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

Introduction

Pre-clinical research on B and NK cell development relies on traditional murine stromal cell-based systems with reduced physiological relevance and clinical applicability.

Methods

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.

Results

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 B and NK cell progenitors. Flow cytometry identified distinct population peaks, confirming temporal control over differentiation.

Conclusion

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.

Graphical Abstract

Significance Statement

This 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.