Abstract

Transplantation of hematopoietic stem cells (HSCs) from human umbilical cord blood (hUCB) holds great promise for treating a broad spectrum of hematological disorders including cancer. However, the limited number of HSCs in a single hUCB unit restricts its widespread use. Although extensive efforts have led to multiple methods for ex vivo expansion of human HSCs by targeting single molecules or pathways, it remains unknown whether it is possible to simultaneously manipulate the large number of targets essential for stem cell self-renewal. Recent studies indicate that N6-methyladenosine (m6A) modulates the expression of a group of mRNAs critical for stem cell-fate determination by influencing their stability. Among several m6A readers, YTHDF2 is recognized as promoting targeted mRNA decay. However, the physiological functions of YTHDF2 in adult stem cells are unknown. Here we show that following the conditional knockout (KO) of mouse Ythdf2 the numbers of functional HSC were increased without skewing lineage differentiation or leading to hematopoietic malignancies. Furthermore, knockdown (KD) of human YTHDF2 led to more than a 10-fold increase in the ex vivo expansion of hUCB HSCs, a fivefold increase in colony-forming units (CFUs), and more than an eightfold increase in functional hUCB HSCs in the secondary serial of a limiting dilution transplantation assay. Mapping of m6A in RNAs from mouse hematopoietic stem and progenitor cells (HSPCs) as well as from hUCB HSCs revealed its enrichment in mRNAs encoding transcription factors critical for stem cell self-renewal. These m6A-marked mRNAs were recognized by Ythdf2 and underwent decay. In Ythdf2 KO HSPCs and YTHDF2 KD hUCB HSCs, these mRNAs were stabilized, facilitating HSC expansion. Knocking down one of YTHDF2′s key targets, Tal1 mRNA, partially rescued the phenotype. Our study provides the first demonstration of the function of YTHDF2 in adult stem cell maintenance and identifies its important role in regulating HSC ex vivo expansion by regulating the stability of multiple mRNAs critical for HSC self-renewal, thus identifying potential for future clinical applications.

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Acknowledgements

We thank K. Tannen for proofreading and editing, and M. Hembree, H. Marshall, K. Zapien, D. Dukes, B. Lewis, A. Moran, M. Durnin, J. McCann, A. Box, J. Park, L. Holmes, J. Blank, and D. DeGraffenreid for technical support. We are grateful to X. Zhuang at University of Chicago for transferring the mice.

Author information

Affiliations

  1. Stowers Institute for Medical Research, Kansas City, MO, 64110, USA

    • Zhenrui Li
    • , Pengxu Qian
    • , Wanqing Shao
    • , Xi C. He
    • , Madelaine Gogol
    • , Zulin Yu
    • , Yongfu Wang
    • , John M. Perry
    • , Kai Zhang
    • , Fang Tao
    • , Kun Zhou
    • , Deqing Hu
    • , Yingli Han
    • , Chongbei Zhao
    • , Richard Alexander
    • , Shiyuan Chen
    • , Allison Peak
    • , Kathyrn Hall
    • , Michael Peterson
    • , Anoja Perera
    • , Jeffrey S. Haug
    • , Tari Parmely
    • , Hua Li
    • , Julia Zeitlinger
    •  & Linheng Li
  2. Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, 66160, USA

    • Zhenrui Li
    •  & Linheng Li
  3. Center of Stem Cell and Regenerative Medicine, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China

    • Pengxu Qian
  4. Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Institute of Hematology, Zhejiang University, Hangzhou, 310058, China

    • Pengxu Qian
  5. Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, 60637, USA

    • Hailing Shi
    •  & Chuan He
  6. Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, 60637, USA

    • Hailing Shi
    •  & Chuan He
  7. State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, 211166, China

    • Meijie Qi
    • , Yunfei Zhu
    •  & Bin Shen
  8. Department of Hematology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200080, China

    • Kun Zhou
  9. Tianjin Medical University School of Basic Medicine, Tian Jin, 300070, China

    • Deqing Hu
  10. Shanghai JiaoTong University School of Medicine, Shanghai, 200025, China

    • Hanzhang Xu

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Contributions

Z.L. and P.Q. conceived the project, performed experiments, analyzed data, and wrote the manuscript. Z.L. conducted m6A-seq and irCLIP-seq. W.S. and J.Z. analyzed the m6A-seq and mRNA stability data. M.G., S.C., and H.L. analyzed irCLIP-seq and RNA-seq data. X.C.H. provided the training and performed the experiments. B.S., M.Q., Y.Z., and X.Z. generated the genetic mouse model. Z.Y., Y.W., J.P., F.T., K.Z., D.H., Y.H., C.Z., and H.X. performed experimental work. R.A., A.P., K.H., M.P., A.P., J.H., and T.P. provided technical assistance. S.H. and C.H. provided key suggestions and plasmids. L.L. supervised the study.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Pengxu Qian or Linheng Li.

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https://doi.org/10.1038/s41422-018-0072-0