T-cell differentiation of multipotent hematopoletic cell line EML in the OP9-DL1 coculture system

Objective. Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakarvocytic, and B-lymphoid lineages, but it remained unknown whether EML, cells have T-cell developmental potential as well. The goal of this study, was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. Materials and Methods. EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. Results. The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML. cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-T alpha, RAG-1, and T-cell receptor-V beta genes; and 3) produced interferon-gamma in response to T-cell receptor stimulation. Conclusions. These results support the notion that EML cell line has the capacity, for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously, described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation. (C) 2009 ISEH - Society for Hematology and Stem Cells. Published by, Elsevier Inc.