ABO: A 3D stroma-supported culture platform enabling full human B-lymphopoiesis for disease modeling and gene therapy development.

Defective B cell development underlies a large proportion of inborn errors of immunity. Progress in understanding disease mechanisms and therapy development remains limited because current human in vitro models incompletely recapitulate B-lymphopoiesis. We present a three-dimensional aggregate culture platform composed of human hematopoietic stem and progenitor cells (HSPCs) and mouse bone marrow (BM) stromal cells as an assay for efficient B cell output (ABO). ABOs support progression through pro-B, pre-B, immature, and transitional B cell stages, with a transcriptional profile and B cell receptor (BCR) repertoire diversity resembling human B cell ontogeny. ABO-derived B cells exhibit functional BCR signaling and differentiate into class-switched memory B cells and antibody-secreting cells following CD40-mediated stimulation. ABOs reproduce the B cell developmental arrest observed in HSPCs from patients with RAG1-deficient severe combined immunodeficiency (SCID), which is corrected by a RAG1 gene therapy lentiviral vector. Thus, ABOs provide a clinically relevant platform for modeling B cell immunodeficiencies and evaluating therapeutic strategies.