Inhibition of Let-7 Processing in Adult Murine Hematopoietic Stem Cells Induces a Fetal-Like High Self-Renewal Pattern in Their Progeny

Abstract

45 Fetal hematopoietic stem cells (HSCs) in mice differ from their adult counterparts in a number of key properties. These include a higher cycling activity, an ability to more rapidly reconstitute the HSC compartment of irradiated recipient mice, a higher output of myeloid as compared to lymphoid progeny, and a greater sensitivity to the self-renewal promoting activity of Steel factor. We have previously shown that most of these features of fetal HSCs are sustained until 3 weeks after birth at which time they are rapidly (within 1 week), completely and permanently replaced with the corresponding properties of adult HSCs. A candidate regulator of this transition, Hmga2, was identified based on its greater expression in highly purified fetal versus adult HSCs (CD45+EPCR+CD48−CD150+; E-SLAM cells) with persistence of this difference in the matching lineage-negative (lin−) compartments. Experiments in which Hmga2 was overexpressed by lentiviral transduction of purified adult HSCs which were then transplanted into irradiated mice provided evidence that this chromatin remodeling factor can activate a fetal-like HSC program in these cells; i.e., more rapidly reconstitute the HSC compartment (increased self-renewal response) and produce clones with a higher proportion of myeloid cells. Based on the known ability of the let-7 family of microRNAs (miRNAs) to target Hmga2 transcripts resulting in their degradation and/or translational repression, we next hypothesized that let-7 miRNAs might be involved in controlling HSC developmental programs. A comparison of the levels of expression of 6 members of the let-7 family in purified fetal and adult HSCs, as well as in lin− hematopoietic cells, showed that transcripts for all of these are higher in the adult subsets, although this difference was significant only for let-7b (p<0.05). Since Lin28 is a natural inhibitor of let-7 miRNA biogenesis we proposed that overexpression of this protein might be used to simultaneously inhibit all let-7 miRNA species and therefore modulate let-7-mediated effects in HSCs. Transduction of BA/F3 cells with a Lin28-YFP lentiviral vector led to an elevated expression of Lin28 and a significant decrease in multiple let-7 miRNAs. To investigate the influence of Lin28 overexpression on adult HSC self-renewal activity in vivo, we used the same Lin28 lentiviral vector (or a control YFP vector) to transduce highly purified HSCs (40 E-SLAM cells, i.e. ∼20 HSCs/group/experiment, 3 experiments) in a 3–4-hour exposure protocol and then transplanted all of the cells directly into irradiated mice (total of 3–4 mice/group). The number of HSCs regenerated 6 weeks later was subsequently measured by performing limiting-dilution transplants in secondary mice (total of 12–16 secondary mice/group/experiment). Interestingly, analysis of the secondary recipients showed that the Lin28-overexpressing adult HSCs had expanded in the primary recipients ∼6-fold more than the control-virus transduced HSCs (p<0.001). These findings support our thesis that alterations in let-7 miRNA levels play a key role in regulating the developmental switch from fetal to adult HSCs programs that occurs between 3 and 4 weeks after birth in mice. Disclosures: No relevant conflicts of interest to declare.

miR-99 regulates normal and malignant hematopoietic stem cell self-renewal

“…Given that ectopic overexpression of the polycistron did not increase HSC function more than overexpression of miR-125 only, the action of miR-99 may depend on the expression of miR-125 or may be further modulated by let7 . Intriguingly, prior studies suggest that let-7 may balance the effects of miR-125 and miR-99 by suppressing HSC function ( Copley et al, 2011 , 2013 ; Zipeto et al, 2016 ), a possibility also suggested by its ability to promote differentiation of AML cells ( Pelosi et al, 2013 ). It will be important to dissect these interactions in more depth in the future, as well as post-transcriptional mechanisms that control expression of each of the miRNAs that constitute this cluster.…”

MicroRNA‐99 family in cancer and immunity

“…Increasing evidence now suggest functional relationship between clustered miRNAs in which cluster members reinforce their repressive effect on their targets by acting cooperatively (Yirong Wang, Luo, Zhang, & Lu, 2016). Members of the same cluster may also perform opposing roles, an example being the case of miR‐125 and let‐7 in which let‐7c promoted differentiation of acute myeloid leukemia (AML) cells while miR‐125 facilitated the self‐renewing property of hematopoietic stem cells (HSCs) (Copley et al, 2011; Khalaj et al, 2017).…”