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Hematopoietic stem cells (HSC) are defined by self-renewal and multilineage differentiation potentials. In order to uncover the genetic program of HSC, we utilized high-density arrays to compare gene expression in highly purified mouse HSC and their mature progeny. One molecule specifically expressed in immature cells is CD27, a member of the TNF receptor family previously shown to play roles in lymphoid proliferation, differentiation, and apoptosis. We show here that the CD27 protein is expressed by about 90% of cells in a purified HSC population. Interestingly, the CD27pos cells are enriched for cells with short-term hematopoietic activities (colony forming potential in vivo and in vitro), while the minority CD27neg population is more effective in clonal long-term transplantation.

Thymic cellularity is maintained throughout life by progenitor cells originating in the bone marrow. In this study, we describe adult mouse bone cells that exhibit several features characteristic of prothymocytes. These include 1) rapid thymic engraftment kinetics following i.v. transplantation, 2) dramatic expansion of thymic progeny, and 3) limited production of hemopoietic progeny other than thymocytes. The adult mouse bone marrow population that is depleted of cells expressing any of a panel of lineage-specific Ags, stem cell Ag-1 positive, and not expressing the Thy1.1 Ag (Thy1.1−) (Thy1.1− progenitors) can repopulate the thymus 9 days more rapidly than can hemopoietic stem cells, a rate of thymic repopulation approaching that observed with transplanted thymocytes. Additionally, Thy1.1− progenitors expand prolifically to generate thymocyte progeny comparable in absolute numbers to those observed from parallel hemopoietic stem cell transplants, and provide a source of progenitors that spans multiple waves of thymic seeding. Nevertheless, the Thy1.1− population yields relatively few B cells and rare myeloid progeny posttransplant. These observations describe the phenotype of an adult mouse bone marrow population highly enriched for rapidly engrafting, long-term thymocyte progenitors. Furthermore, they note disparity in B and T cell expansion from this lymphoid progenitor population and suggest that it contains the progenitor primarily responsible for seeding the thymus throughout life.

Objective-The OP9-DL1 culture system is an in vitro model for T cell development in which activation of the Notch pathway by Delta-like 1 promotes differentiation of mature T cells from progenitors. The roles of specific cytokines in this culture system have not been well defined, and controversy regarding the role of IL7 has recently emerged. We examined the roles played by IL7, Flt3 ligand, and stem cell factor (SCF) in differentiation of adult bone marrow cells in the OP9-DL1 culture system.Methods-Hematopoietic progenitor cells isolated from mouse bone marrow were cultured with OP9 or OP9-DL1 stromal cells and evaluated for T and B lymphocyte differentiation using immunofluorescent staining.Results-IL-7 provided both survival/proliferation and differentiation signals in a dose-dependent manner. T cell development from the CD4/CD8 double negative (DN) stage to the CD4/CD8 double positive (DP) stage required IL-7 provided by the stromal cells, while differentiation from the DP to the CD8 single positive (SP) stage required addition of exogenous IL-7. SCF favored the proliferation of DN lymphoid progenitors and inhibited differentiation to the DP stage in a dose-dependent manner. Conversely, blocking the function of SCF expressed endogenously by OP9-DL1 cells inhibited proliferation of lymphoid progenitors and accelerated T lineage differentiation. Flt3 ligand promoted proliferation without affecting differentiation.Conclusion-These results validate the OP9-DL1 model for the analysis of T cell development from bone marrow-derived progenitor cells, and demonstrate specific roles of SCF, IL-7, and Flt3L in promoting efficient T lineage differentiation.

The stem cell pool can be fractionated by using the mitochondrial dye, rhodamine-123, into Rho low hematopoietic stem cells and Rho high progenitors. Rho low stem cells permanently engraft all lineages, whereas Rho high progenitors transiently produce erythrocytes, without substantial platelet or granulocyte production. We hypothesized that the inability of the Rho high cells to produce platelets in vivo was due to the fact that these cells preferentially engraft in the spleen and lack marrow engraftment. Initially, we demonstrated that Rho high progenitors produced more megakaryocytes in vitro than Rho low stem cells did. To study the activity of the Rho low and Rho high subsets in vivo, we used mice allelic at the hemoglobin and glucose phosphate isomerase loci to track donor-derived erythropoiesis and thrombopoiesis. Rho low stem cells contributed to robust and long-term erythroid and platelet engraftment, whereas Rho high progenitors contributed only to transient erythroid engraftment and produced very low numbers of platelets in vivo. Donorderived megakaryopoiesis occurred at higher densities in the spleen than in the bone marrow in animals receiving Rho low stem cells and peaked around day 28. Blockade of splenic engraftment using pertussis toxin did not affect the peak of splenic megakaryopoiesis, supporting the hypothesis that these megakaryocytes were derived from progenitors that originated in the bone marrow. These data emphasize that in vitro behavior of hematopoietic progenitor cell subsets does not always predict their behavior following transplantation. This study supports a major role for the spleen in thrombopoi IntroductionMurine hematopoietic stem cells have been shown to reside within a population of cells that do not express lineage-specific markers (Lin neg ) and express the antigens Sca-1, c-kit, and Thy-1.1. Cells of this phenotype comprise the stem cell pool in certain strains of mice. 1,2 Rhodamine-123 (Rho) is a mitochondrial dye that stains cells based on their state of activation in which the more metabolically active cells tend to fluoresce brightly and quiescent cells dimly. 3 Rho has been used to subfractionate the stem cell pool into quiescent and metabolically active subsets. The Rho low subset is highly enriched for hematopoietic stem cells, to the degree that as few as 4 of these cells can reconstitute a mouse for the lifetime of the animal. Observations of the behavior of these stem cells have led to the model of the 2-tiered stem-cell pool. 4 According to this model, the Rho low cells make rare contributions to the peripheral compartment, but are mobilized in response to physiologic needs, and represent most of the transplantable stem cells. In contrast, Rho high cells have a high rate of cell division and, thus, make frequent contributions to the peripheral compartment under normal conditions. However, Rho high cells perform poorly in transplantation experiments, producing mainly erythrocytes and contributing little to platelet or granulocyte recovery. 5,6 All previous stu...