Raba, K. | bioRxiv Science

bioRxiv2025

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

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.

developmental biology↗

bioRxiv2020

Umbilical cord blood-derived ILC1-like cells constitute a novel precursor for mature KIR+NKG2A- NK cells

Despite their identification several years ago, molecular identity and developmental relation between human ILC1 and NK cells, comprising group 1 ILCs, is still elusive. To unravel their connection, thorough transcriptional, epigenetic, and functional characterization was performed from umbilical cord blood (CB). Unexpectedly, ILC1-like cells lacked Tbet expression and failed to produce IFNg. Moreover, in contrast to previously described ILC1 subsets they could be efficiently differentiated into NK cells. These were characterized by highly diversified KIR repertoires including late stage NKG2A-KIR+ effector cells that are commonly not generated from previously known NK cell progenitor sources. This property was dependent on stroma cell-derived Notch ligands. The frequency of the novel ILC1-like NK cell progenitor (NKP) significantly declined in CB from early to late gestational age. The study supports a model in which circulating fetal ILC1-like NKPs travel to secondary lymphoid tissues to initiate the formation of diversified NK cell repertoires after birth.

immunology↗