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

Snježana Kutleša, Jennifer Zayas, Alexandra Valle, Robert B Levy, Roland Jurecic

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

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.

Original languageEnglish
JournalExperimental Hematology
Volume37
Issue number8
DOIs
StatePublished - Aug 1 2009

Fingerprint

Coculture Techniques
Cell Differentiation
T-Lymphocytes
Cell Line
Cell Lineage
Stromal Cells
Cytokines
T-Cell Receptor Genes
Interleukin-7
Adult Stem Cells
Stem Cell Factor
Hematopoietic Stem Cells
T-Cell Antigen Receptor
Interferons
Genes
Reverse Transcription
Flow Cytometry
Ligands
Gene Expression
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Cancer Research
  • Cell Biology
  • Genetics
  • Molecular Biology
  • Hematology

Cite this

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system. / Kutleša, Snježana; Zayas, Jennifer; Valle, Alexandra; Levy, Robert B; Jurecic, Roland.

In: Experimental Hematology, Vol. 37, No. 8, 01.08.2009.

Research output: Contribution to journalArticle

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abstract = "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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