CHARACTERIZATION OF THPO-INDEPENDENT HEMATOPOIETIC STEM CELLS WHICH ARE INDUCED TO QUIESCENCE

Abstract

Quiescence is a fundamental property of highly potent hematopoietic stem cells (HSCs). We previously reported that thrombopoietin (Thpo) maintains quiescent HSCs but also stimulates mitochondrial metabolism and proliferation of HSCs. It is not known why and how Thpo orchestrates its contradictory effect of promoting proliferation or quiescence on HSCs. We analyzed the response of adult Thpo-/- mice to injection of Romiplostim (RP), a Thpo mimetic drug. HSCs from Thpo-/- mice increased 5-fold in number after 10 days of RP injection. Interestingly, while HSCs in Thpo-/- mice exhibited a loss in quiescence, RP administration induced quiescence in Thpo-/- HSCs. HSCs from RP-rescued Thpo-/- mice stained low to TMRE and CellROX indicating low mitochondrial function profile. HSCs from Mito-Dendra2;Thpo-/- mice also exhibited lower mitochondrial mass. Furthermore, RP-rescued HSCs from Thpo-/- mice, exhibited higher reconstitution potential than HSCs from RP-treated Thpo+/+ mice. Moreover, highly purified HSCs isolated from steady-state Thpo-/- mice exhibited comparable reconstitution potential to HSCs from Thpo+/+ mice. RNA and ATAC- sequence analyses were also conducted to gain insights on the regulators for the reversal of proliferative to quiescent states in HSCs. Adult Thpo-/- HSCs resembled the proliferative and metabolic characteristics of neonatal HSCs which are not affected with Thpo deficiency. HSCs from neonatal mice treated with 7 doses of RP exhibited a significant increase in the number of quiescent HSCs. Our data suggest a Thpo-independent HSC subpopulation which sustains low mitochondrial metabolic profile, reverts to quiescence and regains stem cell potential with external stimuli. Thpo-independent HSCs may serve as the main pool of HSCs for neonatal hematopoiesis and form a non-quiescent reserve HSC pool from which quiescent HSCs originate in the adult BM.