The ability to study lymphocyte differentiation in culture has been greatly advanced by the availability of the OP9 bone marrow stromal cell line, which was derived from an op/op mouse and thus lacks M-CSF. As a result, the normal default myeloid differentiation from bone marrow-derived stem and progenitor cells does not occur, and lymphocyte differentiation is favored. Introduction of the Notch ligand Delta-like 1 into OP9 cells results in promotion of T cell development and parallel suppression of B cell development. While the OP9-DL1 model of T cell development works quite well when fetal liver-derived progenitors are cultured, the success of T cell development from adult bone marrow-derived progenitors has been more difficult to reproduce. We have undertaken a systematic analysis of variables that can prevent efficient T cell development in OP9-DL1 cultures, and have found that one limiting factor that impacts the efficiency of differentiation of both T and B cell lineages is the accumulation of ammonium ions as a result of the spontaneous decomposition of l-glutamine. L-glutamine, which is present at 2 to 4 mM in standard tissue culture media, is unstable and will spontaneously degrade to form ammonium ions and pyroglutamic acid at a rate of 1%/day at 4°C and at a 10-fold higher rate at 37°C. To evaluate the effects of the two major products of l-glutamine decomposition on lymphoid differentiation, we added each product to differentiation cultures at 3 mM in the presence of a stable source of l-glutamine (l-alanyl-l-glutamine). Cultures were established in 1 ml containing 4×104 stromal cells (OP9 for B cell differentiation, OP9-DL1 for T cell differentiation), 1×103 bone marrow-derived lymphoid progenitors enriched by phenotype (c-kit+LinnegSca-1+Thy-1.1neg), and 5 ng/ml Flt3L plus 5 ng/ml IL-7. Every 3 to 4 days, cultures were harvested and passaged onto fresh stromal cell monolayers; lymphoid cells were counted and evaluated for surface antigen expression at each passage. While addition of pyroglutamic acid had no inhibitory effect on lymphocyte growth or differentiation, addition of ammonium chloride slowed growth and prevented differentiation of both T and B lymphocytes. Growth of the stromal cell monolayers was not affected by ammonium chloride at the concentrations utilized in these studies. We conclude that freshly-prepared culture medium, preferably containing a stabilized form of l-glutamine, is a critical aspect contributing to the success of lymphocyte differentiation cultures established from adult bone marrow cells. We also found that decreasing IL-7 concentrations to 1 ng/ml resulted in more rapid differentiation of T cells and a more balanced representation of CD4 and CD8 single positive cells. Our studies help define optimal conditions for differentiation of bone marrow-derived lymphoid progenitor cells into T and B lineages in vitro, and provide evidence that hematopoietic differentiation displays variable degrees of sensitivity to ammonium ions derived from decomposition of l-glutamine. These results will help define optimal conditions for expansion and differentiation of hematopoietic stem and progenitor cells in vitro.

Author notes

Disclosure: No relevant conflicts of interest to declare.