Jepsen, V. H. | 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↗

bioRxiv2024

Linker histone H1-0 is a specific mediator of the repressive ETV6::RUNX1 transcriptional landscape

ETV6::RUNX1 is the most common oncogenic fusion in pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL). It induces a clinically silent preleukemic state that requires secondary mutations for progression to leukemia. However, the molecular mechanisms contributing to the characteristic quiescence of ETV6::RUNX1+ preleukemic cells remain elusive. Here, we detect factors involved in the preleukemic state by generating human induced pluripotent stem cell (hiPSC) models using CRISPR/Cas9 gene editing. We identified upregulation of linker histone H1-0 in our preleukemic models, which was preserved upon hematopoietic differentiation and transformation to BCP-ALL. ETV6::RUNX1 induces H1-0 promoter activity whereas depletion of H1-0 specifically inhibited ETV6::RUNX1 signature genes, indicating its role as a key mediator of the ETV6::RUNX1 transcriptome. Single-cell gene expression analysis revealed high H1-0 levels in quiescent cells during hematopoiesis and inverse correlation with transcriptional activity. Pharmacologically, H1-0 protein levels correspond to susceptibility of BCP-ALL towards histone deacetylase inhibitors (HDACi). Altogether, our study provides novel insights into ETV6::RUNX1-induced quiescence and suggests that further investigation into combinatorial treatment of BCP-ALL using the H1-0- inducing HDACi Quisinostat may be worthwhile.

cancer biology↗