Generation of TransgenicT Cells from Human CD34+Cord Blood Cells

Hematopoietic stem cells (HSCs), progenitor cells capable of generating all peripheral blood cell lineages, are believed not to express lineage markers (Lin) found on the latter, nor CD38, but may or may not express CD34. Besides HSCs, more committed CD34⁺ progenitors expressing CD38, and lineage markers like CD7 or IL-7R can generate T cells in the thymus (1 ). Cord blood, fetal liver, bone marrow, and peripheral blood CD34⁺ cells can generate T cells. These progenitors differ in their cytokine responsiveness and mitogenic potential, as exemplified by retroviral transduceability. Gene-marked and cultured progenitors can be studied for their ability to generate T cells, either in vivo or in vitro (2 ,3 ). Human fetal thymus fragments are transplanted in severe combined immune deficiency (SCID) mice to establish an in vivo model for T cell generation from these manipulated progenitors (SCID-hu). Alternatively, an in vitro model can be used in which a cultured fetal thymic lobe of nonobese diabetic (NOD)-SCID mice serves as the three-dimensional cellular network that supports human T-cell generation (fetal thymic organ culture [FTOC]). In contrast to the SCID-hu in vivo model, FTOC allows comparing numerous experimental conditions, such as cytokine cocktail titrations, with a manageable work load (3 ). In the FTOC, progenitor cells generate CD4/CD8 double-positive immature and ultimately single-CD4-positive and single-CD8positive mature thymocytes. In this chapter, we will detail 1� isolation of hematopoietic progenitor cells from human cord blood, 2� cytokine-stimulated retroviral transduction of these progenitors, and 3� assay of T-cell generation from gene-marked progenitors in FTOC.