SUMMARY
Hematopoietic stem cells (HSC) need to integrate microenvironmental cues to switch their fate between
quiescence, self-renewal, and differentiation, thereby sustaining hematopoiesis through life. However, the
molecular programs governing HSC stemness become grossly dysregulated during culture, hindering our ability
to expand functional HSCs for treating hematological diseases. Our data uncovered MYCT1 (Myc target 1) as
human HSC regulatory factor that is critical for their expansion and engraftment, but becomes suppressed during
differentiation and culture. We found that MYCT1 localizes in endosomes, interacts with vesicle components and
signaling receptors with critical functions in HSCs (e.g. TGFBR1 and 2), and controls the rate of endocytosis,
which modulates cell signaling and must be tightly regulated to maintain HSC stemness. Many genes
dysregulated upon MYCT1 knockdown (KD) are linked to HSC division and self-renewal. We hypothesize that
MYCT1 governs HSC stemness and their ability to balance between fate options by fine-tuning multiple signaling
cues through the control of endocytosis. We have created a toolbox of MYCT1 knockdown and overexpression
vectors, including various MYCT1 deletion mutants, that can be used in primary human HSPCs as well as HSC-
like and endothelial cell line models to investigate MYCT1 mechanism of action. We will investigate how MYCT1-
mediated control of endocytosis affects signaling in HSCs, and how this influences HSC cell-fate decisions
including quiescence and asymmetric vs symmetric division. We will then assess if rescuing MYCT1 regulated
processes improves the function of cultured human HSC, including those expanded using MLLT3
overexpression. Our work may open up new strategies to maintain HSC transplantability during ex vivo
expansion and ultimately help broaden HSC clinical applications.