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bioRxiv · 10.1101/2025.05.22.655473

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

Bhave, R.·Kath, C.-J.·Ruechel, N.·Vasileiou, E.·Jepsen, V. H.·Raba, K.·Pandyra, A. A.·Fischer, U.·Kogler, G.
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Abstract

IntroductionPre-clinical research on B and NK cell development relies on traditional murine stromal cell-based systems with reduced physiological relevance and clinical applicability. MethodsA 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. ResultsThe 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 B and NK cell progenitors. Flow cytometry identified distinct population peaks, confirming temporal control over differentiation. ConclusionThis 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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/655473v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@427817org.highwire.dtl.DTLVardef@1da616dorg.highwire.dtl.DTLVardef@325ff5org.highwire.dtl.DTLVardef@139f7fb_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance StatementThis article presents a novel, fully humanized, serum-free co-culture system that efficiently directs cord blood-derived hematopoietic stem cells into B and natural killer (NK) cells. By using human bone marrow stromal cells and recombinant human cytokines, it overcomes the limitations of murine-based models and better mimics human blood cell development. This platform enables improved disease modeling and therapeutic testing relevant to human hematopoiesis.

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Bhave, R., Kath, C.-J., Ruechel, N., Vasileiou, E., Jepsen, V. H., Raba, K., Pandyra, A. A., Fischer, U., Kogler, G.. 2025-05-27. Serum-free differentiation platform for the generation of B lymphocytes and natural killer cells from human CD34+ cord blood progenitors. https://doi.org/10.1101/2025.05.22.655473

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