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 N-methyladenosine (mA) modulates the expression of a group of mRNAs critical for stem cell-fate determination by influencing their stability. Among several mA 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 mA 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 mA-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.

IntroductionMalaria caused by infection with protozoan parasites of Plasmodium spp. is one of the most serious infectious diseases of humans and causes about one million deaths annually. Plasmodium infection may induce multiple disease syndromes including severe anemia, metabolic acidosis, and neurological dysfunction, e.g. cerebral malaria (CM) [1]. Sequestration of the Correspondence: Dr. Zhong Su e-mail: su_zhong@gibh.ac.cn parasitized red blood cells (pRBCs) in brain microvessels is a characteristic histopathology feature observed in human and murine CM and is believed to be the primary cause of the neurological syndrome [2,3]. Evidence from more recent studies supports the notion that CM is an immune-driven pathology. The proinflammatory cytokines and mediators produced in the "cytokine storm" during malaria induce activation and recruitment of cytotoxic CD8 + cells in brain blood vessels and upregulate the expression of adhesion receptors by brain endothelial cells, leading to leukocyte accumulation and capillary obstruction, cerebral hemorrhage, and hypoxia in brain tissues [1,[4][5][6]. Therefore, the immune regulatory cells and cytokines may be critical in counteracting the inflammatory response and prevention of malaria-associated www.eji-journal.eu 2908 Yunfeng Liu et al. Eur. J. Immunol. 2013. 43: 2907-2918 neuropathology. The levels of anti-inflammatory cytokines such as IL-10 and TGF-β have been shown to be inversely associated with the occurrence of CM in murine malaria [7,8]. However, no consensus has been reached concerning the role of regulatory T (Treg) cells in regulation of CM pathogenesis [9][10][11].B cells have been traditionally considered antibody-producing cell and APC. Many studies in recent years showed the existence of a subset of B cells that exhibit the immune regulatory functions (reviewed in [12]). An early study by Fillatreau et al.[13] reported a subset of B cells that produce IL-10 and inhibit the immune pathology in murine EAE. Further studies revealed that these regulatory B (Breg) cells play important roles in control of immunopathology in autoimmune diseases [14], cancer [15], and organ transplantation [16]. Breg cells have also been shown to modulate immune responses and immunopathology in infectious diseases. Protective immunities against Salmonella typhimurium and Brucella abortus infections were inhibited by transfer of Breg cells in mouse models [17,18]. Transfer of CD1d + CD5 hi Breg cells rendered the BALB/c mice more susceptible to Leishmania major infection [19]. Adoptive transfer of IL-10-producing B cells isolated from helminth-infected mice inhibited the allergic responses and autoimmune inflammation in recipient mice [20]. Thus, Breg cells are an important component of immunoregulatory system and plays critical roles in immune homeostasis.The immunomodulatory roles of Breg cells have been studied in autoimmune and infectious disease settings, but little is known of its role in regulation of immune response and immunopathology of malaria. In this study, we i...