Neonatal Intracerebral Transplantation for Microglia Replacement and Lineage Analysis In Vivo

Summary

This protocol enables efficient neonatal intracerebral transplantation of defined microglia populations, supporting robust long-term engraftment and analysis of microglia lineage and in vivo integration across multiple donor sources and recipient models.

Abstract

Microglia replacement via myeloid cell transplantation has emerged as a promising experimental approach for studying neurological processes and disease. However, existing transplantation strategies are often limited by inefficient engraftment, reliance on irradiation-based conditioning, or restricted flexibility across donor and recipient systems. Here, we describe a neonatal intracerebral transplantation protocol for introducing purified microglia populations (Hoxb8 and non-Hoxb8 microglia) into the developing mouse brain. This method enables efficient donor cell engraftment and integration within the brain parenchyma under physiologically permissive conditions. Importantly, the protocol is compatible with multiple donor sources, including embryonic hematopoietic progenitors and postnatal brain-derived microglia, and can be applied across distinct recipient models with reduced or absent endogenous microglia, including conditional Csf1r-deficient mice and Csf1r^ΔFIRE mice. Donor cells are purified by fluorescence-activated cell sorting and delivered bilaterally into neonatal mouse brains using glass micropipettes for efficient and minimally invasive delivery. Recipient mice are aged to allow donor cells to expand and populate the microglia niche, and outcomes are assessed by histological analysis of donor cell distribution and marker expression. This approach provides a robust and scalable platform for studying microglia ontogeny and in vivo integration across diverse experimental contexts.

Introduction

Microglia replacement through transplantation of myeloid cells has emerged as a powerful strategy for probing microglia biology and as a potential therapeutic paradigm for neurological disease. Over the past several years, the field has advanced to strategies capable of widespread microglia replacement in the central nervous system¹. Recent studies have demonstrated that donor-derived macrophages and progenitors can engraft the brain, adopt microglia-like transcriptional programs, and in some cases improve disease-associated phenotypes^2,3,4,....

Protocol

All methods and experiments in this study have been performed on mice. Experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Utah, Public Health Service Assurance #D16-00018 (A3031-01).

1. Preparing breeding pairs for donor and recipient litters

1. Place breeder pairs in the same cage in the evening to initiate timed matings.
2. Inspect females each morning for the presence of a vaginal plug to confirm successful mating.
3. Remove plugged females from males and house them individually until parturition, which takes approximately 18–....

Results

Neonatal intracerebral transplantation of purified microglia populations results in long-term survival and widespread engraftment of donor-derived cells within the recipient brain parenchyma. Purified donor microglia populations were isolated by fluorescence-activated cell sorting using fluorescent lineage reporters (Hoxb8 microglia: tdTomato⁺ GFP⁺; non-Hoxb8 microglia: tdTomato⁻ GFP⁺) prior to transplantation (Figure 1A). Following neonatal intracerebral transplantation, d.......

Discussion

Neonatal intracerebral transplantation of purified microglial progenitors or defined microglial subpopulations provides a robust approach for reconstructing the microglial compartment in vivo and examining lineage-specific properties of brain macrophages. This method enables selective reconstitution using genetically defined donor cells, allowing direct assessment of donor cell engraftment and maturation within the brain environment.

