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Updated: Mar 18, 2026
Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors
Published on: July 8, 2012
Phosphoproteomic profiling of mouse primary HSPCs reveals new regulators of HSPC mobilization
Leo D Wang1, Scott B Ficarro2, John N Hutchinson3
1Joslin Diabetes Center, Boston, MA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA; Dana-Farber Boston Children's Cancer and Blood Disorders Center, Boston, MA;
Abstract:
Protein phosphorylation is a central mechanism of signal transduction that both positively and negatively regulates protein function. Large-scale studies of the dynamic phosphorylation states of cell signaling systems have been applied extensively in cell lines and whole tissues to reveal critical regulatory networks, and candidate-based evaluations of phosphorylation in rare cell populations have also been informative. However, application of comprehensive profiling technologies to adult stem cell and progenitor populations has been challenging, due in large part to the scarcity of such cells in adult tissues. Here, we combine multicolor flow cytometry with highly efficient 3-dimensional high performance liquid chromatography/mass spectrometry to enable quantitative phosphoproteomic analysis from 200 000 highly purified primary mouse hematopoietic stem and progenitor cells (HSPCs). Using this platform, we identify ARHGAP25 as a novel regulator of HSPC mobilization and demonstrate that ARHGAP25 phosphorylation at serine 363 is an important modulator of its function. Our approach provides a robust platform for large-scale phosphoproteomic analyses performed with limited numbers of rare progenitor cells. Data from our study comprises a new resource for understanding the molecular signaling networks that underlie hematopoietic stem cell mobilization.
Insights
Researchers developed a new method to study protein phosphorylation in rare hematopoietic stem and progenitor cells (HSPCs). This phosphoproteomic analysis identified ARHGAP25 as a key regulator of HSPC mobilization.
Area of Science:
- Cellular signaling
- Proteomics
- Hematopoiesis
Background:
- Protein phosphorylation is crucial for signal transduction and protein function regulation.
- Previous phosphoproteomic studies were limited in rare cell populations like adult stem cells due to cell scarcity.
- Analyzing phosphorylation in hematopoietic stem and progenitor cells (HSPCs) is vital for understanding blood formation and related disorders.
Purpose of the Study:
- To develop a robust platform for quantitative phosphoproteomic analysis in limited numbers of rare progenitor cells.
- To identify novel regulators of hematopoietic stem and progenitor cell (HSPC) mobilization.
- To investigate the role of protein phosphorylation in HSPC function and signaling.
Main Methods:
- Combined multicolor flow cytometry with 3-dimensional high-performance liquid chromatography/mass spectrometry (3D-HPLC/MS).
- Performed quantitative phosphoproteomic analysis on 200,000 highly purified primary mouse hematopoietic stem and progenitor cells (HSPCs).
- Utilized a novel platform for large-scale phosphoproteomic analysis with limited cell numbers.
Main Results:
- Successfully enabled quantitative phosphoproteomic analysis from a small number of primary mouse HSPCs.
- Identified ARHGAP25 as a novel regulator of hematopoietic stem and progenitor cell (HSPC) mobilization.
- Demonstrated that ARHGAP25 phosphorylation at serine 363 is a critical modulator of its function.
Conclusions:
- The developed phosphoproteomic platform is effective for analyzing rare progenitor cells.
- ARHGAP25 and its phosphorylation at serine 363 play a significant role in hematopoietic stem and progenitor cell (HSPC) mobilization.
- This study provides a valuable resource for understanding molecular signaling networks in hematopoietic stem cell mobilization.
Related Concept Videos
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01:19Multipotency of Hematopoietic Stem Cells
01:21Hematopoiesis