Monocytes Can Efficiently Replace All Brain Macrophages and Fetal Liver Monocytes Can Generate Bona Fide Sall1+ Microglia

Academic Background

The homeostasis of the central nervous system (CNS) relies on two key types of macrophages: microglia and border-associated macrophages (BAMs). Traditionally, microglia are believed to originate from the embryonic yolk sac and possess lifelong self-renewal capabilities, while bone marrow (BM)-derived monocytes in adults cannot replace their functions. This limitation restricts the potential for cell transplantation therapies in neurodegenerative diseases. However, recent studies have found that under certain pathological conditions (e.g., Alzheimer’s disease), monocytes may infiltrate the brain parenchyma, though their differentiation fate and functional properties remain unclear. This study aims to address the following core questions:
1. Can monocytes fully replace brain macrophages?
2. Does the developmental origin of monocytes (e.g., fetal liver vs. adult bone marrow) influence their ability to differentiate into microglia?
3. Do human monocytes exhibit similar properties in xenotransplantation models?

Source of the Paper

This study was conducted by an international team led by Jonathan Bastos (Vrije Universiteit Brussel) and Carleigh O’Brien (University of Pennsylvania), with Kiavash Movahedi (Vrije Universiteit Brussel) as the corresponding author. It was published in Immunity (May 2025, Volume 58, Pages 1269–1288) and supported by grants from the European Research Council (ERC) and the Research Foundation Flanders (FWO), among others.

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Research Process and Results

1. Validation of Monocyte Replacement of Embryonic BAMs

Experimental Design:
- Model: Flt3Cre:YFP mice (labeling BM-derived cells) were treated with PLX3397 (a CSF1R inhibitor) to induce macrophage depletion.
- Samples: Control group (n=6) vs. PLX-treated group (n=7), with a 2-week depletion followed by 7 weeks of recovery.
- Methods: Flow cytometry analyzed YFP+ cell distribution in meninges and brain parenchyma, while immunofluorescence confirmed LYVE1+ BAM replacement.

Key Results:
- After PLX treatment, LYVE1+ BAMs in the meninges were almost entirely replaced by YFP+ monocyte-derived cells (Figures 1a–c).
- Competition Mechanism: Rapid self-renewal of endogenous microglia in the parenchyma hindered monocyte engraftment, but localized irradiation (600 rad) disrupted this barrier, enabling replacement of 83.3% of brain macrophages (Figures 1d–e).

2. Monocyte Differentiation into Long-Lived Microglia

Innovative Methods:
- Genetic Model: Microglia depletion was induced in neonatal CX3CR1CreER:CSF1Rfl/fl mice, followed by intracranial injection of GFP+ monocytes (Figure 2a).
- Long-Term Tracking: Engraftment efficiency was compared between monocyte-derived and embryonic microglia over 20 months.

Findings:
- Monocyte-derived mo-microglia exhibited similar expansion capacity and longevity as embryonic-derived microglia (em-microglia) (Figures 2c–d).
- Phenotypic Differences: Mo-microglia had fewer branches and expressed MS4A7 (a BM marker) but not SALL1 (an embryonic marker) (Figure 2g).

3. Monocyte Ontogeny Determines Brain Macrophage Identity

Single-Cell RNA Sequencing (scRNA-seq) Analysis:
- Samples: Clec12a+ cells from PLX-treated mouse meninges were sorted for scRNA-seq (n=15).
- Clustering Results:
- Embryonic BAMs (loBAM1) highly expressed COLEC12 and CD163, with functional enrichment in endocytosis.
- Monocyte-derived BAMs (loBAM2) upregulated inflammation-related genes (e.g., H2-Aa), suggesting functional divergence (Figures 4f–h).

4. Fetal Liver Monocytes Generate Genuine SALL1+ Microglia

Key Experiment:
- E14 fetal liver (FL) monocytes were transplanted into neonatal mouse brains, and GFP+ cells were sorted for scRNA-seq after 8 weeks.
- Epigenetic Analysis: snATAC-seq revealed higher chromatin accessibility at the SALL1 locus in FL monocytes compared to BM monocytes (Figures 6k–l).

Breakthrough Discovery:
- FL monocytes differentiated into SALL1+ microglia, with transcriptomes indistinguishable from embryonic-derived cells (Figures 5l–n), whereas BM monocytes only generated SALL1− cells.

5. Behavior of Human Monocytes in Xenotransplantation Models

Clinical Relevance Experiment:
- Samples: Human umbilical cord blood or adult peripheral blood CD14+ monocytes were transplanted into the brains of hCSF1KI immunodeficient mice (n=17).
- Single-Cell Analysis:
- Human cells formed KCNQ3+ microglia-like cells but lacked SALL1 expression (Figure 7g).
- In Alzheimer’s disease (AD) patient brains, a similar ZNF804A+ microglial cluster was identified, with abundance correlating positively with disease severity (Figure 7l).

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Conclusions and Significance

1. Theoretical Implications:

   • Challenges the traditional view that “only embryonic-derived macrophages can colonize the brain parenchyma,” demonstrating monocytes’ potential for whole-brain macrophage replacement.
     
   • Reveals that developmental ontogeny determines cell fate through epigenetic regulation (e.g., SALL1 accessibility).
     

2. Translational Potential:

   • Offers novel cell therapy strategies for neurodegenerative diseases (e.g., AD) by using umbilical cord blood monocytes to replace dysfunctional microglia.
     
   • Proposes CD163 and MS4A7 as markers to distinguish endogenous from monocyte-derived macrophages.
     

Highlights of the Study

• Methodological Innovation: Combines genetic depletion models, single-cell multi-omics (scRNA-seq + snATAC-seq), and xenotransplantation.
  
• Cross-Species Validation: Confirms the pathological relevance of monocyte-derived microglia in AD, from mice to human data.
  
• Clinical Translation: First demonstration that fetal liver monocytes overcome the “SALL1 expression barrier,” providing a new cell source for regenerative medicine.