A two-step culture process for generating abundant CD14⁺ monocytes from human hematopoietic stem cells

Background: Monocytes are differentiated from hematopoietic stem cells (HSCs) and can furtherly differentiate into dendritic cells (DCs). Monocytes and DCs both have ability to perform endocytosis, secrete cytokines and activate T cell immune response. Therefore, monocytes are one of crucial immune cells in immunotherapy, and it is an important issue to develop a culture system for generating abundant CD14⁺ monocytes from HSCs for clinical application. Methods: In this study, a two-step culture process utilizing a serum-free expansion medium for HSCs and a cytokine cocktail for monocyte induction was employed, resulting in the successful generation of CD14⁺ cells with a classical monocyte phenotype, characterized by high CD14 expression and low CD16 expression. Comprehensive analysis of monocyte-related surface markers confirms the attainment of a monocyte profile. The differentiated monocytes exhibit significant immunostimulatory capabilities, as demonstrated by their cytokine secretion profiles and their ability to stimulate allogeneic T cells. Significant findings: A two-step culture process involving a 7-day HSC expansion followed by a 14-day monocyte differentiation can yield more than 3,000 monocytes from a single freshly isolated HSC and can provide a promising monocyte source for immunotherpay.