Dynamic Changes in Proteostasis Network Activity Influence Hematopoietic Stem Cell Ontogeny and Human Umbilical Cord Blood Fitness

Hematopoietic stem cells (HSCs) establish hematopoiesis and maintain regeneration of blood and immune cells to meet shifting demands for blood cell production during development and throughout life. The protein homeostasis (proteostasis) network is uniquely configured in adult HSCs to preserve stem cell fitness and longevity. However, the regulation of proteostasis in developing HSCs is largely unexplored. Here, we comprehensively analyzed proteostasis network activity throughout fetal and neonatal development. Fetal HSCs exhibited up to 7-fold higher protein synthesis rates than their adult counterparts but contained similarly low amounts of unfolded and misfolded proteins as adult HSCs, which depend on unusually low protein synthesis rates to preserve proteome quality. Surprisingly, this was not accomplished through enhanced protein degradation as fetal and adult HSCs exhibited similarly low protein degradation activity. However, we found that fetal HSCs preferentially activated Heat shock factor 1 (Hsf1), a key proteostasis sensor and master regulator of the heat shock response, and preferentially expressed multiple Hsf1 target genes. Conditional deletion of Hsf1 in the developing hematopoietic system resulted in compensatory suppression of protein synthesis and aberrant expansion of fetal liver HSCs but had no effect when deleted in young adult HSCs. These data indicate that fetal and adult HSCs utilize distinct mechanisms to preserve proteostasis.

Strikingly, as part of these studies, we discovered that HSCs exhibit a dramatic spike in unfolded protein abundance at birth. This raised the question of whether proteostasis disruption limits the efficacy and regenerative potential of human umbilical cord blood-derived hematopoietic stem and progenitor cells, which are collected immediately after birth. To test this, we fractionated umbilical cord blood-derived CD34⁺ cells based on unfolded protein content and assessed their function. CD34⁺ cells with low unfolded protein content had up to 9-fold higher colony-forming efficiency than CD34⁺ cells with high unfolded protein. Furthermore, CD34⁺ cells with high unfolded protein content exhibited diminished reconstituting activity in xenotransplantation assays in vivo.

These findings indicate that the HSC proteostasis network undergoes dynamic changes throughout ontogeny and that HSCs depend on distinct age-specific mechanisms to maintain proteostasis throughout life. Stratifying CD34⁺ cells by variations in proteostasis identifies populations with varied fitness and engraftment potential. As a result, uncovering distinct proteostasis dependencies could offer a unique window for uncovering new therapeutic avenues for enhancing HSC fitness that leverage developmental stage-specific proteostasis programs and reveal proteostasis-based biomarkers as predictors of stem cell quality and transplantation outcomes.

Signer:Vir: Current equity holder in publicly-traded company; Illumina: Current equity holder in publicly-traded company; Syndax: Current equity holder in publicly-traded company; Exact Sciences: Current equity holder in publicly-traded company, Other: Spouse employment.