Figure 6. T lineage differentiation from marrow progenitors is influenced by IL-7 concentration and subculture.

A. Lin⁻c-Kit^loIL-7Rα⁺ pro-lymphocytes were cultured first on OP9-DL1 with 5 ng/ml IL-7 and 5 ng/ml Flt3-Lfor two weeks. They were then subcultured to fresh stromal cells with the indicated concentrations of IL-7 plus Flt3-L and subcultured again one week later. After a total of four weeks of culture, cells were harvested and stained for flow cytometry analysis.

B. HSC, LSP, ELP and CLP fractions were cultured on OP9-DL1 with 1 ng/ml IL-7 and 5 ng/ml Flt3-L for eight days. They were then subcultured under the same conditions for another 16 days before harvest and staining for CD4 and CD8.

C. Total yields of CD4⁺ CD8⁺ (DP) cells were calculated per input HSC, LSP, ELP or CLP in cultures that were maintained for the whole time on OP9-DL1 with Flt3-L and either 1 ng/ml IL-7 or 0.2 ng/ml IL-7. After the first eight days, all groups were subcultured with the original conditions for another 16 days before harvest and analysis. The absolute numbers of DP cells per sorted progenitor were calculated by multiplying yields of the first culture interval with that of the second interval and the frequency of DP cells.

D. HSC were cultured on OP9-DL1 with Flt3-L and 1 ng/ml IL-7 for 8 days, and then subcultured in two groups . One group (shown by open bars) was harvested and stained on day 27, The parallel group (closed bars) was maintained in the same way, but passaged one additional time on day 24. Numbers of total cells, CD4⁻ CD8⁻ ( DN) and DP cells are shown.