Identification and characterization of in vitro expanded hematopoietic stem cells

James L C Che, Daniel Bode, Iwo Kucinski, Alyssa H Cull, Fiona Bain, Hans J Becker, Maria Jassinskaja, Melania Barile, Grace Boyd, Miriam Belmonte, Andy G X Zeng, Kyomi J Igarashi, Juan Rubio-Lara, Mairi S Shepherd, Anna Clay, John E Dick, Adam C Wilkinson, Hiromitsu Nakauchi, Satoshi Yamazaki, Berthold GöttgensDavid G Kent*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved > 200-fold expansion of functional HSCs, but their molecular characterization has not been possible since the majority of cells are non-HSCs and single cell-initiated cultures have substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with the EPCR surface marker, we report exclusive identification of HSCs from non-HSCs in expansion cultures. By directly linking single-clone functional transplantation data with single-clone gene expression profiling, we show that the molecular profile of expanded HSCs is similar to proliferating fetal HSCs and reveals a gene expression signature, including Esam, Prdm16, Fstl1, and Palld, that can identify functional HSCs from multiple cellular states. This "repopulation signature" (RepopSig) also enriches for HSCs in human datasets. Together, these findings demonstrate the power of integrating functional and molecular datasets to better derive meaningful gene signatures and opens the opportunity for a wide range of functional screening and molecular experiments previously not possible due to limited HSC numbers.

Original languageEnglish
Article numbere55502
Pages (from-to)e55502
Number of pages19
JournalEMBO Reports
Issue number10
Early online date16 Aug 2022
Publication statusPublished - 6 Oct 2022

Bibliographical note

© 2022The Authors


  • Animals
  • Cells, Cultured
  • Endothelial Protein C Receptor/metabolism
  • Follistatin-Related Proteins/metabolism
  • Hematopoietic Stem Cells/metabolism
  • Humans
  • Mice
  • Transcription Factors/metabolism

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