A key advantage of this strategy is its compatibili.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bell jar	Various	N/A	Used for euthanasia
Borosilicate glass capillaries with filament	Sutter Instrument	BF100-78-15	Used for injection needles
Bovine serum albumin (BSA)	Sigma Aldrich	A8806	Used to prepare PB buffer. Various brands are available. The one listed here is just an example.
CD11b Alexa Fluor 700	BioLegend	101222	Myeloid cells
CD45 APC	BioLegend	103112	Hematopoietic cells
Cell strainers (70 µm)	Falcon	352350	Used to generate single- cell suspensions. Various brands are available. The one listed here is just an example.
Chicken anti-GFP antibody	Aves Labs	GFP-1020	Immunofluorescence staining
c-Kit PE-Cy7 antibody	BioLegend	105814	Hematopoietic progenitors
Confocal microscope	Leica	TCS SP5	Used for fluorescence imaging
Conical tubes (15 mL/ 50 mL)	Eppendorf	https://www.eppendorf.com/us-en/Products/Lab-Consumables/Lab-Tubes/Eppendorf-Tubes-BioBased-p-PF-4440301	Tissue/cell processing. Various brands are available. The one listed here is just an example.
Cryostat machine	Leica	https://www.leicabiosystems.com/en-de/histology-equipment/cryostats/	Generate frozen tissue sections. Various brands are available. The one listed here is just an example.
DAPI nuclear stain	Thermo Fisher Scientific	D1306	Used for viability and nuclear staining
Dissecting microscope	Leica	https://www.leica-microsystems.com/products/light-microscopes/stereo-microscopes/	Used for trimming and preparing injection needles. Various brands are available. The one listed here is just an example.
FACSAria Flow Cytometer Sorter	BD Bioscience	FACSAria	Cell purification
Fc block (purified CD16/32)	Biolegend	101302	Blocks unwanted antibody binding to mouse cells expressing Fc receptors
Fetal bovine serum (FBS)	ThermoFisher Scientific	https://www.thermofisher.com/in/en/home/life-science/cell-culture/mammalian-cell-culture/fbs.html	Used in cell preparation buffers. Various brands are available. The one listed here is just an example.
Flexible tubing for mouth pipette	Various	N/A	Component of manual injection apparatus
Flow cytometry analysis software	FlowJo	v10.8.1	Used for FACS data analysis
Flow cytometry cell sorter	BD Biosciences	FACSAria	Used for cell purification
gentleMACS Octo Dissociator with heaters	Miltenyi Biotec	130-096-427	Used for brain tissue dissociation
Goat anti-chicken Alexa Fluor 488 secondary antibody	Thermo Fisher Scientific	A-11039	Secondary antibody
Goat anti-guinea pig Alexa Fluor 555 secondary antibody	Thermo Fisher Scientific	A-21428	Secondary antibody
Goat anti-rabbit Alexa Fluor 647 secondary antibody	Thermo Fisher Scientific	A-21245	Secondary antibody
Goat anti-rat Alexa Fluor 647 secondary antibody	Thermo Fisher Scientific	A-48265	Secondary antibody
Guinea pig anti-tdTomato antibody	Frontier Institute	AB_2631185	Immunofluorescence staining
Hanks’ balanced salt solution (HBSS)	Gibco	14175-079	Used during cell preparation
Hemocytometer	Various	N/A	Cell counting
Inline filter for mouth pipette	Various	N/A	Prevents contamination
Microgrinder	Narishige	EG-400	Used to bevel glass injection needles
Micropipette puller	Sutter Instrument	P-1000	Used to generate fine injection needles
Mouth pipette mouthpiece	Various	N/A	Used for manual control of injection
Neural Tissue Dissociation Kit (P)	Miltenyi Biotec	130-092-628	Used for enzymatic dissociation of brain tissue
Petri dishes	Thermo Fisher Scientific	https://www.thermofisher.com/search/browse/category/us/en/90111022	Used for storing pulled needles or tissue harvest. Various brands are available. The one listed here is just an example.
Rabbit anti-Iba1 antibody	Wako	019-19741	Microglia marker
Rabbit anti-TMEM119 antibody	Abcam	209064	Microglia-specific marker
Rat anti-CD206 antibody	BioLegend	141712	Used to identify macrophage populations
Rat anti-P2RY12 antibody	BioLegend	848002	Microglia marker
Razor blades/scalpels	Various	N/A	Used to trim needle tips or tissue harvest
RBC lysis buffer	ChemCruz	sc-296258	Used for removal of red blood cells
Stereotaxic apparatus	Kopf Instruments	https://kopfinstruments.com/	Used for surgical implantation. Various brands are available. The one listed here is just an example.
Ter119 PerCP-Cy5.5 antibody	BioLegend	116228	Erythroid lineage cells