T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system.
OBJECTIVE
Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, 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.


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-Talpha, RAG-1, and T-cell receptor-Vbeta 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.
Kutlesa
Snjezana
S
Department of Microbiology and Immunology, Miller School of Medicine, University of Miami, Miami, Fla. 33136, USA.
Zayas
Jennifer
J
Valle
Alexandra
A
Levy
Robert B
RB
Jurecic
Roland
R
D015415:Biomarkers / Q000032:analysis; D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D018920:Coculture Techniques; D016207:Cytokines / Q000494:pharmacology; D020869:Gene Expression Profiling; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D016130:Immunophenotyping; D039902:Multipotent Stem Cells / Q000166:cytology*; D017154:Stromal Cells / Q000166:cytology; D013601:T-Lymphocytes / Q000166:cytology*
D015415:Biomarkers; D016207:Cytokines