Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route
May 31st, 2022
The protocol presented here allows the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain via a transnasal route in immunocompetent mice. The method for the preparation and transnasal transplantation of cells and the administration of cytokine mixture for the maintenance of iPSMG is shown.
This protocol allows a non-invasive transplantation of iPSMG into immunocompetent mouse brain by combining pharmacological on/off of a CSF1 receptor antagonist PLX5622 with transnasal transplantation. The main advantage of this technique is that iPSMG can be transplanted without inducing brain damage even in immunocompetent mice. Microglial transplantation may serve as a potential therapy to exchange dysfunctional microglia with functional ones in various neurodegenerative diseases in the future.
Demonstrating the procedure will be Bijay Parajuli, a researcher from my laboratory. Begin by quickly thawing the frozen induced pluripotent stem cell-derived human microglia cells in a 37-degree Celsius water bath. Swirl the samples until all visible ice has melted.
Add the thawed iPSMG to the culture media, warmed to 37 degrees Celsius. Add one milliliter of cells to 10 milliliters of culture media. Centrifuge the cells at 300 times g for five minutes to obtain a cell pellet.
Remove the supernatant without disturbing the cell pellet. Add the transplantation medium to obtain a cell concentration of 10 to the fifth cells per microliter. Place the iPSMG on ice, and immediately proceed to transplantation.
To prepare mice for transnasal transplantation, feed the male mice with a diet containing PLX for seven days. At the end of the seventh day, cease the PLX diet and feed the mice with a normal diet. One hour before transnasal transplantation of iPSMG, administer 2.5 microliters of hyaluronidase in PBS to each nostril twice using a 10-microliter pipette tip to increase the permeability to the nasal mucosa.
Place the mice in the supine position after the hyaluronidase application. Administer 2.5 microliters of hyaluronidase ten minutes before the transnasal transplantation of iPSMG. Apply 2.5 microliters of cell suspension into one nostril of the mouse using a 10-microliter pipette tip.
Place the mouse in the supine position for five minutes before administration of cell suspension to the other nostril. Administer the cell suspension four times, applying a total volume of 20 microliters per animal. 48 hours after the cessation of PLX feeding, repeat the administration of hyaluronidase and cell suspension on the same mice.
For administration of cytokines, apply 2.5 microliters of the transplantation medium into one nostril of the mouse using a 10-microliter pipette tip. After two months of transnasal transplantation, the number of transplanted cells can be determined by counting cells that are positive for both human-specific and pan-microglial markers. Endogenous mouse microglia are positive for pan-microglial marker only.
In control mice, only mouse microglia were detected in both cortex and hippocampus. In the cortex of iPSMG transplanted mice, only mouse microglia were detected, whereas in the hippocampus iPSMG were detected. When attempting this protocol, complete removal of supernatant is essential to prevent the dilution of cytokines.
It is crucial to apply cytokine every 12 hours for the viability of transplanted cells. Depletion of endogenous mouse microglia is required for the transplantation of iPSMG. This procedure allows the transplantation of iPSMG into a normal or diseased mouse brain.
Thus, characteristic of iPSMG as well as the response to disease can be determined. This technique is suitable to determine the in vivo characteristic of iPSMG in the mouse brain. Thus, transplanting iPSMG into disease model mice will help us understand the response and may pave the way for a new therapeutic target.
Overview
This study presents a non-invasive method for the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain of immunocompetent mice via a transnasal route. The protocol leverages cytokine administration and the deletion of endogenous microglia to enable effective integration of iPSMG, potentially paving the way for therapeutic approaches in neurodegenerative diseases.
Key Study Components
Area of Science
- Neuroscience
- Stem Cell Research
- Microglial Biology
Background
- Induced pluripotent stem cells can be differentiated into functional microglia.
- Microglial dysfunction is implicated in various neurodegenerative diseases.
- Transnasal delivery presents a novel approach to cell transplantation.
- Administering cytokines is crucial for cell viability post-transplantation.
Purpose of Study
- To develop a method for non-invasive microglial transplantation.
- To assess the feasibility of transnasal delivery in maintaining normal brain function.
- To evaluate the potential of iPSMG in replacing dysfunctional microglia.
Methods Used
- The primary platform involves transnasal transplantation of iPSMG in immunocompetent mice.
- Male mice were pre-treated with CSF1 receptor antagonist PLX5622 and a hyaluronidase solution to enhance nasal permeability.
- Cell suspensions were administered in multiple doses over specified timelines to ensure proper integration.
- Post-transplant monitoring involved assessing the presence and viability of transplanted cells.
Main Results
- Transplanted iPSMG were successfully integrated into the mouse hippocampus without damaging endogenous microglia.
- Cytokine administration every 12 hours was critical for maintaining cell viability after transplantation.
- This method allows the examination of iPSMG responses in both normal and diseased models.
Conclusions
- This protocol facilitates the study of iPSMG in vivo, which could inform future gene therapies for neurodegenerative diseases.
- Understanding the behavior of transplanted microglia can help delineate their role in brain health.
- This approach may open avenues for novel therapeutic targets in treating microglial-related pathologies.
Frequently Asked Questions
Executive Industry Relevance
Non-invasive transplantation of human iPSC-derived microglia into immunocompetent mouse brains enables direct in vivo study of human microglial function and disease response. This approach addresses translational gaps between rodent and human microglia, supporting more predictive preclinical models for neurodegenerative disease research. The protocol enhances target validation and mechanistic de-risking at early discovery and preclinical inflection points.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables in vivo interrogation of human microglial biology within a mammalian brain environment.
- Supports mechanistic de-risking by distinguishing human-specific microglial responses from murine models.
- Facilitates functional target validation for neuroinflammation and neurodegeneration pathways.
Screening & Assay Development
- Provides a validated system for evaluating human microglial integration and viability in vivo.
- Enables reproducible quantification of transplanted versus endogenous microglia using specific markers.
- Supports downstream screening of compounds targeting human microglial function in a physiological context.
Translational & Preclinical Research
- Aligns preclinical models with human disease relevance by incorporating human microglia into mouse brains.
- Enables assessment of human microglial response in both normal and disease model mice.
- Improves predictive confidence for translational biomarker discovery and therapeutic hypothesis testing.
Pipeline & Workflow Integration
This protocol bridges early discovery and preclinical research by enabling functional studies of human microglia in vivo, supporting lead identification and translational continuity.
- Discovery Biology: Supports hypothesis testing on human microglial roles in brain physiology and pathology.
- Screening: Delivers quantitative readouts of cell integration and viability post-transplantation.
- Analytics: Enables marker-based discrimination between human and mouse microglia for robust data analysis.
- Translational Research: Facilitates disease-relevant modeling by transplanting iPSMG into disease model mice.
- Enterprise Reuse: Establishes a reusable platform for evaluating human microglial function across multiple disease contexts.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces species-specific ambiguity in neuroinflammation research.
- Operational Value: Standardizes non-invasive cell delivery and quantification workflows for cross-study reproducibility.
- Strategic Value: Enables better go/no-go decisions for microglia-targeted therapeutics by providing human-relevant data.
- Portfolio Impact: Supports risk-adjusted prioritization of neurodegenerative disease programs by improving model fidelity.
Implementation Considerations
- Requires expertise in iPSC culture, microglial differentiation, and in vivo transplantation techniques.
- Needs access to pharmacological agents for endogenous microglia depletion and cytokine support.
- Demands standardized protocols for cell preparation, transplantation, and post-transplant analysis.
- Adaptation to other model systems may require optimization of delivery and depletion parameters.
- Continuous cytokine administration is critical for transplanted cell viability, as supported by the protocol.
