Development of a Method of Thymocyte Differentiation of Bone Marrow‐Enriched CD34+CD38−Cells in Postnatal Allogeneic Cultured Thymic Epithelia to Evaluate Immunodeficiency Disorders

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Abstract
An in vitro model of CD34+CD38− stem cell (SC) differentiation in postnatal cultured thymic epithelia fragment (CTEF) cocultures is described. Sequential phenotypic analysis of the progeny of the SC-CTEF demonstrated predominantly thymocytes and minor populations of promyelocytes, monocytes and natural killer cells. Triple-positive CD3+CD4+CD8+, double-positive CD4+CD8+, and mature single-positive CD4+ and CD8+ T cells, which were TCRαβ+, were identified indicating normal thymocyte maturation. In kinetic studies, mature single-positive CD4+ T cells increased from 29% of total cells at one week to 54% at four weeks of coculture. These findings demonstrate that coculture of bone marrow-derived SC and allogeneic cultured thymic epithelia in vitro results in continuous normal predominantly thymocyte differentiation. The SC-CTEF cocultures were then infected with two different strains of human immunodeficiency virus. CD4+ thymocytes were markedly decreased. However, inhibition of early thymocyte maturation steps was also suggested by the presence of increased triple-negative and CD44+CD25−CD3− thymocytes and decreased CD44+CD25+ thymocytes. This model system of thymocyte maturation will be useful in the evaluation of primary T cell immunodeficiency disorders, gene therapy of SC and pharmacological augmentation of thymic function.