of Hematopoietic Stem Cells