A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System
August 30th, 2019
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We describe a novel method for generating double humanized BLT-mice that feature a functional human immune system and a stable engrafted human-like gut microbiome. This protocol can be followed without the need for germ-free mice or gnotobiotic facilities.
Overview
This article presents a novel method for generating double humanized BLT-mice with a functional human immune system and a stable human-like gut microbiome. The protocol allows for reproducible creation of these mice without the need for germ-free conditions.
Key Study Components
Area of Science
- Neuroscience
- Immunology
- Microbiome Research
Background
- Double humanized mice are essential for studying human immune responses.
- They provide insights into the interactions between the human gut microbiome and immune system.
- Traditional methods require germ-free or gnotobiotic facilities.
- This new method simplifies the process of creating these models.
Purpose of Study
- To develop a reliable protocol for generating double humanized mice.
- To facilitate research on the human gut microbiome and immune system interactions.
- To enhance the applicability of mouse models in biomedical research.
Methods Used
- Implantation of human liver and thymus tissues into the mouse kidney capsule.
- Whole body sublethal irradiation of mice prior to surgery.
- Monitoring of engraftment and functionality of human tissues.
- Assessment of gut microbiome stability post-implantation.
Main Results
- Successful engraftment of human immune system components in mice.
- Establishment of a stable human-like gut microbiome.
- Demonstration of the model's utility in studying human biology.
- Potential applications in both basic and translational research.
Conclusions
- The protocol provides a new avenue for creating double humanized mice.
- This model can significantly advance research on human microbiome and immune interactions.
- Future studies can leverage this model for various biomedical applications.
Frequently Asked Questions
What are double humanized mice?
Double humanized mice are genetically engineered mice that possess both a functional human immune system and a human-like gut microbiome.
Why is this research important?
It allows researchers to study the complex interactions between the human immune system and gut microbiome in a living organism.
What are the applications of this model?
This model can be used for basic research as well as translational studies in immunology and microbiome research.
Do you need special facilities to create these mice?
No, this protocol does not require germ-free mice or gnotobiotic facilities.
Who conducted this research?
The research was conducted by Jianshui Zhang and Yilun Chang from the laboratory.
What is the significance of using whole body sublethal irradiation?
Whole body sublethal irradiation is used to prepare the mice for the implantation of human tissues, ensuring better engraftment.
Executive Industry Relevance
The double humanized BLT-mice model addresses a critical gap in preclinical research by enabling the study of human immune system and gut microbiome interactions in a single in vivo system. This capability supports mechanistic de-risking in early discovery by providing a disease-relevant system for evaluating microbiome-modulating therapeutics and immune-mediated pathways. The model enhances predictive confidence in target validation and lead identification efforts where microbiome-immune crosstalk is implicated.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses involving microbiome-immune axis mechanisms in a humanized context.
- Operational Value: Provides a stable, reproducible system for functional target validation without requiring gnotobiotic facilities.
- Predictive Value: Supports portfolio triage by modeling human-like microbiome engraftment and immune reconstitution over extended periods.
Screening & Assay Development
- Scientific Value: Generates validated biological systems with quantifiable 16S rRNA profiles and immune cell readouts for assay standardization.
- Operational Value: Delivers microbiome and immune outputs that are stable for up to 14.5 weeks, enabling longitudinal screening campaigns.
- Scalability: Facilitates platform reuse across multiple human donor samples to capture inter-individual variability in preclinical screening.
Translational & Preclinical Research
- Translational Continuity: Models human-relevant microbiome-immune interactions to support biomarker discovery and mechanism of action studies.
- Preclinical Model: Serves as a disease-relevant system for evaluating therapeutics where gut microbiome modulation impacts immune function.
- Risk-Adjusted Advancement: Enables evaluation of lead candidates in a system that reflects human microbiome diversity and immune competence.
Pipeline & Workflow Integration
The model integrates into the discovery workflow from early target validation through preclinical evaluation, particularly for therapeutics targeting immune-microbiome interfaces.
- Discovery Biology: Supports hypothesis testing and pathway clarification in microbiome-driven immune pathologies.
- Screening: Enables assay readiness through stable engraftment of human immune cells and donor-specific microbiome profiles.
- Analytics: Provides quantitative dependent variables including flow cytometry immune profiling and 16S rRNA sequencing for condition comparison.
- Translational Research: Connects discovery findings to preclinical validation via measurable immune reconstitution and microbiome stability metrics.
- Enterprise Reuse: Establishes a reusable platform for studying diverse human donor microbiomes in immunology and infectious disease programs.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity in microbiome-immune target validation through a physiologically relevant humanized system.
- Operational Value: Eliminates dependency on germ-free animal facilities, improving accessibility and reducing operational complexity.
- Strategic Value: Improves go/no-go decisions by de-risking targets with known microbiome dependencies early in the pipeline.
- Portfolio Impact: Enables risk-adjusted prioritization of candidates based on efficacy in a model with stable human-like microbiome and immune function.
Implementation Considerations
- Requires expertise in surgical implantation of human tissues and hematopoietic stem cell injection.
- Dependent on access to human fetal liver, thymus, and CD34+ stem cells, as well as sterile fecal transplant material.
- Necessitates standardized protocols for antibiotic pretreatment, oral gavage, and longitudinal monitoring via blood and fecal sampling.
- Requires adaptation across different human donor samples to account for microbiome variability in study design.
- Practical limitations include the need for aseptic technique during tissue implantation and microbiome transfer to prevent contamination.
Biopharma FAQs
Why does limiting bacterial contamination matter in double hu-BLT mice?
Limiting unwanted bacterial introduction is critical to maintain mouse health and ensure the stability of the transplanted human-like gut microbiome throughout the study period.
How does antibiotic pretreatment support human microbiome engraftment?
Broad spectrum antibiotics are used to deplete the pre-existing murine gut microbiome, creating a niche for successful engraftment of transplanted human fecal microbiota.
What enables longitudinal tracking of immune reconstitution in the model?
Peripheral blood sampling and flow cytometry analysis using antibodies against human CD45, CD3, CD4, CD8, CD19 and mouse CD45 allow assessment of human immune cell levels over time.
Why is 16S rRNA sequencing used to characterize the gut microbiome?
16S rRNA gene sequencing reveals unique profiles based on the human donor sample and confirms the establishment of a human-like gut microbiome in double hu-BLT mice.
How does the model support studies of human disease impacts on the microbiome?
The model allows researchers to examine how human disease affects the gut microbiome while controlling for genetic and environmental variables through immune system humanization.
Chapters in this video
0:04
Title
0:41
Bone Marrow, Liver, Thymus-Humanized (hu-BLT) Mouse Generation
3:43
Human Immune Cell Level Evaluation and Fresh Fecal Sample Collection
5:20
Fecal Transplant
6:05
Results: Representative Double Humanized BLT-Mouse Gut Microbiome Analyses
7:02
Conclusion
