TY - JOUR
T1 - T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system
AU - Kutleša, Snježana
AU - Zayas, Jennifer
AU - Valle, Alexandra
AU - Levy, Robert B.
AU - Jurecic, Roland
N1 - Funding Information:
Cell analysis and sorting were done at the UM Sylvester Cancer Center Flow Cytometry Core Lab. We are indebted to Dr. Zúñiga-Pflücker for sharing with us the OP9-DL1 and OP9 stromal cell lines, and for critical advice and very helpful comments. We also thank our colleagues Drs. Adkins, Strbo, and Malek for helpful comments and for sharing some of the reagents. This work was supported by the National Institutes of Health RO1 RR15242 grant (Bethesda, MD, USA) (R.J.) and the University of Miami Pilot Study Grant (Miami, FL, USA) (R.J.).
PY - 2009/8
Y1 - 2009/8
N2 - 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. 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-Tα, RAG-1, and T-cell receptor - Vβ genes; and 3) produced interferon-γ 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.
AB - 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. 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-Tα, RAG-1, and T-cell receptor - Vβ genes; and 3) produced interferon-γ 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.
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U2 - 10.1016/j.exphem.2009.05.002
DO - 10.1016/j.exphem.2009.05.002
M3 - Article
C2 - 19447159
AN - SCOPUS:67650074833
VL - 37
SP - 909-923.e1
JO - Experimental Hematology
JF - Experimental Hematology
SN - 0301-472X
IS - 8
ER -