Blockade of Ceramide-Dependent Extracellular Vesicle Secretion Improves Hematopoietic Stem Cell Transplantation

Hematopoietic stem cell (HSC) transplantation serves as a curative therapy for numerous benign and malignant hematopoietic diseases, and as a platform for HSC gene therapy. A main constraint is the limited quantity of HSCs collected from patients or donors and the inherent risk of graft failure, or oligocolonal reconstitution, especially after ex vivo manipulation. Current efforts to mitigate these limitations have predominantly focused on the discovery of extrinsic factors to maintain or expand hematopoietic stem and progenitor cells (HSPCs), whereas the role of the HSPC secretome in modulating niche function and improving occupancy have not been extensively studied. Recently, classes of nanometer-sized extracellular vesicles (EVs) have been shown to regulate tissue function by providing potent proangiogenic signals and remodeling cellular microenvironments. EV secretion additionally regulates homeostasis and stemness properties of HSPCs; however, in-depth studies into HSPC EV biogenesis and the impact on homeostatic and regenerative potency have been hindered by numerous technical challenges.

Here, we utilize an optimized ex vivo expansion protocol for human and murine HSPCs to examine the impact of different EV biogenesis pathways on vesicle secretion. Our studies rapidly focused on ceramide-dependent vesicle secretion; blockade of ceramide biosynthesis with a widely used neutral sphingomyelinase inhibitor, GW4869 significantly reduced HSPC EV secretion. Treatment of HSPCs with GW4869 further inhibited mTOR signaling, altered autophagic flux, and decreased cell cycle entry. Increased ratios of lymphoid (MPP4):myeloid (MPP2/3) primed progenitors accumulated in culture. Upon competitive transplantation into myeloablated recipients, GW4869-exposed murine HSPCs demonstrated superior long-term engraftment potential (>8 steady-state fold change, p < 0.001), independent of bone marrow or splenic homing. Ceramide inhibition did not significantly alter long-term lineage output from transplanted HSPCs. Confirmatory genetic knockout (KO) of neutral sphingomyelinase (nSMase2) in murine and human HSPCs using Cas9 ribonucleoprotein complex delivery similarly demonstrated enhanced colony forming potential. To understand the impact of ceramide inhibition on vesicle cargo, comparative mass spectrometry was performed on HSPC nSMase2 KO EVs (EV^nSmase2-KO). Nearly 900 proteins were depleted (fold change <2) in EV^nSmase2-KO; these underrepresented proteins were noted to be involved in myeloid differentiation and activation, immune response, and exocytosis, suggesting vesicle modulation of these processes. These results suggest that ceramide inhibition not only leads to a reduction in EV release, but also impacts selective trafficking of EV cargo, ultimately altering HSPC function.

While other EV biogenesis pathways have been demonstrated to promote HSC-self renewal, these findings implicate ceramide-dependent EV secretion as a novel negative regulator of HSPC repopulation. Transient disruption of ceramide-dependent EV secretion results in a more quiescent and less metabolically active HSPC pool, and additionally prevents bias toward a myeloid-primed progenitor pool seen with ex vivo expansion. These effects culminate in dramatically improved repopulation potential of HSPCs. Furthermore, unlike other small molecular inhibitors proposed to enhance engraftment of HSPCs, including rapamycin, no significant hinderance of ex vivo expansion was seen with GW4869 treatment. We therefore propose broadened exploration of ceramide inhibition for ex vivo maintenance of HSPCs prior to transplantation.

No relevant conflicts of interest to declare.