Regenerative Therapy (Dec 2024)
Synergistic effect and molecular mechanism of nicotinamide and UM171 in ex vivo expansion of long-term hematopoietic stem cells
Abstract
Introduction: Several approaches to expand human hematopoietic stem cells (HSCs) have been reported, but the ability of these methods to expand long-term hematopoietic stem cells (LT-HSCs) remains to be improved, which limits the application of HSCs-based therapies. Methods: CD34+ cells were purified from umbilical cord blood using MacsCD34 beads, and then cultured for 12 d in a serum-free medium. Flow cytometry was used to detect phenotype, cell cycle distribution, and apoptosis of the cultured cells. Colony-forming cell (CFC) assays can evaluate multi-lineage differentiation potential of HSCs. Real-time polymerase chain reaction was employed to detect the expression of genes related to self-renewal programs and antioxidant activity. DCFH-DA probes were used to evaluate intracellular production of reactive oxygen species (ROS). Determination of the effect of different culture conditions on the balance of cytokine by cytometric bead array. Results: Here, we show a combination, Nicotinamide (NAM) combined with pyrimidoindole derivative UM171, can massively expand LT-HSCs ex vivo, and the expanded cells maintained the capability of self-renewal and multilineage differentiation. Additionally, our data indicated that UM171 promoted self-renewal of HSCs by inducing HSCs entry into the cell cycle and activating Notch and Wnt pathways, but the infinite occurrence of this process may lead to mitochondrial metabolism disorder and differentiation of HSCs. NAM kept HSCs in their primitive and dormant states by reducing intracellular ROS levels and upregulating the expression of stemness related genes, so we believed that NAM can act as a brake to control the above process. Conclusions: The discovery of the synergistic effect of NAM and UM171 for expanding LT-HSCs provides a new strategy in solving the clinical issue of limited numbers of HSCs.
