Controlling metabolic fluxes for better human haematopoietic stem cell function

Hematopoietic stem cells (HSCs) ex vivo culture is a fundamental step for HSC-based Gene Therapy (GT), the only curative treatment for more than 10 distinct monogenic diseases worldwide. However, a major clinical challenge for HSC GT arises from the loss of HSC function during ex vivo culture, posing a high risk of delayed or failed blood recovery in patients. Recent work from Laurenti laboratory has demonstrated that more than 50% of functional HSCs are lost within the early adaptation to ex vivo culture with significant transcriptional remodelling in genes linked to metabolism. Metabolic switches are known key regulators of HSC function in vivo. Therefore, the central hypotheses of this proposal are that i) metabolic switches contribute to the early loss of human HSCs regenerative capacity during ex vivo culture, ii) targeting metabolic switches pharmacologically can improve HSC function ex vivo. This proposal will first generate a kinetically resolved map of metabolomic switches occurring during human HSC ex vivo adaptation. For this, I will integrate metabolomic analysis on highly purified human HSCs cultured in preclinical GT conditions with scRNA-seq data. My preliminary work, using computational methods on time-course scRNA-seq and functional assays with small molecule inhibitors, has identified purinergic metabolism fluxes as dynamically changed and pharmacologically targetable during HSC ex vivo adaptation. Therefore, in my second aim, I will screen the effects of pharmacological inhibition of key purinergic enzymes to determine how the purinergic metabolic switch: i) regulates HSC function ex vivo and ii) affects HSC regeneration using a preclinical model of HSC GT. This project will generate new insights into the interplay between stem cell metabolism, stress and regeneration. In addition, it will identify new preclinical strategies to improve HSC function ex vivo, with direct relevance to HSC GT and other clinical applications requiring HSC culture.