We report here the successful selective cultivation of murine thymic mesenchymal reticular cells (MTMC) and murine thymic epithelial cells (MTEC) grown on extracellular matrix in the presence of defined medium. The selective growth of these two cell types was based on 1) conditions of tissue disruption and 2) differential growth requirements. Both cell types were dependent on transferrin, high density lipoproteins, insulin, hydrocortisone, and epidermal growth factor, whereas MTMC was dependent also on selenium and 3,5,3'-triiodothyronine. The elimination of single factors or extracellular matrix resulted in specific and different changes in the growth pattern of each cell subpopulation. Cells of both types exhibited the ultrastructural features of high metabolic activity. The epithelial nature of MTEC cultures was defined by bundles of tonofilaments and desmosomes and by positive staining to keratins and negative to vimentin. In addition MTEC were positively stained with mAb to thymic medullary epithelial cells and by Ulex europeus agglutinin, and were able to form Hassall's corpuscles, suggesting their medullary origin. MTEC were also H-2 and Ia positive. In contrast MTMC were positive for vimentin and periodic acid-Schiff, low positive for H-2, and negative for keratin and Ia. Both cells did not contain nonspecific esterase, nor did they phagocytize latex beads. With the use of all these criteria we classified MTEC as epithelial cells from the medullary compartment of the thymus and MTMC as reticular cells of mesenchymal origin.
Two morphologically distinct primary cultures of murine thymic stroma were established and found to be of epithelial (MTEC) and mesenchymal (MTMC) origin. These cultures were generated by selective conditions of tissue disruption and were maintained on extracellular matrix in defined medium. Culture supernatants (CS) from these cultures (EC-CS and MC-CS respectively), were tested for cytokine production and for effects on thymocyte maturation. Both supernatants displayed the activities of IL-3 and of granulocyte/macrophage-CSF and not of IL-1, -2, -4, or IFN. In addition they were found to be mitogenic to murine thymocytes in a "spontaneous" [3H]TdR incorporation assay. The two supernatants differed, however, in their effect on Con A stimulation. EC-CS had a strong enhancing effect, both when used for preincubation (18 h) before Con A stimulation or when present simultaneously with it. MC-CS had a small inconsistent effect under these conditions. Also EC-CS enhanced IL-2 and IL-3 production by thymocytes. The responsive thymocyte subpopulation was the one that does not bind peanut agglutinin. CS of an established thymic epithelial cell line displayed only part of these activities at a considerably lower level. CS from primary kidney cell culture was completely devoid of activity. The results suggest that primary thymic stromal cell cultures, cultivated under the defined conditions described here, may better preserve physiologic secretory activities, and probably also other cell functions, compared with established cell lines. Furthermore, the results are compatible with the hypothesis that the soluble factors, secreted by thymic stromal cells, are active on either very early or late stages of thymic differentiation, whereas the main intrathymic stages of differentiation are conceivable dependent primarily on direct contact with stromal cells.
The thymic extract TP-1 significantly enhanced the production of immune interferon (IF) by human peripheral blood mononuclear cells in Con A- or Raji- but not PHA-induced cultures. TP-1 effect was more pronounced under conditions of low IF production. The effect was evident before cell proliferation took place and without necessary concurrent effect of TP-1 on the blastogenic response. TP-1 had no detectable effect on nonactivated cultures. The enhancement of IF production by TP-1 to the degree demonstrated here necessitates consideration of its possible effects in every assay of thymic hormones that involves lectin or allogeneic stimulation of lymphocytes.
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