Serum-free differentiation platform for the generation of B lymphocytes and natural killer cells from human CD34+ cord blood progenitors

Pre-clinical research on B and NK cell development relies on murine stromal cell-based systems with reduced physiological relevance and clinical applicability. A serum-free, fully humanized co-culture system utilizing human bone marrow-derived mesenchymal stromal cells (BM-MSCs) was developed to differentiate CB-CD34+ cells towards B and NK cell lineages. Differentiation dynamics were monitored via flow cytometry, with immunophenotypic analysis tracking progression from progenitors to mature cells. The system generated CD19+ IgM+ immature B cells and CD56+ CD16+ NK cells, recapitulating fetal stages of human lymphopoiesis. Serum-free media conditions ensured reproducibility and high overall yield of CD19+ B (35 ± 5.32%) and CD56+ NK (28.46 ± 7.01%) cell progenitors. Flow cytometry identified distinct population peaks, confirming temporal control over differentiation. This clinically relevant platform addresses the limitations of traditional models by providing a more physiologically accurate human microenvironment. The serum-free system supports applications in disease modeling, genotoxic compound screening, and mutational studies of hematopoiesis. By enabling scalable production of B and NK cells it aims to accelerate translational research for immunodeficiencies, cancer immunotherapy, and hematopoietic disorders.