Suppression of m6A reader Ythdf2 promotes hematopoietic stem cell expansion

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, but the limited number of HSCs in a single hUCB unit restricts its widespread use. Although extensive efforts have developed multiple methods for ex vivo expansion of human HSCs by targeting single molecules or pathways, it remains unknown whether simultaneously manipulating a large number of targets essential for stem cell self-renewal could be achievable. Recent studies have emerged that N6-methyladenosine (m6A) modulates expression of a group of mRNAs critical for stem cell fate determination by influencing their stability. Among several m6A readers, Ythdf2 is well recognized to promote the targeted mRNA decay. However, the physiological functions of Ythdf2 on adult stem cells are still elusive. Here we show that conditional knockout (KO) mouse Ythdf2 increased phenotypic and functional HSC numbers, but neither skewed lineage differentiation nor led to hematopoietic malignancies. Furthermore, knockdown (KD) of human YTHDF2 led to over 10-fold increase in ex vivo expansion of hUCB HSCs, 5-fold increase in colony-forming units (CFUs), and more than 8-fold increase in functional hUCB HSCs in the secondary serial of limiting dilution transplantation assay. Mechanistically, m6A mapping of RNAs from mouse hematopoietic stem and progenitor cells (HSPCs) as well as from hUCB HSCs revealed m6A enrichment on mRNAs encoding transcription factors critical for stem cell self-renewal. These m6A-marked mRNAs were recognized by Ythdf2 and underwent mRNA decay. In Ythdf2 KO HSPCs and YTHDF2 KD hUCB HSCs, these mRNAs were stabilized, leading to an increase in protein levels and facilitating HSC expansion. Knockdown one of the Ythdf2 key targets, Tal1 mRNA, partially rescued the phenotype. Therefore, our study for the first time shows the function of Ythdf2 in adult stem cell maintenance and identifies an important role of Ythdf2 in regulating HSC ex vivo expansion via the mechanism of controlling the stability of multiple mRNAs critical for HSC self-renewal, thus having a strong potential for future clinical applications.