Identification and characterization of in vitro expanded hematopoietic stem cells

Area of Science:

• Stem cell biology
• Cellular therapy
• Molecular genetics

Background:

• Hematopoietic stem cells (HSCs) are crucial for cellular and gene therapies.
• Expanding functional HSCs ex vivo presents challenges due to non-HSC contamination and clonal variability.
• Accurate identification of functional HSCs in expanded cultures is essential for therapeutic applications.

Purpose of the Study:

• To develop a method for exclusive identification of HSCs in expansion cultures.
• To characterize the molecular profile of expanded functional HSCs.
• To identify a gene expression signature that defines functional HSCs.

Main Methods:

• Utilized the Fgd5 reporter mouse and EPCR surface marker for HSC identification.
• Integrated single-cell functional transplantation data with gene expression profiling.
• Analyzed gene expression profiles of expanded HSCs and compared them to fetal HSCs.

Main Results:

• Successfully identified HSCs exclusively from non-HSCs in expansion cultures.
• Demonstrated that expanded HSCs share molecular profiles with proliferating fetal HSCs.
• Discovered a gene expression signature (RepopSig), including Esam, Prdm16, Fstl1, and Palld, that identifies functional HSCs across different cellular states.
• Showed that RepopSig also enriches HSCs in human datasets.

Conclusions:

• The combination of Fgd5 and EPCR enables precise identification of HSCs in expanded cultures.
• The identified RepopSig provides a molecular tool to distinguish functional HSCs.
• This approach facilitates future functional screening and molecular experiments with expanded HSCs, advancing cellular therapy development.