In Vitro Lymphoid Differentiation of Human Hematopoietic Progenitor Cells from Co-cultures in Hypoxia

Understanding the identity of cells from which lymphoid differentiation occurs can be addressed using specific cell assays. Ex vivo conditions have been established based on co-culture systems between human hematopoietic stem/progenitor cells or early lymphoid progenitors and stromal cells. These systems provide a functional approach to carefully assess cell potential at both qualitative and quantitative levels. This chapter describes a two-step co-culture system for isolated hematopoietic stem/progenitor cells, using a murine bone marrow stromal cell line (MS-5), a hypoxic environment (3.5% O₂), defined lymphoid medium and cytokine conditions, and limiting dilution settings. This setup enables a robust characterization of the intrinsic lymphoid potential of progenitor cells, closely mimicking physiological condition. Importantly, this approach can be instrumental in investigating how intrinsic or microenvironmental alterations in early hematopoiesis contribute to immune dysregulation and the development of autoimmune diseases.