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 Göttgens; David G Kent

doi:10.15252/embr.202255502

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

Biology

Research output: Contribution to journal › Article › peer-review

Abstract

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 language	English
Article number	e55502
Pages (from-to)	e55502
Number of pages	19
Journal	EMBO Reports
Volume	23
Issue number	10
Early online date	16 Aug 2022
DOIs	https://doi.org/10.15252/embr.202255502 https://doi.org/10.15252/embr.202255502
Publication status	Published - 6 Oct 2022

Bibliographical note

Keywords

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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10.15252/embr.202255502Licence: CC BY
10.15252/embr.202255502

Identification and characterization of in vitro expanded hematopoietic stem cells
© 2022The Authors
Final published version, 1.78 MBLicence: CC BY

Tracking ex vivo hematopoietic stem cell function using Fgd5 and EPCR reveals molecular regulators of expansion.
Che, J. (Creator), Bode, D. (Creator), Kucinski, I. (Creator), Cull, A. H. (Creator), Bain, F. (Creator), Barile, M. (Creator), Boyd, G. (Creator), Belmonte, M. (Creator), Rubio-Lara, J. (Creator), Shepherd, M. S. (Creator), Clay, A. (Creator), Wilkinson, A. C. (Creator), Yamazaki, S. (Creator), Gottgens, B. (Creator) & Kent, D. G. (Creator), Gene Expression Omnibus (GEO), 19 Aug 2022
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE175400
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Cite this

Che, J. L. C., Bode, D., Kucinski, I., Cull, A. H., Bain, F., Becker, H. J., Jassinskaja, M., Barile, M., Boyd, G., Belmonte, M., Zeng, A. G. X., Igarashi, K. J., Rubio-Lara, J., Shepherd, M. S., Clay, A., Dick, J. E., Wilkinson, A. C., Nakauchi, H., Yamazaki, S., ... Kent, D. G. (2022). Identification and characterization of in vitro expanded hematopoietic stem cells. EMBO Reports, 23(10), e55502. Article e55502. https://doi.org/10.15252/embr.202255502, https://doi.org/10.15252/embr.202255502

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title = "Identification and characterization of in vitro expanded hematopoietic stem cells",

abstract = "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.",

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Che, JLC , Bode, D, Kucinski, I, Cull, AH, Bain, F, Becker, HJ, Jassinskaja, M, Barile, M, Boyd, G, Belmonte, M, Zeng, AGX, Igarashi, KJ, Rubio-Lara, J, Shepherd, MS, Clay, A, Dick, JE, Wilkinson, AC, Nakauchi, H, Yamazaki, S, Göttgens, B & Kent, DG 2022, 'Identification and characterization of in vitro expanded hematopoietic stem cells', EMBO Reports, vol. 23, no. 10, e55502, pp. e55502. https://doi.org/10.15252/embr.202255502, https://doi.org/10.15252/embr.202255502

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T1 - Identification and characterization of in vitro expanded hematopoietic stem cells

AU - Che, James L C

AU - Bode, Daniel

AU - Kucinski, Iwo

AU - Cull, Alyssa H

AU - Bain, Fiona

AU - Becker, Hans J

AU - Jassinskaja, Maria

AU - Barile, Melania

AU - Boyd, Grace

AU - Belmonte, Miriam

AU - Zeng, Andy G X

AU - Igarashi, Kyomi J

AU - Rubio-Lara, Juan

AU - Shepherd, Mairi S

AU - Clay, Anna

AU - Dick, John E

AU - Wilkinson, Adam C

AU - Nakauchi, Hiromitsu

AU - Yamazaki, Satoshi

AU - Göttgens, Berthold

AU - Kent, David G

PY - 2022/10/6

Y1 - 2022/10/6

N2 - 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.

AB - 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.

KW - Animals

KW - Cells, Cultured

KW - Endothelial Protein C Receptor/metabolism

KW - Follistatin-Related Proteins/metabolism

KW - Hematopoietic Stem Cells/metabolism

KW - Humans

KW - Mice

KW - Transcription Factors/metabolism

U2 - 10.15252/embr.202255502

DO - 10.15252/embr.202255502

M3 - Article

C2 - 35971894

SN - 1469-221X

VL - 23

SP - e55502

JO - EMBO Reports

JF - EMBO Reports

IS - 10

M1 - e55502

ER -