多能造血细胞系EML在OP9-DL1共培养系统中的T细胞分化
T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system.
作者信息
Kutlesa Snjezana, Zayas Jennifer, Valle Alexandra, Levy Robert B, Jurecic Roland
机构信息
Department of Microbiology and Immunology, Miller School of Medicine, University of Miami, Miami, Fla. 33136, USA.
出版信息
Exp Hematol. 2009 Aug;37(8):909-23. doi: 10.1016/j.exphem.2009.05.002. Epub 2009 May 15.
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-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.
目的
多能造血细胞系EML可分化为髓系、红系、巨核系和B淋巴细胞系，但EML细胞是否也具有T细胞发育潜能尚不清楚。本研究的目的是确定与表达Notch配体Delta样1的OP9基质细胞（OP9-DL1）共培养是否能诱导EML细胞分化为T细胞系。
材料与方法
将EML细胞与对照OP9或OP9-DL1基质细胞在细胞因子（干细胞因子、白细胞介素-7和Fms样酪氨酸激酶3配体）存在的情况下共培养。通过流式细胞术以及对表征T细胞发育特定阶段并与之相关的细胞表面标志物和基因进行逆转录聚合酶链反应表达分析，评估它们的T细胞系分化情况。
结果
表型、分子和功能分析表明，在添加细胞因子的EML/OP9-DL1共培养物中，而非对照EML/OP9共培养物中，EML细胞系经历了T细胞系的定向分化和分化。在OP9-DL1共培养物中，EML细胞系已分化为以下细胞：1）类似于T细胞发育的双阴性、双阳性和单阳性阶段；2）开始表达GATA-3、前Tα、RAG-1和T细胞受体-Vβ基因；3）在T细胞受体刺激下产生干扰素-γ。
结论
这些结果支持EML细胞系具有T细胞分化能力这一观点。值得注意的是，EML细胞中T系基因表达的诱导以及其分化为T细胞发育的不同阶段与之前描述的成年造血干细胞和祖细胞在OP9-DL1共培养物中的T细胞分化非常相似。因此，EML/OP9-DL1共培养可能是一个有用的实验系统，用于研究特定基因在T细胞系特化、定向分化和分化中的作用。
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