# Multicolor mouse models to resolve hematopoietic stem and progenitor cells in vivo

> **NIH NIH R21** · UNIVERSITY OF WISCONSIN-MADISON · 2024 · $217,533

## Abstract

PROJECT SUMMARY: Hematopoietic stem cells (HSCs) are at the top of a hierarchy of differentiating progenitor
cells that can reconstitute the entire blood and immune system. Proper regulation of HSC differentiation is critical
during homeostasis and response to stress or infection. Dysregulation of HSCs naturally occurs during aging,
and aberrant differentiation can cause blood cancers and other diseases. Decades of research have been
devoted to understanding and defining the hierarchy of HSC differentiation. However, the limitations of current
biological tools and animal models has left key questions unanswered. Foremost, we have not been able to track
individual HSCs and multipotent progenitors (MPPs), directly and continuously, in their endogenous
microenvironment. Previously, HSCs and MPPs in mouse models could only be identified by dissociation of bone
marrow, labeling with antibodies to detect markers of differentiated lineages (Lin) and at least four other markers;
for example, c-Kit (aka Kit or CD117), Sca-1 (aka Ly6a), CD48, CD150 (aka Slamf1). More recently, single color
transgenic HSC reporter lines have labeled a proportion of HSCs that could be detected by intravital imaging,
but without additional markers for MPPs, the dynamics of HSC fate decisions are lost. We hypothesize that newly
discovered combinations of cell type-specific markers will allow live tracking of HSC/MPP behaviors and key
lineage decisions. The convergence of multiple cutting-edge technologies makes it possible to address this
hypothesis by generating a multi-color, multi-loci mouse reporter system in the following aims. Aim 1) Generate
an HSC/MPP reporter mouse by targeting knock-in 2A fusion reporters to four loci. We will use
CRISPR/Cas9-dependent homology directed repair (HDR) in mouse zygotes, with repair template delivered by
adeno-associated virus (AAV). Each locus will retain functional proteins that cleave to express distinct fluorescent
proteins (e.g., BFP, GFP, tdTomato, iRFP), representing a positive or negative marker for HSCs and/or MPPs.
Aim 2) Characterize the HSC/MPP reporter line using complementary assays to assess faithful labeling
of HSCs and MPPs. After knock-in validation, we will interbreed the lines to generate four color transgenics.
Labeled HSCs and MPPs will be quantified by flow cytometry and functionally validated by limiting dilution
transplantation. Bones will be imaged ex vivo to confirm that spatial maps of endogenously labeled HSCs and
MPPs are consistent with published immunolabeling data. Outstanding questions about the migration of HSCs
and MPPs will be answered by intravital microscopy. Our research proposal fits this funding opportunity because
it represents a significant “improvement of animal models for stem cell-based regenerative medicine.” It
addresses the research interests of multiple NIH Institutes and Centers because the models generated would
apply to the basic biology of the blood system (NHLBI), immune cells (NIAI...

## Key facts

- **NIH application ID:** 11036635
- **Project number:** 1R21OD037861-01
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** Owen James Tamplin
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $217,533
- **Award type:** 1
- **Project period:** 2024-09-15 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11036635

## Citation

> US National Institutes of Health, RePORTER application 11036635, Multicolor mouse models to resolve hematopoietic stem and progenitor cells in vivo (1R21OD037861-01). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/11036635. Licensed CC0.

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