# Investigating the mechanisms of lysosome segregation and cell fate regulation in dividing hematopoietic stem cells

> **NIH NIH R01** · ST. JUDE CHILDREN'S RESEARCH HOSPITAL · 2024 · $773,257

## Abstract

PROJECT SUMMARY
Hematopoietic stem cell (HSC) transplantation is the only curative therapy for many diseases, but low HSC
numbers and their tendency to differentiate in vitro are limiting factors for the more widespread application of this
life-saving therapy. Knowing how HSCs decide between self-renewal and differentiation is critical to develop
efficient culture systems that manipulate HSC fate decisions to either expand or differentiate HSCs into specific
mature blood cells for transplantation. However, HSC decision-making processes are incompletely understood
and our ability to expand HSCs is limited. HSC self-renewal and differentiation are regulated by the symmetric
and asymmetric segregation of cell fate determinants (CFD) during division. After asymmetric segregation of
CFDs, only one daughter retains HSC properties, while symmetric segregation of CFDs can create two HSCs.
An exciting prediction of this model is, that factors promoting symmetric segregation of CFD during division
expand HSCs. However, identifying these factors has been hampered because of major technical challenges
including: 1) HSCs are rare and divide infrequently, making transient events like divisions hard to capture in fixed
samples, and 2) cell fate acquisition after HSC division takes many days. Understanding how the segregation of
CFD influences cell fate acquisition thus requires precise quantifications of living HSCs over many days with
single-cell resolution. To address this challenge, we developed novel long-term live-cell imaging tools, that allow
us to track and quantify single HSC behaviors and marker expression over several weeks and cell generations.
This allowed us to visualize for the first time that lysosomes are asymmetrically segregated during HSC divisions
and act as CFD to instruct later changes in metabolism and differentiation in HSC daughter cells. Here, we
propose to leverage this novel technology to identify factors regulating lysosome segregation and to investigate
how RhoGTPases, which are known polarity proteins in invertebrates, regulate lysosome segregation, and HSC
self-renewal and differentiation. Studies of non-dividing cells showed that Tubulin, Actin and Vimentin filaments
of the cytoskeleton regulate lysosome motility. However, which of these filaments regulates lysosome motility
during HSC division remains unclear. To address this knowledge gap, we will study lysosomal motility and
segregation in dividing HSCs using our live-cell imaging tools. We will assess the polarization and colocalization
of lysosomes with Tubulin, Actin and Vimentin filaments and functionally interrogate how pharmacological and
genetic manipulation of known cytoskeletal transport mechanisms and polarity regulators, such as CDC42, affect
lysosomal segregation and daughter cell fate acquisition. Our preliminary data, show that lysosome segregation
is regulated via ARP2/3-mediated actin nucleation, which lies downstream of the Integrin/Focal Adhesion Kinase
(FA...

## Key facts

- **NIH application ID:** 10940054
- **Project number:** 1R01HL174644-01
- **Recipient organization:** ST. JUDE CHILDREN'S RESEARCH HOSPITAL
- **Principal Investigator:** Dirk Loeffler
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $773,257
- **Award type:** 1
- **Project period:** 2024-09-01 → 2029-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10940054

## Citation

> US National Institutes of Health, RePORTER application 10940054, Investigating the mechanisms of lysosome segregation and cell fate regulation in dividing hematopoietic stem cells (1R01HL174644-01). Retrieved via AI Analytics 2026-06-02 from https://api.ai-analytics.org/grant/nih/10940054. Licensed CC0.

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