Metabolic adaptation pilots the differentiation of human hematopoietic cells (scRNA-Seq and CITE-Seq)
In order to get insight in the role of metabolic perturbations during the first steps of cell differentiation, the phenotype, gene expression profile and chromatin accessibility of CD34+ cells were examined using bulk- and single-cell techniques during the initial 96 hours of in vitro differentiation. Specific enzyme inhibitors targeting key steps of the energy metabolism were employed to induce perturbations and assess their effect. The results support a model whereby energy requirements play the role of initiator of differentiation. According to it, metabolic perturbations induce rapid and non-specific, enabling a state of multi-primed gene expression. A selective re-compaction of the chromatin constrained by the availability of key metabolites essential for repressive epigenetic modifications and by the action of transcription regulator proteins consolidates the phenotypic response of the cells. Overall design: Human hematopoietic CD34+ cells were grown in Xvivo medium supplemented with cytokines with or without energy metabolism inhibitor (DON, 2-DG or AOA) during 96h. Some conditions were supplemented with alpha-ketoglutarate for rescue experiments. Control condition contains two biological replicates (CTRL and CTRL2), they were merged for downstream bioinformatic analysis. Each sample is derived from a pool of at least seven healthy donors. The cells were stained with CITE-seq antibodies (ADTs and HTOs) and inserted in a microfluidic chip for single cell analysis. ADT/HTO barcode sequence hashtagged sample TotalSeq™ A0251 anti-human hashtag1 GTCAACTCTTTAGCG CTRL, DON, DON+aK TotalSeq™ A0252 anti-human hashtag2 TGATGGCCTATTGGG 2DG, CTRL+aK, 2DG+aK TotalSeq™ A0126 anti-human CD133  TGGTAACGACAGTCC TotalSeq™ A0054 anti-human CD34 GCAGAAATCTCCCTT