Propagation of common lymphoid progenitors in OP9-DL1 co-culture results in generation of cells incapable of β-selection (36.54)

Abstract

Abstract Common lymphoid progenitors derived from murine bone marrow have previously been demonstrated to be capable of long-term propagation in OP9-DL1 co-cultures. Cultures of this type initially display a wave of differentiation to the double positive stage of T cell development that is observable at three to four weeks post-culture initiation. Continued propagation eventually produces a heterogeneous population of cells that collectively resemble thymocytes derived from animals incapable of β-selection. As TCRβ gene rearrangement occurs at this stage in normal thymocytes, we evaluated the rearrangement status of the co-culture derived cells. Sequential, weekly analysis revealed that TCRβ rearrangements occurred as expected in cultures that differentiated to the DP stage. After the initial wave of DP production however, TCRβ rearrangements were no longer detectable. Further analysis revealed that portions of the CD3 complex were expressed on the derived cells at levels equivalent to or higher than wild-type thymocytes at a similar maturational stage while pre-Tα levels were found to be expressed at a lower level. Finally, by using a novel in vitro β-selection assay, we demonstrate that the cultured cells still do not initiate differentiation past the DN3a stage. These data indicate that while ex vivo derivation and maintenance of T cell precursors is possible, standard OP9-DL1 culture conditions lead to development of cells resistant to induction of the β-selection program.