Differentiation of normal human pre-B cells in vitro

Abstract

The differentiation of surface Ig- pre-B cells into surface Ig+ B cells is a critical transition in mammalian B cell ontogeny. Elucidation of the growth factor requirements and differentiative potential of human pre-B cells has been hampered by the absence of a reproducible culture system that supports differentiation. Fluorescence-activated cell sorting and magnetic bead depletion were used to purify fetal bone marrow CD10+/surface μ- cells, which contain 60-70% cytoplasmic μ+ pre-B cells. CD10+/surface μ- cells cultured for 2 d were observed to differentiate into surface μ+ cells. Analysis by Southern blotting provided direct evidence that rearrangement of κ light chain genes occurs in culture, and flow cytometric analysis revealed the appearance of surface Ig+ B cells expressing μ/κ or μ/λ. Unexpectedly, the κ/λ ratio in differentiated cells was the inverse of what is normally observed in adult peripheral blood. Differentiation occurs in the absence of exogenous growth factors or cytokines, suggesting that a stimulus-independent differentiative inertia might characterize pre-B cells in vivo. Future use of this model will facilitate our understanding of normal and abnormal human pre-B cell differentiation.