Differentiation in culture of murine primitive lymphohematopoietic progenitors toward T-cell lineage

Earlier, we described a stromal cell-free two-step clonal culture system in which murine primitive lymphohematopoietic progenitors produce myeloid and B-lymphoid lineage cells. In the same culture T-cell potential of the prog enitors was maintained. We now report that, in addition to myeloid and B-ly mphoid cells, putative T-cell progenitors are also produced in culture. Lin eage-negative (Lin(-)) Ly-6A/E+ c-kit(+) bone marrow cells from 5-fluoroura cil-treated mice were cultured in methylcellulose in the presence of SF (St eel factor), interleukin (IL)-11, and IL-7, and the resulting primary colon ies were picked and pooled. When injected into severe combined immune defic iency (scid) mice, the pooled cells reconstituted the T-cell compartment of the scid mice earlier than freshly prepared primitive marrow cells. This r econstitution activity of the pooled primary colony cells was enriched in t he Ly-6A/E+ and Fc gamma RII/III-/low cell fractions. Reverse transcriptase -polymerase chain reaction (RT-PCR) and DNA-PCR analyses showed that some o f the primary colony cells are differentiated sufficiently to express messe nger RNA (mRNA) of T-cell receptor (TCR) beta-chain and pre-TCR alpha (pT a lpha) and, although not frequently, to perform D beta-J beta rearrangement of the TCR gene. Micromanipulation studies confirmed the clonal origin of m yeloid lineage cells and the cells positive for the T-cell-specific transcr ipts and D-J rearrangement of TCR beta-chain. These results suggested that, in the presence of SF, IL-11, and IL-7, primitive lymphohematopoietic prog enitors differentiate toward T-cell lineage in addition to myeloid and B-ce ll lineages. (C) 1999 by The American Society of Hematology.