Freshly isolated thymic stromal cells in monolayer cultures (TSMCs) cannot support T-cell differentiation. Until the development of the OP9-DL1 monolayer culture system — in which OP9 bone-marrow stromal cells that are stably transfected with the gene encoding the Notch ligand Delta-like 1 (DL1) can support T-cell differentiation — it was widely accepted that in vitro T-cell differentiation could only occur in a three-dimensional system, such as fetal thymic organ cultures (FTOCs) or reaggregate thymic organ cultures (RTOCs). So, in comparison to three-dimensional culture systems, what is it that TSMCs lack in terms of support for T-cell differentiation?

Mohtashami and Zúñiga-Pflücker set out to address this question. The authors used RT-PCR (PCR after reverse transcription of RNA) to look at gene expression by various thymic stromal cells in TSMCs and thymocyte-depleted FTOCs. Because OP9 cells cannot support T-cell differentiation but OP9-DL1 cells can, the authors focused on expression of the various Notch ligands. TSMCs failed to express mRNA transcripts encoding DL1 and DL4, whereas these transcripts were expressed in FTOCs. Moreover, TSMCs that were retrovirally transduced with the genes encoding either DL1 or DL4 regained expression of these ligands and could support T-cell differentiation, indicating that DL1 and DL4 have similar abilities to direct haematopoietic progenitor cells towards a T-cell fate. By contrast, forced expression of either DL1 or DL4 in NIH-3T3 fibroblast was not sufficient to induce and support T-cell development, indicating that signals between Notch and its ligands alone are not sufficient.

These results show that, in the absence of a three-dimensional thymic environment, TSMCs must express either DL1 or DL4 to retain the ability to support T-cell-lineage commitment.