Abstract
Hematopoietic stem cells (HSCs) differentiate in the thymus in to T cells along precisely defined steps. In this process the interaction between the thymic stroma, the epithelium and the hematopoietic precursors is indispensable. Previously it has been shown that human CD34+ cells from fetal liver, umbilical cord blood and adult bone marrow could differentiate meaningful numbers of T cells when seeded either into fetal thymic organ cultures (FTOCs) or human and Rhesus fetal thymic stromal monolayers. In severe combined immunodeficient (SCID) mouse models, development of T cells from transplanted human CD34+ cells requires previous engraftment of a human fetal thymus. All the mentioned assays require fetal tissues and are technically cumbersome. We set out to explore the possibility of using murine thymic stromal microenvironment to support differentiation of lymphocytes from human HSCs. Here we report that highly purified CD34+ or AC133+CD34+ human umbilical cord blood cells generate T cells and natural killer (NK) cells when cultured on irradiated murine thymic stromal monolayers in the presence of recombinant human IL-12 and recombinant murine SCF. In this system, the human stem cells differentiate sequentially into CD4+CD8−CD3−, CD4+CD8+CD3−, CD4+CD8+ CD3+, and finally, CD4+CD8−CD3+ and CD4−CD8+CD3+ cells expressing T cell receptor α/β. CD56+CD3− NK cells were also differentiated and all cells strongly expressed human leucocyte-common antigen CD45. The kinetics of differentiation from HSCs to mature T cells were completed in about 6 weeks. Mature T-cells appeared to be functional as assessed by their normal response to mitogenic stimuli. This novel chimeric human-mouse thymic stromal culture offers a tool to study human T-cell ontogeny in vitro and is a rapid assay for T-cell precursor activity of genetically modified human stem cells. It also provides a preclinical model for analyzing the stability of transgene expression in differentiating lymphocytes.
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