Enrichment of c-kit+ Lin- haemopoietic progenitor cells that commit themselves to extrathymic T cells in in vitro culture of appendix mononuclear cells

Abstract

The appendix as well as the small intestine have recently been found to carry c-kit+ stem cells which give rise to extrathymic T cells. In this study, the properties of c-kit+ stem cells in the appendix of mice were further characterized. When appendix mononuclear cells (MNC) were cultured in the presence of stem cell factor, interleukin-3, interleukin-6 and erythropoietin on a methylcellulose culture plate, the population of c-kitdull Lin- and that of c-kithi Lin- cells expanded. Morphological study revealed that these c-kithi Lin- cells were basophilic granular cells (possibly mast cells). Both populations of cultured appendix MNC were then injected into severe combined immunodeficient mice or cultured with Tst-4 thymic stroma cells. These in vivo and in vitro studies demonstrated that c-kitdull Lin- cells were oligopotent haemopoietic progenitor cells which gave rise to extrathymic T cells, while c-kithi Lin- cells lacked haemopoietic progenitor cell activity. In contrast to c-kit+ stem cells in the bone marrow, those in the appendix did not give rise to myeloid cells and conventional thymic T cells under any of the conditions tested. The present results suggest that the appendix primarily comprises c-kit+ cells which give rise to basophilic granular cells and extrathymic T cells and that such c-kit+ cells have the ability to replicate themselves in culture in vitro.

Figure 1

Phenotypic characterization of leucocytes isolated from the appendix and bone marrow before and after culture. Two-colour staining for that for lineage markers and c-kit (a), that for Mac-1 and Gr-1(b), that for CD3 and IL-2Rβ (c), and that for CD3 and B220 (d), were conducted. Numbers in the figure represent the percentages of fluorescence-positive cells in corresponding areas. The data shown are representative of three experiments. The low density as well as the high density of c-kit⁺ Lin⁻ cells were generated in the in vitro culture of appendix leucocytes.

Figure 2

Number of cells yielded by the in vitro culture of bone marrow cells and appendix MNC in a methylcellulose culture plate. Number of cells were enumerated at the indicated days. The mean and one SD were produced from four experiments.

Figure 3

A comparison of the light scatter between c-kit^hi Lin⁻ cells of cultured appendix leucocytes. The c-kit^hi Lin⁻ cells had high side-scatter (SSC) and forward-scatter (FSC) whereas c-kit^dull Lin⁻ cells had low SSC and FSC.

Figure 4

Morphology of cultured appendix leucocytes in May–Grünwald–Giemsa staining. Purified c-kit^hi Lin⁻ cells were estimated to be basophilic granular cells while purified c-kit^dull Lin⁻ cells were estimated to be possible stem cells with cytoplasmic vacuoles.

Figure 5

Constitution of T cells in scid mice by transfer of c-kit⁺ cells of cultured appendix MNC and bone marrow cells. Two-colour staining (a) for CD3 and B220, (b) for TCR-αβ and TCR-γδ, (c) for CD4 and CD8, and (d) for CD3 and IL-2Rβ. The c-kit⁺ cells (c-kit^dull and c-kit^hi cells in the case of cultured appendix MNC) were obtained by culturing leucocytes isolated from the appendix and bone marrow. Such c-kit⁺ cells of cultured appendix MNC and bone marrow cells were transferred to scid mice. Lymphocytes were obtained from various immune organs of scid mice 4 weeks after c-kit⁺ cell transfer. Numbers in the figure represent the percentages of fluorescence-positive cells in corresponding areas. The data shown are representative of four experiments.

Figure 6

In vitro induction of T cells from cultured c-kit⁺ cells in the appendix under various conditions. The c-kit⁺ Lin⁻ cells and c-kit^dull Lin⁻ cells were isolated from both cultured bone marrow cells and cultured appendix MNC, respectively. Thymic stroma cells (Tst-4) were used for T-cell induction. Numbers in the figure represent the percentage of fluorescence-positive cells in corresponding areas. The data shown are representative of three experiments.

Figure 7

Expression of RAG-1 and RAG-2 mRNAs by culturing c-kit⁺ Lin⁻ cells in the appendix in the presence of Tst-4 stroma cells. Culture-induced c-kit^dull(∼low) Lin⁻ cells and c-kit^hi Lin⁻ cells from the appendix were again cultured in the presence of Tst-4 stoma cells. Thymocytes were used as a positive control for the expression of RAG-1 and RAG-2 mRNAs.