Metabolite Patterns in Human Myeloid Hematopoiesis Result from Lineage-Dependent Active Metabolic Pathways

Abstract

Assessment of hematotoxicity from environmental or xenobiotic compounds is of notable interest and is frequently assessed via the colony forming unit (CFU) assay. Identification of the mode of action of single compounds is of further interest, as this often enables transfer of results across different tissues and compounds. Metabolomics displays one promising approach for such identification, nevertheless, suitability with current protocols is restricted. Here, we combined a hematopoietic stem and progenitor cell (HSPC) expansion approach with distinct lineage differentiations, resulting in formation of erythrocytes, dendritic cells and neutrophils. We examined the unique combination of pathway activity in glycolysis, glutaminolysis, polyamine synthesis, fatty acid oxidation and synthesis, as well as glycerophospholipid and sphingolipid metabolism. We further assessed their interconnections and essentialness for each lineage formation. By this, we provide further insights into active metabolic pathways during the differentiation of HSPC into different lineages, enabling profound understanding of possible metabolic changes in each lineage caused by exogenous compounds.

Lineage-Selective Disturbance of Early Human Hematopoietic Progenitor Cell Differentiation by the Commonly Used Plasticizer Di-2-ethylhexyl Phthalate via Reactive Oxygen Species: Fatty Acid Oxidation Makes the Difference

“…Human cord blood CD34 + cells were expanded in Stempro-34 serum-free medium (SFM) containing 2 mM L-glutamine, 100 ng/mL stem cell factor (SCF), 100 ng/mL Flt-3 ligand (Flt-3L), 50 ng/mL interleukin (IL)-6, 40 ng/mL thrombopoietin (TPO), 1 µM Stem-Regenin (SR)-1, 0.1 µM BML-210, and 0.2 mM valproic acid as previously described [31].…”

“…Erythroid differentiation was induced in Stempro-34 SFM containing 2 mM L-glutamine, 100 ng/mL SCF, 100 ng/mL Flt-3L, 50 ng/mL erythropoietin (EPO), and 20 ng/mL IL-3 for up to 6 days as previously described [31]. Dendritic cell differentiation was induced in Stempro-34 SFM containing 2 mM L-glutamine, 10 ng/mL SCF, 10 ng/mL TPO, and 10 ng/mL IL-3 for up to 11 days as previously described [31].…”

“…Erythroid differentiation was induced in Stempro-34 SFM containing 2 mM L-glutamine, 100 ng/mL SCF, 100 ng/mL Flt-3L, 50 ng/mL erythropoietin (EPO), and 20 ng/mL IL-3 for up to 6 days as previously described [31]. Dendritic cell differentiation was induced in Stempro-34 SFM containing 2 mM L-glutamine, 10 ng/mL SCF, 10 ng/mL TPO, and 10 ng/mL IL-3 for up to 11 days as previously described [31]. Neutrophil differentiation was induced in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% fetal bovine serum, 50 ng/mL SCF, and 50 ng/mL IL-15 for up to 10 days as previously described [31].…”

“…Dendritic cell differentiation was induced in Stempro-34 SFM containing 2 mM L-glutamine, 10 ng/mL SCF, 10 ng/mL TPO, and 10 ng/mL IL-3 for up to 11 days as previously described [31]. Neutrophil differentiation was induced in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% fetal bovine serum, 50 ng/mL SCF, and 50 ng/mL IL-15 for up to 10 days as previously described [31]. DEHP, MEHP, or 2-ethylhexanol (2-EH), all dissolved in ethanol, pure ethanol (vehicle control), or agonists/antagonists as indicated, was added to the differentiating cultures and maintained at constant concentrations during the experiments.…”

“…We recently established and characterized a model of myeloid HSPC differentiation capable of undergoing differentiation into erythrocyte, dendritic cell, and neutrophil lineages. Metabolic activities differed markedly between these lineages; thus, this model will be ideal for use in experiments designed to characterize the effects of individual compounds on specific signaling and metabolic pathways and will also facilitate the identification of common mechanisms [31]. The use of this model could also lead to the identification of potentially unique protective mechanisms; this would not be possible in experiments featuring single cell lines.…”