Observations of residual differentiation potential during lineage commitment.

We have recently described a subset of the multipotent progenitor pool that contains a common lymphoid progenitor. This subset of cells is lineage negative and expresses c-kit and Sca-1, but lacks expression of Thy 1.1 (Thyneg). Based on the observation that lethally irradiated mice transplanted with these cells die from anemia unless supported with competitor marrow, we hypothesized that these progenitors lacked erythroid potential. We analyzed the erythroid potential of these cells by transplanting them into mice allelic at the hemoglobin locus and compared their erythroid potential with the Thy-1.1low (Thylow) subset that contains hematopoietic stem cells. We also performed CFU-C assays in methylcellulose containing recombinant cytokines and determined erythroid contribution to colonies using in situ benzidine staining. Donor-derived hemoglobin was observed following transplant of Thyneg cells, even though 19 of 20 of these animals died from anemia. In contrast, recipients of Thylow cells showed complete donor-derived engraftment 30 days following transplant. While approximately 60% of day 4 colonies derived from Thyneg cells expressed hemoglobin, by day 11 less than 5% were hemoglobinized. In contrast, greater than 70% of the Thylow subset contained hemoglobinized cells at the end of the observation period. A similar transient appearance of myeloid progeny was also observed in colonies derived from c-kitlow Thyneg lymphoid progenitor cells. We conclude that these studies demonstrate commitment to the lymphoid lineage at the Thylow-to-Thyneg interface, and that the loss of erythroid and myeloid potential is gradual rather than abrupt. Hemoglobinized colonies may be undergoing apoptosis because of down-regulation of GATA-1 or because of a death signal from surrounding nonerythrocytic cells.

Phenotypic distinction and functional characterization of pro-B cells in adult mouse bone marrow.

A lymphoid-committed progenitor population was isolated from mouse bone marrow based on the cell surface phenotype Thy-1.1(neg)Sca-1(pos)c-Kit(low)Lin(neg). These cells were CD43(pos)CD24(pos) on isolation and proliferated in response to the cytokine combination of steel factor, IL-7, and Flt3 ligand. Lymphoid-committed progenitors could be segregated into more primitive and more differentiated subsets based on expression of AA4.1. The more differentiated subset generated only B lymphoid cells in 92% of total colonies assayed, lacked T lineage potential, and expressed Pax5. These studies have therefore defined and isolated a B lymphoid-committed progenitor population at a developmental stage corresponding to the initial expression of CD45R.

Rapid, B lymphoid-restricted engraftment mediated by a primitive bone marrow subpopulation.

Utilizing multiparameter flow cytometry, we have defined a subset of bone marrow cells containing lymphoid-restricted differentiation potential after i.v. transplantation. Bone marrow cells characterized by expression of the Sca-1 and c-kit Ags and lacking Ags of differentiating lineages were segregated into subsets based on allele-specific Thy-1.1 Ag expression. Although hematopoietic stem cells were recovered in the Thy-1.1low subset as previously described, the Thy-1.1neg subset consisted of progenitor cells that preferentially reconstituted the B lymphocyte lineage after i.v. transplantation. Recipients of Thy-1.1neg cells did not survive beyond 30 days, presumably due to the failure of erythroid and platelet lineages to recover after transplants. Thy-1.1neg cells predominantly reconstituted the bone marrow and peripheral blood of lethally irradiated recipients with B lineage cells within 2 weeks, although a low frequency of myeloid lineage cells was also detected. In contrast, myeloid progenitors outnumbered lymphoid progenitors when the Thy-1.1neg population was assayed in culture. When Thy-1. 1low stem cells were rigorously excluded from the Thy-1.1neg subset, reconstitution of T lymphocytes was rarely observed in peripheral blood after i.v. transplantation. Competitive repopulation studies showed that the B lymphoid reconstitution derived from Thy-1.1neg cells was not sustained over a 20-wk period. Therefore, the Thy-1. 1neg population defined in these studies includes transplantable, non-self-renewing B lymphocyte progenitor cells.

B cell development is perturbed in bone marrow from c-fos/v-jun doubly transgenic mice.

c-fos and c-jun gene products form a heterodimeric complex (AP-1) that regulates target gene expression by binding to a specific DNA sequence motif. In order to study a role of AP-1 (Fos/Jun) in growth and differentiation of immature B lineage cells, we have established and mated two independent transgenic mice carrying the mouse c-fos gene or the viral v-jun gene fused to the H-2K promoter. IL-7 dependent bone marrow cell culture from doubly transgenic (H2-fos/jun) mice demonstrated severe delay of early B cell development. Proliferation of pre-B cells in the fresh bone marrow from H2-fos/jun mice to IL-7 stimulation was very low. These results suggest that the deregulated production of AP-1 perturbs IL-7 mediated proliferation and differentiation of immature B cells.