2002 - EX VIVO ACTIVATION OF HEAT SHOCK FACTOR 1 (HSF1) PROMOTES SUSTAINED HEMATOPOIETIC STEM CELL SELF-RENEWAL

Abstract

The inability to maintain and expand hematopoietic stem cells (HSCs) in culture represents a major barrier to their expanded use in cell-based therapies. We recently discovered that HSCs exhibit low protein synthesis in vivo, regardless of whether they are quiescent or undergoing self-renewing divisions. In the present study, we determined that cultured HSCs rapidly upregulated genes that promote translation and exhibited a ∼2000% increase in protein synthesis. Increased protein synthesis overwhelmed protein quality control systems within HSCs, caused an imbalance in protein homeostasis, and was associated with nuclear translocation of Hsf1. Hsf1 is the master regulator of the heat shock pathway, and induces transcription of heat shock proteins that coordinate proteotoxic stress response to maintain protein homeostasis. Inactive Hsf1 is typically sequestered in the cytoplasm, and is rarely seen in the nucleus of HSCs in vivo. Genetic deletion of Hsf1 exacerbated HSC depletion in vitro, but had little effect on HSC function in vivo. These data indicated that Hsf1 promotes ex vivo HSC maintenance, and raised the possibility that increasing Hsf1 activation could enhance HSC self-renewal. To test this, we developed a new serum- and stroma-free platform to culture purified HSCs. In 10-day cultures initiated with just 10 purified HSCs, small molecules that promoted Hsf1 nuclear translocation supported extensive proliferation and complete retention of long-term multilineage reconstituting activity in serial transplantation assays. The positive effect of these small molecules on HSC growth was completely ablated in the absence of Hsf1. At the molecular level, Hsf1 activation reduced the unfolded protein load and partially rebalanced protein homeostasis in HSCs. These findings indicate that maintaining protein homeostasis is a key factor in promoting ex vivo HSC self-renewal.